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A great deal of additional information on the European Union is available on the Internet.
It can be accessed through the Europa server (http://europa.eu.int).
Cataloguing data can be found at the end of this publication.
Luxembourg: Office for Official Publications of the European Communities, 1998
ISBN 92-828-2437-3
European Communities, 1998

Reproduction is authorised, provided the source is acknowledged.
Printed in Belgium
The rules governing medicinal products
in the European Union
Volume 3A
Guidelines
Medicinal products for human use
Quality and biotechnology
1998 Edition
EUROPEAN COMMISSION
Directorate General III - Industry
Pharmaceuticals and cosmetics
THE RULES GOVERNING MEDICINAL PRODUCTS
IN THE EUROPEAN UNION
Volume 1 Pharmaceutical legislation

Volume 2 Notice to applicants

Volume 3 Guidelines
Volume 4 Good manufacturing practices

Medicinal products for human and veterinary use
Volume 5 Pharmaceutical legislation

Veterinary medicinal products
Volume 6 Notice to applicants

Volume 7 Guidelines
Volume 8 Maximum residue limits

Volume 9 Pharmacovigilance
Medicinal products for human use and veterinary medicinal products
FOREWORD
Directive 75/318/EEC describes the requirements for the demonstration of the quality, safety
and efficacy of medicinal products. The conduct of tests and studies for such demonstration
has been harmonised, both within the European Union and internationally.
Volume 3 of "The Rules Governing Medicinal Products in the European Union" incorporates
testing guidelines prepared within the European Union including those which have been
developed in the International Conference on Harmonisation (ICH) process. It is presented
in three parts:
Volume 3A - Quality and biotechnology
Volume 3B - Safety and the environment
Volume 3C - Efficacy and information on the medicinal product
These guidelines serve a two-fold objective. Firstly, they are intended to provide a basis for a
practical harmonisation of the manner in which the Member States and the European
Agency for the Evaluation of Medicinal Products interpret and apply the detailed
requirements for the demonstration of quality, safety and efficacy contained in the
Community directives. Secondly, they are intended to facilitate the preparation of
applications for marketing authorization which will be recognized as valid by all Member
States and the European Agency for the Evaluation of Medicinal Products.
The use of guidelines, which are not legally binding, rather than a formal legal instrument,
such as a directive, has been preferred in order to maintain an element of flexibility and not
to place undue legislative restraints on scientific progress. It is recognized that in some
cases, as a result of scientific developments, an alternative approach may be appropriate.
However, where an applicant chooses not to apply a guideline, that decision must be
explained and justified in the Expert Reports submitted by the company in support of the
application.
By their very nature, the guidelines must be updated in the light of scientific and technical
progress. Moreover, further guidelines are currently under discussion, thus it is intended
that this volume should be updated and revised as necessary.
These Notes for Guidance, which have no legal force, have been prepared by the Committee
for Proprietary Medicinal Products of the European Agency for the Evaluation of Medicinal
Products, in consultation with the competent authorities of the Member States, to assist
applicants for a marketing authorization for a medicinal product. In case of doubt, reference
should be made to the text of the relevant EEC Directives.
Ill
TABLE OF CONTENTS
QUALITY GUIDELINES
DEVELOPMENT PHARMACEUTICS AND PROCESS VALIDATION 3
MANUFACTURE OF THE FINISHED DOSAGE FORM 11
LIMITATIONS TO THE USE OF ETHYLENE OXIDE IN THE MANUFACTURE OF
MEDICINAL PRODUCTS 19
THE USE OF IONISING RADIATION IN THE MANUFACTURE OF MEDICINAL
PRODUCTS 23
CHEMISTRY OF ACTIVE SUBSTANCES 31
REQUIREMENTS IN RELATION TO ACTIVE SUBSTANCES 39
EUROPEAN DRUG MASTER FILE PROCEDURE FOR ACTIVE SUBSTANCES 47
IMPURITIES IN NEW ACTIVE SUBSTANCES *) 57
EXCIPIENTS IN THE DOSSIER FOR APPLICATION FOR MARKETING
AUTHORISATION OF A MEDICINAL PRODUCT 67
PLASTIC PRIMARY PACKAGING MATERIALS 75
SPECIFICATIONS AND CONTROL TESTS ON THE FINISHED PRODUCT 83
IMPURITIES IN NEW MEDICINAL PRODUCTS *) 95
VALIDATION OF ANALYTICAL PROCEDURES: METHODOLOGY *) 107
VALIDATION OF ANALYTICAL PROCEDURES: DEFINITIONS AND
TERMINOLOGY *) 119
STABILITY TESTING OF NEW ACTIVE SUBSTANCES AND MEDICINAL
PRODUCTS *) 127
STABILITY TESTING ON ACTIVE INGREDIENTS AND FINISHED PRODUCTS 143
STABILITY TESTING: REQUIREMENTS FOR NEW DOSAGE FORMS *) 153
PHOTOSTABILITY TESTING OF NEW ACTIVE SUBSTANCES AND MEDICINAL
PRODUCTS*) 157
QUALITY OF PROLONGED RELEASE ORAL SOLID DOSAGE FORMS 167
RADIOPHARMACEUTICALS 175
RADIOPHARMACEUTICALS BASED ON MONOCLONAL ANTIBODIES 185
QUALITY OF HERBAL REMEDIES 195
Table of contents
BIOTECHNOLOGY GUIDELINES 203

PRODUCTION AND QUALITY CONTROL OF MEDICINAL PRODUCTS DERIVED
BY RECOMBINANT DNA TECHNOLOGY 205
QUALITY OF BIOTECHNOLOGICAL PRODUCTS: ANALYSIS OF THE
EXPRESSION CONSTRUCT IN CELLS USED FOR PRODUCTION OF RDNA
DERIVED PROTEIN PRODUCTS*) 217
PRODUCTION AND QUALITY CONTROL OF CYTOKINE PRODUCTS DERIVED
BY BIOTECHNOLOGICAL PROCESSES '. 223
PRODUCTION AND QUALITY CONTROL OF MONOCLONAL ANTIBODIES 237
QUALITY OF BIOTECHNOLOGICAL PRODUCTS: STABILITY TESTING OF
BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS*) 263
GENE THERAPY PRODUCT QUALITY ASPECTS IN THE PRODUCTION OF
VECTORS AND GENETICALLY MODIFIED SOMATIC CELLS 275
USE OF TRANSGENIC ANIMALS IN THE MANUFACTURE OF BIOLOGICAL
MEDICINAL PRODUCTS FOR HUMAN USE 287
VIRUS VALIDATION STUDIES: THE DESIGN, CONTRIBUTION AND
INTERPRETATION OF STUDIES VALIDATING THE INACTIVATION AND
REMOVAL OF VIRUSES 295
VALIDATION OF VIRUS REMOVAL/INACTIVATION PROCEDURES: CHOICE OF
VIRUSES 311
MINIMISING THE RISK OF TRANSMITTING AGENTS CAUSING SPONGIFORM
ENCEPHALOPATHY VIA MEDICINAL PRODUCTS 315
TESTS ON SAMPLES OF BIOLOGICAL ORIGIN. 323
PLASMA DERIVED MEDICINAL PRODUCTS 333
PLASMA POOL TESTING 351
HARMONISATION OF REQUIREMENTS FOR INFLUENZA VACCINES 355
ALLERGEN PRODUCTS 373
ASSESSING THE EFFICACY AND SAFETY OF HUMAN PLASMA DERIVED
FACTOR VIILC AND FACTOR IX:C PRODUCTS IN CLINICAL TRIALS IN
HAEMOPHILIACS BEFORE AND AFTER AUTHORISATION 381
ASSESSING THE EFFICACY AND SAFETY OF NORMAL INTRAVENOUS
IMMUNOGLOBULIN PRODUCTS FOR MARKETING AUTHORISATIONS 393
INDEX 405
VI
QUALITY GUIDELINES
3AQ1a
DEVELOPMENT PHARMACEUTICS AND PROCESS

VALIDATION
Guideline Title Development P h a r m a c e u t i c s a n d Process Validation

Legislative basis Directive 75/318/EEC as a m e n d e d
Date of first adoption April 1988
Date of entry i n t o October 1988
force
Status Last revised 1988
P r e v i o u s titles/other None
references
Additional Notes This note for guidance c o n c e r n s t h e application of Part
2, sections A.4 and of t h e Annex to D i r e c t i v e
75/318/EEC as a m e n d e d w i t h a view to the granting of a
m a r k e t i n g a u t h o r i s a t i o n for a medicinal product.
CONTENTS
DEVELOPMENT PHARMACEUTICS
1. INTRODUCTION
2. CONSTITUENTS
3. COMPOSITION
4. CONTAINER
II PROCESS VALIDATION
3AQ1a
DEVELOPMENT PHARMACEUTICS AND PROCESS

VALIDATION
I. DEVELOPMENT PHARMACEUTICS
L INTRODUCTION
Pharmaceutical development studies may need to be carried out to establish that the type of
dosage form selected and the formulation proposed are satisfactory for the purpose specified
in the application. They also aim to identify those formulation and processing aspects that
are crucial for batch reproducibility and which therefore need to be monitored routinely.
Because of the great variety in active substances and dosage forms, this note for guidance is
only an illustration of the type of information which has been found useful in establishing
the factors which affect quality of a finished product.
2. CONSTITUENTS
2.1 Active substances
2.1.1 Compatibility
The compatibility of the active substance(s) with the excipients should, where necessary, be
demonstrated.
2.1.2 Physical characteristics

It may be necessary to study the effect of such parameters as e.g. crystal form, moisture
content and particle size of the active substance on the formulation. The latter may be of
importance in bioavailability, content uniformity, suspension properties, stability and for
eye irritation studies. Having identified a parameter as being critical, its control should
then be reflected in the active substance specification, or dealt with by other appropriate
means.
2.1.3 Overage
Overages are primarily employed to cover losses during manufacture, i.e. manufacturing
overage, and/or during shelf life, stability overage. The inclusion of any overage should be
justified.
2.2 Excipients
2.2.1 An explanation should be provided with regard to the function of the excipients in the
formulation.
2.2.2 Excipient compatibility should be established where relevant.
2.2.3 Where unusual excipients are used in the manufacture of the product, e.g. the matrix
of a slow release preparation, full information on the composition and function of the
3AQ1a
excipient in the formulation of the product should be furnished together with any
documentation which may be available to demonstrate safety of the raw material. A new
substance introduced as an excipient will be regarded in the same way as a new active
substance, unless it is already approved for use in food by the same route of administration.
3. COMPOSITION
3.1 Liquid dosage forms
3.1.1 Physical parameters
a) pH
Evidence should be presented to show that the effect of pH within the specified range of the
formulation has been investigated. Consideration should be given to the effect of pH on active
constituent(s), and, where relevant, on the antimicrobial efficacy. Should such a study show
positive results any long-term effects would need to be investigated during stability studies.
b) others
Depending on the formulation, such parameters as ease of dissolution and redispersion,
particle size, aggregation, rheological properties, etc. should also be considered during
pharmaceutical development studies.
In the formulation of parenteral products, consideration may have to be given to such factors
as tonicity adjustment, globule size of emulsions, particle size and shape as well as changes
in crystal form, etc.
3.1.2 Additives
- preservatives,
- antioxidants,
- others.
The concentration of additive(s) incorporated into the formulation should be shown by
experimental results to be optimum for the intended usage. Consideration should therefore be
given to such factors as storage, reconstitution and dilution before use and frequency of
opening the pack when choosing suitable level(s) of additive(s) and designing tests to
establish efficacy of a preservative system. Large packs intended for dispensing purpose
may require more stringent testing. Both antibacterial and antifungal efficacy should be
demonstrated and the test should include suitable positive and negative controls. Testing
conditions and the results thereby obtained must be reported.
3.1.3 Compatibility with other products

This is of particular importance for products to be administered intravenously.
Where the data sheet gives instructions for dilution before administration, data should be
presented to demonstrate physical and chemical compatibility with the recommended
diluents over the recommended or anticipated period of use.
3AQ1a
3.2 Semi-solid dosage forms

3.2.1 Physical parameters
a) pH
Evidence should be presented to show that the effect of pH within the specified range of the
formulation, where relevant, including preservative activity, has been investigated. Where
a significant effect is observed, any long-term effects would need to be investigated during
stability studies.
b) others
Where the active substance is suspended rather than dissolved, particle size control and
particle size aggregation should be taken into consideration during development studies..
Rheology studies may also need to be carried out during the development of semi-solids.
3.2.2 Additives
- preservatives,
- antioxidants,
- others.
The concentration of additive(s) incorporated into the formulation should be shown by
experimental results to be optimum for the intended usage. Consideration should therefore be
given to such factors as storage, reconstitution, dilution before use and frequency of opening
the pack when choosing suitable level(s) of additive(s) and designing tests to establish the
efficacy of the preservative system. In such tests, both antibacterial and anti fungal efficacy
should be demonstrated and the test should include suitable positive and negative controls.
Testing conditions and the results thereby obtained must be reported.
3.3 S o l i d d o s a g e f o r m s
3.3.1 Dissolution
The dissolution apparatus used in the testing of both unmodified and modified release
preparations should be either of those described in the European Pharmacopoeia. Where these
prove unsuitable, dissolution test equipment described in the National Pharmacopoeia of the
Member State should be adopted as second choice, or, failing this, any other method.
However, justification for the use of a method other than European Pharmacopoeia must be
put forward.
a) Unmodified release preparations
Dissolution tests must be performed during development and stability studies in order to
establish whether such testing would need to be done during stability studies and routinely as
part of the finished product specification.
b) Modified release preparations
The choice of dissolution test conditions and release rates adopted for assessing batch
reproducibility needs to be justified. This should take account of in vivo studies carried out to
establish the release and absorption profile of the product and would, if feasible, consist of a
study correlating in vitro release rates to in vivo results to allow meaningful batch
reproducibility evaluation. Such a correlation would be of particular importance for
3AQ1a
medicinal products containing active substances with a narrow therapeutic window. A

significant change in composition, method or site of manufacture or equipment, control tests
on starting materials or finished product may however necessitate further in vitro
correlation studies or in vivo bioavailability studies.
3.3.2 Homogeneity
The European Pharmacopoeia includes a requirement for uniformity of content of single-
dose preparations where the amount of active constituent is less than 2 mg per dose or less
than 2% by mass of the total mass. Notwithstanding this requirement, the adequacy of the
mixing process in obtaining the required homogeneity of the mixture ought to be considered
for all solid dose forms i.e., tablets, powders, etc.
4. CONTAINER
Appropriate studies should be performed to demonstrate the integrity of the container and
closure. A possible interaction between product and container may need to be considered.
4.1 Sorption to container

Data should be presented to show that consideration has been given to the possibility of
sorption of the active constituent(s) and additive(s) from liquid or semi-solid formulations if
relevant to safety or stability. These phenomena are known to occur with rubber closures and
with both glass and plastic containers and administration sets. Where evidence exists for
significant sorption to administration sets, the data sheet should include an appropriate
reference to this fact.
4.2 Leaching
Data should be presented to show that there is no significant leaching of any pack
component, including label adhesive, into liquid or finely divided solid preparations over
the shelf life period, where relevant.
4.3 Dose reproducibility

If a dosing device is used, evidence should be presented that a reproducible dose of the product
is delivered under testing conditions, which, as far as possible, are relevant for the use of the
product by the patient.
8
3AQla
II. PROCESS VALIDATION

Whereas development pharmaceutics is concerned with establishing that the proposed
formulation is satisfactory for the purpose specified, process validation is intended to
establish that the proposed manufacturing process is a suitable one and yields consistently a
product of the desired quality. While process validation is generally a concept more closely
associated with Good Manufacturing Practice (GMP) and therefore falling into the area of
inspections, if a non-standard method of manufacture is used or if certain aspects of the
method of manufacture are crucial for product quality, efficacy or safety but cannot
necessarily be detected by analytical means, data on process validation may be required in
applications for marketing authorisation for a medicinal product. Areas mostly concerned
are process environment, process equipment and the manufacturing process itself, the latter
being the most important one. Thus data may be required to establish e.g. that:
- non-standard sterilisation conditions provide an acceptable level of assurance of
product sterility,
or
- the manufacturing process for a modified release system will only vary to an extent
that will still yield a product of the desired quality and not have any effect on product
efficacy or safety.
3AQ2a
MANUFACTURE OF THE FINISHED DOSAGE FORM
Guideline Title Manufacture of t h e Finished Dosage F o r m

Date of first adoption September 1995
Date of entry i n t o May 1996

force
Status This version re-issued in April 1996
P r e v i o u s titles/other N o n e /CPMP/QWP/486/95
references
Additional Notes This note for guidance concerns the application of Part
2, section D of t h e Annex to Directive 75/318/EEC as
amended, with a view to the g r a n t i n g of a m a r k e t i n g
a u t h o r i s a t i o n for a new medicinal product.
CONTENTS
1. INTRODUCTION
2. THE APPLICATION FOR MARKETING AUTHORISATION AND GMP
3. MANUFACTURING FORMULA
4. DESCRIPTION OF THE MANUFACTURING PROCESS
5. DESCRIPTION OF THE MANUFACTURING CHAIN
6. VALIDATION OF THE MANUFACTURING PROCESS
7. SPECIAL ITEMS
11
3AQ2a
MANUFACTURE OF THE FINISHED DOSAGE FORM
L INTRODUCTION
According to Directive 65/65/EEC, an application for a marketing authorisation shall
contain a brief description of the method of preparation.
This is described in more detail in the Annex, Part 2 of Directive 91/507/EEC, which states:
"The description of the method of preparation [...] shall be drafted in such a way as to give an
adequate synopsis of the nature of the operations employed.
For this purpose it shall include at least:

mention of the various stages of manufacture, so that an assessment can be made of
whether the processes employed in producing the pharmaceutical form might have
produced an adverse change in the constituents,
in the case of continuous manufacture, full details concerning precautions taken to
ensure the homogeneity of the finished product,
the actual manufacturing formula, with the quantitative particulars of all the
substances used, the quantities of the excipients, however, being given in approximate
terms in so far as the pharmaceutical form makes this necessary; mention shall be
made of any substances that may disappear in the course of manufacture; any overage
shall be indicated and justified,
a statement of the stages of manufacture at which sampling is carried out for in-
process control tests, where other data in the documents supporting the application show
such tests to be necessary for the quality control of the finished medicinal product,
experimental studies validating the manufacturing process, where a non-standard
method of manufacture is used or where it is critical for the product,
for sterile products, details of the sterilisation processes and/or aseptic procedures
used."
This note for guidance provides guidance on the background and the interpretation of some
aspects of the text of the Directive.
This note for guidance does not pertain to biological medicinal products such as vaccines,
sera, toxins and allergens, products derived from human blood and plasma as well as
medicinal products prepared biotechnologically.
2. THE APPLICATION FOR MARKETING AUTHORISATION

AND GMP
Medicinal products on the market in the EC should be produced under the EC Rules for Good
Manufacturing Practice (GMP), see Directive 91/356/EEC.
Many general elements of GMP and quality assurance do not need to be described in the
application for marketing authorisation. Examples are qualifications of key personnel,
13
3AQ2a
cleaning procedures for the production equipment and production areas, final packaging
and labelling procedures etc.
In general, the dossier for marketing authorisation should contain only those elements of the
quality assurance which are specific for the medicinal product, whereas non product related
elements of the quality assurance fall within the field of GMP, consequently, no description
is necessary in the application for a marketing authorisation.
This note for guidance addresses the items that should be presented in the application for a
marketing authorisation. For items not to be covered by the application for a marketing
authorisation, the obligation for adherence to the EC GMP principles is implicit.
3. MANUFACTURING FORMULA
The intended batch size should be indicated.
An application for a variable and/or alternative batch size should be justified. Consistent
conformity of the finished product to all the specifications should be made plausible.
The names and quantities of all ingredients used in the course of the manufacture should be
stated. This includes ingredients which are removed from the product during the production
process, such as solvents. Substances that may not always be used should also be mentioned,
such as acids and alkalis for pH adjustment. Overages must be indicated in quantitative
terms and justified in the section on Development Pharmaceutics.
For each ingredient, the allowed upper and lower acceptance limits for the actual quantity of
each ingredient from the nominal quantity of the batch manufacturing formula should be
stated.
For active ingredients, these acceptance limits should be within 95 to 105% of the nominal
quantity; for excipients, acceptance limits of 90 to 110% of the nominal quantity are
acceptable without further justification.
Wider acceptance limits may be acceptable but should be justified by showing that batches
with a composition close to the upper and lower proposed acceptance limits remain within the
finished product specifications.
When that the quantity of an active ingredient to be used is calculated from the actual assay
value of the batch of that active ingredient ("factorisation"), this has to be indicated. If
another ingredient is used to keep the total mass per batch equal to the quantity provided for
in the batch manufacturing formula, this should also be indicated.
4. DESCRIPTION OF THE MANUFACTURING PROCESS

A description of the manufacturing process should be given.
A proposal to allow alternative steps in the manufacturing process (for instance: two
alternative sterilisation methods for the container) should be accompanied by evidence
showing that all processes proposed will consistently produce a finished product in
compliance with the specifications.
If relevant (see below), the apparatus to be used has to be described. The in-process controls
and corresponding acceptance limits need to be described as well, when relevant (see below).
14
3AQ2a
The various steps in the manufacturing process and corresponding in-process controls
should also be shown in a flow-chart.
The presented data on the manufacturing process, apparatus and in-process controls are
binding for the future manufacturing of the medicinal product, unless authorisation for
changes is given by the Competent Authority.
It is in the interest of both the applicant and the regulatory authorities to avoid unnecessary
applications for variations. Very detailed descriptions of the manufacturing process,
apparatus and in-process controls should therefore be avoided.
In selecting the necessary level of detail the following should be considered:
the testing at release of the finished product,
the description of the manufacturing process and apparatus,
the in-process controls and validated acceptance limits.
Together these should provide a high degree of probability that each unit of every batch of the
finished product, will be in conformity with the specifications.
So, if the consistent quality of a medicinal product can be fully safeguarded by the "implicit"
production under GMP and testing of the finished product at release, the description of the
manufacturing process need not be comprehensive, and apparatus and in-process controls
need not to be described.
However, many quality parameters that are tested at release do not provide sufficient
certainty of the quality of the whole batch from a statistical point of view, because the quality
parameter may not necessarily be homogeneous within the batch.
An example is the homogeneous distribution of the active ingredient in solid and semi-solid
dosage forms, i.e. content uniformity. Testing at release alone does not provide sufficient
certainty for the content uniformity of the whole batch from a statistical point of view.
So, the apparatus to be used and the appropriate in-process controls (i.e. mixing time, mixing
speed etc.) and the validated acceptance limits for these in-process controls (see below) must
be proposed in the application file.
Another example is sterilisation. For all sterilisation processes, appropriate in-process
controls and their acceptance limits are to be described in the application file, see below.
5. DESCRIPTION OF THE MANUFACTURING CHAIN

An account shall be given of the sites at which each stage of the manufacturing and
assembly operations takes place. Different manufacturing sites belonging to the same
company shall be mentioned as separate units. The company responsible for the final
approval of the release of the product onto the market shall be specified.
6. VALIDATION DATA OF THE MANUFACTURING PROCESS

Validation studies that are used to identify critical steps in non-standard manufacturing
processes are part of the Development Pharmaceutics, and should be described in Part IIA of
the application file.
15
3AQ2a
Examples are: new dosage forms, the manufacturing of liposomes, etc.

Irrespective of these Development Pharmaceutical process validations, process Validation
results of the actual production process must be described in Part IIB if conformity to the
finished product specifications cannot be guaranteed to an acceptable degree of statistical
certainty by testing the finished product at release. This holds also for standard
manufacturing processes.
Examples are mixing, granulation and emulsifying processes of solid and semi-solid
dosage forms and non-pharmacopoeial sterilisation procedures, see below.
Process validation data obtained with closely related medicine products may be acceptable.
Please note that notwithstanding a successful process validation, the quality parameters
related to the validated process should be specified under the release specifications and end
of shelf life specifications. For instance, sterility should always be specified at release and
end of shelf life, notwithstanding a successful validation of the sterilisation process. Also,
the content uniformity of solid and semi-solid dosage forms should be specified in the
release and end of shelf life specifications, notwithstanding a successful process validation
with respect to homogeneity.
It may be acceptable to refrain from the routine testing at release of such a specification
("parametric release"), see the note for guidance Specifications and Control Tests on the
Finished Product. With respect to parametric release in relation to sterilisation, the text of the
Ph. Eur. "Methods of preparation of sterile products" is to be observed.
7. SPECIAL ITEMS
7.1 Method of sterilisation
The choice of the method of sterilisation should be justified under Development
Pharmaceutics, Part IIA.
According to the text of the Ph. Eur.: "Methods of preparation of sterile products", terminal
sterilisation in the final container is to be preferred. Refraining from terminal sterilisation
in the final container should be justified in the application file.
In Part IIB the actual sterilisation process to be applied should be described.
All sterilisation processes should be carried out according to the instructions of the Ph. Eur.
In the application file, an explicit statement should be made that the instructions of the Ph.
Eur. are followed.
According to the Ph. Eur., all sterilisation procedures should be validated and be carried out
under the EC GMP-rules. However, in the application file for marketing authorisation for
some sterilisation procedures no, or only limited validation data and data on the bioburden
of the product prior to the sterilisation need to be presented, see below.
In the case of terminal sterilisation in the final container by heat using a reference
condition of the Ph. Eur., only the time and temperature of the cycle and the acceptance
limits of the corresponding in-process controls need to be provided in the application file.
So, this holds for sterilisation by saturated steam at a minimum of 121 C for 15 min. and by
dry heat at a minimum of 160 C for at least 2 hours.
16
3AQ2a
In accordance with the Ph. Eur., these conditions should be met within all units. However,
the validation data showing that all units are subjected to these conditions are normally not
required in the application file. They may be requested by the competent authorities in
certain circumstances.
For terminal sterilisation cycles in the final container by heat with a time and/or
temperature below the values of the reference conditions of the Ph. Eur., not only the
acceptance limits for the in-process controls for time and temperature should be stated, but
also the maximum acceptable bioburden before sterilisation.
The results of the validation of the sterilisation cycle with regard to the effectiveness i n
terms of the Sterility Assurance Level (SAL) obtained should be presented in the application
file.
For sterilisation by filtration the maximum acceptable bioburden prior to the filtration must
be stated in the application. In most situations NMT 10 CFU's/100 ml will be acceptable,
depending on the volume to be filtered in relation to the diameter of the filter. If this
requirement is not met, it is necessary to use a pre-filtration through a bacteria-retaining
filter to obtain a sufficiently low bioburden.
The type of bacteria-retentive filter, and its pore size should also be described in the
application. Pore sizes of 0.22 um or less are acceptable without further justification, in
accordance with the Ph. Eur. A proposal to use a larger pore size in combination with an
additional sterilisation step has to be validated and justified in the application file.
Results of media filling fall within the field of GMP and need not be presented routinely i n
the application for marketing authorisation but may be requested by the competent authorities
in certain circumstances.
For sterilisation by gamma and electron radiation, see the note for guidance The Use of
Ionisation Radiation in the Manufacture of Medicinal Products.
For sterilisation by ethylene oxide, the provisions laid down in the note for guidance
Limitations to the use of Ethylene Oxide in the Manufacture of Medicinal Products should be
followed, i.e. its use as a sterilisation method is only acceptable if no other method of
sterilisation is available.
The application for marketing authorisation should contain a description of the apparatus,
quantitative data on the mixture of gases to be used, data on the bioburden prior to
sterilisation, the time of exposure of the gas, the temperature and humidity prior to and
during the sterilisation cycle, and the conditions for the removal of ethylene oxide. All these
conditions should be monitored by suitable in-process controls that are to be described
together with the acceptance limits for these in-process controls.
Results of the process validation should be presented to justify these acceptance limits for the
in-process controls. The results should demonstrate both an acceptable assurance, of sterility
and removal of ethylene oxide to an acceptable level.
Limits of NMT 1 ppm of ethylene oxide (if applicable, measured by means of a simulated use
extraction method) and NMT 50 ppm of ethylene chlorhydrin (or any other halogenated
ethylenehydrin) are acceptable without further justification, once sterilisation by ethylene
oxide has been justified.
Notwithstanding successful process validation, a limit for residual ethylene oxide, and the
corresponding validated analytical method, should be included in the product release- and
end of shelf life specifications.
17
3AQ2a
7.2 Re-processing of residual product

Procedures for the re-processing of residual product of non-biological medicinal products fall
within the field of GMP and need not to be described in the marketing authorisation dossier.
7.3 Removal of solvents or gases

If toxic gases or solvents are used in the manufacture of the finished product, release and
end of shelf life specifications for maximum acceptable residues of these solvents or gases
should be proposed for the product. A justification for the proposed limits can be presented i f
required in Part HA, Part IIB or HE of the file. Both toxicological and technological aspects
should be discussed in this justification.
Stages of the manufacturing process which affect the levels of such materials in the product
should be controlled by in-process controls and the acceptance limits for these in-process
controls should be validated. The results of these process validations should be presented i n
the application for marketing authorisation; see the paragraph in 7.1 above on ethylene oxide
sterilisation.
7.4 Cleaning of primary packaging material

Washing procedures of the primary containers and closures normally fall within the field
of GMP and are not needed routinely in the application for marketing authorisation but
may, in certain circumstances, be requested by the Competent Authority.
7.5 Sterilisation of primary packaging material

Where applicable, the sterilisation procedure of the primary containers and closures should
be described, and, when necessary, validated according to the paragraph on sterilisation.
7.6 Production areas

Details on the production area, i.e. specifications for the microbiological quality of the areas
and freedom from particles in the air normally fall within the field of GMP and are not
needed routinely in the application for marketing authorisation but may, in certain
circumstances, be requested by the Competent Authority.
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3AQ3a
LIMITATIONS TO THE USE OF ETHYLENE OXIDE IN

THE MANUFACTURE OF MEDICINAL PRODUCTS
Guideline Title Limitations to the use of Ethylene Oxide in the

Manufacture of Medicinal Products
Legislative basis Directive 75/318/EEC as amended
Date of first adoption December 1993
Date of entry into June 1994
force
Status Last revised February 1994
Previous titles/other None/III/9261/90
references
Additional Notes This note for guidance deals with the use of ethylene
oxide in the manufacture of medicinal products. It
should be read in conjunction with Volume IV of "The
Rules Governing Medicinal Products in the European
Union", particularly the annex on manufacture of sterile
medicinal products
CONTENTS
1 TOXICOLOGICAL BACKGROUND
2 CATEGORIES OF USE OF ETHYLENE OXIDE
3 SPECIFICATIONS/TEST PROCEDURES
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3AQ3a
LIMITATIONS TO THE USE OF ETHYLENE OXIDE IN

THE MANUFACTURE OF MEDICINAL PRODUCTS
This note for guidance deals with the use of ethylene oxyde in pharmaceutical raw
materials, finished products and containers.
1 TOXICOLOGICAL BACKGROUND
Ethylene oxide is a substance which, due to its epoxide structure, is counted among the very
reactive compounds. This reactivity also includes organic structures within cells and cell
nuclei. In this case, alkylation and reactions with DNA, RNA and proteins occur.
Cytotoxicity, carcinogenicity and mutagenicity of ethylene oxide, which have been
demonstrated by many in vitro and in vivo tests, are attributed to these properties.
Epidemiological data from many sources indicated that workers exposed to ethylene oxide at
their working place had an increased incidence of leukaemia and other tumours.
In view of the known positive potential of ethylene oxide for genotoxic carcinogenicity, it is
recommended that use is acceptable only when pharmaceutically absolutely necessary, and
then at a limit of 1 ppm. This limit is based on the current limit of detection for ethylene
oxide.
Any deviation upwards from this limit must be justified and defended, taking into account
the clinical risk/benefit assessment for the particular products under consideration.
2. CATEGORIES OF USE OF ETHYLENE OXIDE

Ethylene oxide is used in the synthesis of pharmaceutical raw materials and as a sterilant.
Since it is effective only as a surface sterialnt it should be used only for sterilising justified
and validated on an individual basis.
Ethylene oxide sterilisation should be used only where safer alternatives cannot be used.
3. SPECIFICATIONS/TEST PROCEDURES
Due to the above mentioned considerations, the limits are fixed on a mass/mass basis and
not on a daily intake basis. If no official test procedure (e.g. Pharmacopoeia) is available a
validated test procedure must be proposed by the applicant (see also note for guidance on
Validation of Analytical Procedures: Methodology).
3.1 Raw materials

Specification:
Ethylene oxide: 1 g/g
Ethylene chlorhydrin (or any other halogenated ethylenehydrine): 50 ug/g.
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3AQ3a
3.2 Finished product

If the residual ethylene oxide originates from its use in the raw material, its content must be
limited in the raw material.
Specification (when used on the finished product):
Ethylene oxide: 1 g/g
Ethylene chlorhydrin (or any other halogenated ethylenehydrine): 50 g/g.
3.3 Containers
Specification (based on simulated use):
Ethylene oxide: 1 /1 (container volume)
Ethylene chlorhydrin (or any other halogenated ethylenehydrine): 50 g/ml (container
volume).
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3AQ4a
THE USE OF IONISING RADIATION IN THE

MANUFACTURE OF MEDICINAL PRODUCTS
Guideline Title The use of Ionising R a d i a t i o n in t h e Manufacture of

Medicinal P r o d u c t s
Date of entry into J u l y 1992
force
Status Last revised December 1991
P r e v i o u s titles/other None/III/9109/90
references
Additional Notes This note for guidance deals w i t h t h e use of ionising
radiation in t h e m a n u f a c t u r e of medicinal p r o d u c t s . It
should be r e a d in conjunction with Volume TV of "The
Rules Governing Medicinal P r o d u c t s in t h e E u r o p e a n
Union", particularly t h e a n n e x on ionising r a d i a t i o n .
CONTENTS
1. INTRODUCTION
2. ADMINISTRATIVE DATA
3. MANUFACTURING PROCESS
4. VALIDATION OF THE IRRADIATION PROCEDURE
GLOSSARY
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THE USE OF IONISING RADIATION IN THE

MANUFACTURE OF MEDICINAL PRODUCTS
L INTRODUCTION
This note for guidance is intended for applicants wishing to use ionising radiation in the
manufacture of medicinal products. Irradiation may be used for microbial decontamination,
sterilisation or other treatments. Different materials or products may be irradiated: starting
materials, packaging materials, intermediate products, bulk products and finished products.
Information should be given in sufficient detail to enable the competent authority to evaluate
whether or not the manufacturing subprocess is effective and the product is safe for the
patient.
Manufacturers using ionising radiation in the manufacture of medicinal products should
refer to the Guide to Good Manufacturing Practice (Volume IV of "The Rules Governing
Medicinal Products in the European Union") and in particular to the annex on ionising
radiation used in the manufacture of medicinal products and, where relevant, to the annex
on manufacture of sterile medicinal products.
2. ADMINISTRATIVE DATA
a) The name and description of the product (including its packaging material) to be
irradiated should be given. Its shape, size and composition (type and quantity of
substances) should be described in detail. Furthermore, it should be made clear whether
starting materials, packaging materials, intermediate products, bulk products or the
finished product are irradiated. Sizes of production batches and of irradiation batches
should be defined. In the case of a continuous process, a batch comprises all the units
processed in a given period of time.
b) The purpose of the irradiation should be stated. Both the minimum dose to achieve this
purpose and the maximum permissible dose should be stated.
c) In addition to the names and addresses of all manufacturers involved in the
manufacture of the product, the name and address of the irradiation plant should be
given, making clear which operations are to be conducted at which site.
d) A copy of the authorisation referred to in Directive 75/319/EEC as amended and
covering the irradiation plant should be attached to the application.
3. MANUFACTURING PROCESS
Irradiation of a medicinal product is part of its manufacturing process and the description of
that part of processing should be sufficiently detailed. The application should include the
following information:
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3AQ4a
3.1 Description of the irradiation plant

a) Type (radionuclide source, electron generator) and builder of the plant;
b) working mode (batch- or continuous mode);
c) authorised and actual activity of the radionuclide in the radiation source (GBq), or the
maximum and minimum electron energy (MeV) of the generator as appropriate;
d) concise description of the plant including drawings, showing clearly the course of the
product within the plant, the position and geometry of the irradiation source and the
conveyor system including the source pass mechanism.
3.2 Description of the irradiation process

a) a description of the material to be irradiated should be given, including limits (if a n y )
on bioburden and any process aimed to limit or control the bioburden. Action to be
taken when particular bioburden limits are exceeded should be stated;
b) the number and positions of the irradiation containers in relation to the position of the
source during the whole dwelling time, and the method of moving them through the
chamber, should be described;
c) the material and dimensions of the irradiation container should be described;

d) the maximum total irradiation time and the maximum dwelling time of the product i n
the irradiation chamber should be stated;
e) results of dose mapping studies using a "dummy product" are required;
f) the loading pattern of the product must be stated for each irradiation container. If the
load consists of mixed products, the composition of the load m u s t be described
including their stated position in the irradiation container. The m e a n density of the
load and the acceptable m a x i m u m density should be given. A modification of the
loading p a t t e r n may be acceptable provided a new dose mapping is performed, showing
that the stated minimum and maximum doses are not exceeded;
g) when the loading pattern of the product within the irradiation container has been
defined, dose mapping should be performed with a sufficient number of appropriate
dosimeters to show the distribution of the absorbed dose within the loaded i r r a d i a t i o n
container and to show the places of m i n i m u m and m a x i m u m doses. This dose
mapping should be carried out for a representative number of irradiation containers to
determine the variability of the absorbed dose in the load of one container and the
differences between several containers.
Note: Separate dose mapping exercises should be carried out for each product or distinct
category of products and each pathway to be used for processing products.
h) a written standard operating procedure should be established including the following

m i n i m u m items:
- the loading pattern of product(s) within the irradiation container;
- the type, number and location of routine dosimeters within one irradiation batch
or within a stated period of time in the case of a continuous process;
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3AQ4a
- any adjustments to be applied to the routine dosimeter measurements to convert

them into the absorbed dose at both minimum and maximum positions;
- the stated minimum and maximum absorbed dose including experimentally
determined errors of dosimeters;
- whether or not repeated treatment is acceptable; for the product concerned, the
circumstances in which such repeated treatment is allowed, and the number of
occasions on which it is allowed for a particular batch;
- in the case of electron beam irradiators electron energy, average beam current,
beam width and conveyor speed should be stated with acceptable limits.
Note: The stated minimum dose is that required for the intended purpose, the stated
maximum dose is limited by unacceptable changes induced by irradiation in the product
and/or the packaging, or imposed by official restrictions.
A minimum absorbed dose of 25 kGy may be regarded as adequate for the purpose of
sterilising pharmaceutical components or products which have a low initial bioburden and
no radioresistant spores. Other doses may be used provided that a biological validation has
been performed.
4. VALIDATION OF THE IRRADIATION PROCEDURE

4.1 Validation with regard to the irradiation procedure and dose
a) with electron irradiation, if the maximum electron energy exceeds 10 MeV, it should be
demonstrated that no radionuclides develop in the product;
b) information derived from experimental investigations into the acceptable variation in
the loading pattern should be given;
c) information should be included on the errors due to the type of dosimeters used and on
the influence of their position;
d) information on the relationship between the absorbed doses in the extreme positions
within the load and the positions of routine dosimeters should be given.
4.2 Validation with regard to the purpose of irradiation (see section

2.b)
For reduction of bioburden and/or sterilisation:
a) Where appropriate, information on the bioburden of the product before irradiation
should be given with data from several batches to show the usual bioburden levels and
types of organisms usually present;
b) data on the reduction of bioburden during the irradiation with different doses,
including the minimum dose, should be given for at least 2 batches;
c) an inactivation curve derived from the above data should be submitted. If the test
specimen itself has a low bioburden, it should be artificially contaminated with
> 107 cfu/single unit preferably with a microorganism originally occurring in the
product and with a minimum D-Value of 3 kGy;
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3AQ4a
d) the bioburden limit on the product prior to irradiation should be based on data derived
from a) - c).
In other cases, experimental results should show that the purpose of irradiation has been
achieved.
4.3 Validation with regard to the quality of the product

a) Information should be given about any qualitative and quantitative changes in the
product, including its packaging, as a result of irradiation;
Note: Methods used for quantitative determinations should be validated in
accordance with the note for guidance Validation of Analytical Procedures:
Methodology.
b) Information should be given about the formation of radiolysis products or other
degradation or interaction products. Whenever possible, the radiolysis products should
be identified;
c) the results of the studies carried out with high doses of radiation to determine the
maximum dose should be given;
d) as assessment of the significance of any observed changes should be included;
e) information should be given about the effect of irradiation on the stability of the product
and therefore stability studies should be performed on products which have received the
maximum absorbed dose.
Note: The relevance of any changes in the product induced by irradiation as
regards quality of the product as well as health and safety of the patient should be
discussed. The toxicological risks caused by products of irradiation (see section 3.3.b)
should be evaluated. Safety of the irradiated product for the patient should be discussed
in the expert report.
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3AQ4a
GLOSSARY
Absorbed Dose
The quantity of radiation energy imparted per unit mass of material. The unit of absorbed
dose is the Gray (Gy) where 1 Gray is equivalent to absorption of 1 Joule per kilogram (J.kg-
1).
Batch
A defined quantity of starting material, packaging material or product processed in one
process or series of processes so that.it could be expected to be homogeneous.
Note: To complete certain stages of manufacture, it may be necessary to divide a batch into
a number of subbatches, which are later brought together to form a final homogeneous batch.
In the case of continuous manufacture, the batch must correspond to a defined fraction of the
production, characterised by its intended homogeneity.
For control of the finished product, the following definition has been given in Directive
75/318/EEC as amended: 'For the control of the finished product, a batch of a proprietary
medicinal product comprises all the units of a pharmaceutical form which are made from
the same initial mass of material and have undergone a single series of manufacturing
operations or a single sterilisation operation or, in the case of a continuous production
process, all the units manufactured in a given period of time'.
Bioburden
The total number of all viable aerobic bacteria, yeasts and moulds expressed as colony
forming units (cfu) per unit or gram of product.
Bulk Product
Any product which has completed all processing stages up to, but not including, final
packaging.
Dose Mapping
An exercise conducted within the irradiation equipment to determine the distribution of
absorbed dose throughout a load of product or simulated product of specified density ("dummy
product") arranged in the irradiation container in a defined configuration.
Dosimeter
A device or system having a reproducible measurable response to radiation, which can be
used to measure the absorbed dose in a given material.
Dummy Product
Homogeneous material of known density for filling the irradiation container for the purpose
of carrying out dose distribution experiments with ionising radiation.
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3AQ4a
Finished Product
A medicinal product which has undergone all stages of production including packaging.
Intermediate Product
Partly processed material which must undergo further manufacturing steps before it becomes
a bulk product.
Irradiation Container
The outermost container in which the products are irradiated.
Packaging Material
Any material employed in the packaging of a product, excluding any outer packaging used
for transportation or shipment. Packaging materials are referred to as primary or
secondary according to whether or not they are intended to be in direct contact with the
product.
Starting Material
Any substance used in the production of a product, but excluding packaging materials.
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3AQ5a
CHEMISTRY OF ACTIVE SUBSTANCES
Guideline Title Chemistry of Active S u b s t a n c e s

Date of first adoption October 1987
Date of entry i n t o October 1987
force
Previous titles/other Chemistry of Active Ingredients
references
Additional Notes This note for guidance concerns t h e application of P a r t
2, section C of t h e Annex to Directive 75/318/EEC as
amended with a view to t h e g r a n t i n g of a m a r k e t i n g
authorisation for a medicinal product. The section on
impurities is r e p l a c e d by t h e guideline Impurities in
New Active Substances for new active substances. F o r
a b r i d g e d a p p l i c a t i o n s , biotechnological/biological
products a n d o t h e r p r o d u c t s exempted from t h e
" i m p u r i t i e s " guideline, t h e s e r e q u i r e m e n t s c o n t i n u e to
apply.
CONTENTS
1. INTRODUCTION
2. IDENTITY OF MATERIALS
3. MANUFACTURE
4. DEVELOPMENT CHEMISTRY
5. IMPURITIES
6. ACTD7E SUBSTANCE SPECIFICATION
7. BATCH ANALYSIS
8. REFERENCE STANDARDS
9. RADIOLABELLED PRODUCT
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3AQ5a
CHEMISTRY OF ACTIVE SUBSTANCES
1 INTRODUCTION
The purpose of this note for guidance is to set out the type of information required for the
control of new active substances used for the first time in a medicinal product, which are not
described in the European Pharmacopoeia or a pharmacopoeia of a Member State.
2. IDENTITY OF MATERIAL
This section deals with the identity, nomenclature and chemical structure of the active
substance which is the subject of the application for marketing authorisation. Only brief
details of physical characteristics should be stated, as full details and proof of structure are
required later.
2.1 Nomenclature
- International Non-Proprietary Name (INN),
- National Approved Names, ( )
US Adopted Name (USAN),
- Laboratory Code(s),
- Systematic Chemical Name(s),
- Other Names (e.g. Proprietary).
2.2 Description
- physical form,
- structural formula,
- molecular formula,
- relative molecular mass.
A brief description should be given of the appearance of the material. Where possible, the
structural formula should be given diagrammatically with all known stereochemistry
indicated conventionally, with molecular formula and relative molecular mass; otherwise a
detailed description of the nature of the substance should be given.
The relative molecular mass of the therapeutically active moiety should also be included,
where appropriate.
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3AQ5a
3. MANUFACTURE
A concise but comprehensive account of the manufacture of the active substance should be
provided. The headings given below should be followed where the active substance concerned
is a totally synthetic product. Some modification may be required where the molecule is only
partially synthetic e.g. penicillin-derivatives.
3.1 Manufacturing process

When a complete or partial chemical synthesis is involved, this should be represented by
diagrams of the chemical reactions in the form of a flow sheet.
3.2 Description of process

An appropriate description should be given of each stage of the manufacture, including,
where applicable:
- solvents and reagents used,
- catalysts used,
- conditions of reactions where these are critical,
- information on intermediates which are isolated and purified,
- details of the final purification and solvents involved.
The description of the process should indicate the scale of manufacture. It is often helpful if
an indication of the yield produced at each stage is given.
The description must normally fully define the method of synthesis. However, if alternative
steps or solvents are proposed these should be justified and show that the final quality of
material obtained does not differ significantly.
3.3 Quality control during synthesis

3.3.1 Starting materials
Describe the analytical controls which are applied to ensure that the starting materials,
which make a significant contribution to the molecular formula, and any reagents are
correctly identified and are shown to be of a satisfactory quality. An indication of the content
of significant impurities in starting materials should be given. Specifications for solvents
used in the final stages of synthesis, crystallisation and/or washing should be submitted.
The criteria for accepting or rejecting batches of these materials should be indicated. The
control of starting materials should be designed to detect isomeric or other impurities which
are potentially reactive and could be carried through to the final product of the synthesis.
3.3.2 Intermediate control

The quality control checks which are carried out at each stage of the process and on the
isolated intermediates should be described. A statement of the test procedure(s) and criteria
for acceptance should be given for each stage, where appropriate.
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3AQ5a
4. DEVELOPMENT CHEMISTRY
This section should indicate the research and development programme which h a s been
undertaken on the new active substances to investigate the evidence of structure and the
chemical and physico-chemical properties.
The findings described in this section should be reflected in the control tests on the active
substance by which batch-to-batch uniformity is controlled.
4.1 Evidence of chemical structure

A scientific discussion of the chemistry of the active substances molecule should be g i v e n
and should include, where applicable, unequivocal proof of structure, configuration,
conformation and potential isomerism. This should include a presentation of the stereo
chemical properties of the molecule, e.g. geometric isomerism (cis/trans, E/Z), number of
chiral centres and configuration at each centre. A s u m m a r y and discussion of the
unequivocal proof of structure by the experts involved in the Expert Report can often provide
useful additional background information. Care should be taken that the visual evidence of
spectra is completely legible when reproduced in the copies of the application. It is important
that the evidence of structure should be related to the actual material to be used in the
marketed product, especially for highly complex molecular structures. Where the data
included in this sub-heading are from a source of synthetic process other t h a n t h a t covered by
the application (i.e. different routes),evidence may be required to confirm the structural
identity of the different materials. This is particularly important where toxicity work h a s
been carried out on material from a different source (see also item 7). Where the synthetic
route and structure of the intermediates are cited as evidence of structure, references to
relevant published papers in the literature would be helpful. Where relevant, the i n f o r m a t i o n
might include such evidence as:
- elemental analysis with theoretical values,
- infra-red spectra with interpretation,
nuclear magnetic resonance spectra with interpretation including C13 data where
relevant,
- discussion on UV characteristics including pH dependent shifts,

- mass spectrum with interpretation and discussion of results,
- discussion of the synthetic route as evidence of structure,

- evidence of structure of key intermediates of synthesis (e.g. using IR, NMR, etc.),
characteristic chemical reactions which are diagnostic of the structure of the molecule,
- X-ray crystallography with interpretation and discussion of results(refer to 4.2.3),
optical rotation with discussion of optical purity in the case of isomerism. (Absence of
optical rotation should be reported when this serves to illustrate that an a s y m m e t r i c
molecule is racemic),
evidence of the indicated relative molecular mass.
The relevance of the isomer to activity should be discussed.
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3AQ5a
4.2 Physico-chemical characteristics

Information set out under the relevant headings below should cover aspects of physico-
chemical characteristics which have been investigated, whether or not they are include in the
monograph for the active substance.
4.2.1 Solubility
The solubility in water, including pH dependence, and in other solvents should be given as
numerical values with particular reference to the formulation and test procedures.
4.2.2 Physical characteristics
An indication should be given as to whether the substance is crystalline, amorphous, etc. and
where relevant, information on particle size, solvation, melting point, boiling point etc.
4.2.3 Polymorphism
Where relevant, the presence of polymorphic forms and the methods of detection and control
should be discussed, or their absence confirmed.
4.2.4 pKa and pH values
Where relevant, the plia values of the active substance and the pH in solutions of defined
concentration should be given. In the case of a salt, this information for the corresponding
base or acid should be given.
4.2.5 Other characteristics
Any other relevant information should be given (for oil/water partition coefficient,
numerical values should be presented).
4.3 Analytical development

Any critical aspects of analytical development relevant to the active substance monograph
should be mentioned. The discussion here should highlight any unusual aspects of the tests
for identity, physico-chemical characteristics and content which are used in the monograph.
(Tests for purity and freedom from contamination can be discussed under the section on
impurities). Discussion of the precision and accuracy of test procedures is particularly
applicable to substances where biological control is necessary.
5. IMPURHTES
A broad outline should be given of the research programme which has been undertaken to
demonstrate that the test procedures used for impurity control in the active substance
specification are valid including limit of detection and limit of quantification. Negative
information can sometimes be important.
5.1 Impurities
- by-products of the synthesis arising from side reactions, impurities in the starting
materials or isomerisation,
- residual solvents and reagents,
- trace elements arising from the use of catalysts or from other sources,
36
3AQ5a
- degradation products (see note for guidance Stability Tests on Active Substances and
Finished Products).
A list and brief description of the products which have been considered as potential
impurities arising from the synthesis should be given. In each case, it should be stated
whether actual samples of such impurities have been synthesised for test purposes and which
of the analytical methods described under 5.2 have been used to detect those impurities.
Possible routes of degradation should also be discussed on the basis of results of
investigations on exposure of the substances to stress conditions (such as heat, light, pH,
moisture and other relevant factors).
5.2 Test procedures

The analytical methods with limits of detection of the test procedures which have been used to
detect each of the likely impurities considered in 5.1 above or other related impurities, the
exact identities of which may be unknown, should be described. Copies of relevant
chromatograms should be provided.
5.3 Summary of results

A summary should be given of the nature and levels of impurities which have been detected
in the batch samples of the material. The Expert Report should provide a justification for
selecting the limits (based on findings from toxicity testing) and methods used for impurity
control in the specification.
6. ACTIVE SUBSTANCE SPECIFICATION

6.1 The tests applied and the limits thereby imposed should be stated for:
- physical characteristics,
- tests for identity,
- standards for purity and limitation of impurities,
- standards and tests for potency.
6.2 Analytical methods employed should be described in detail.
7. BATCH ANALYSIS
Data should be provided in this section to illustrate the actual results which have been
obtained from routine quality control of the active substance. Results should be given, if
possible, for:
- batches of material used in the toxicity tests and clinical trials reported in support of
the application,
- recent consecutive batches (5) which are representative of the product which will be
supplied for the purposes covered by the marketing authorisation to show that the
proposed methods will give routine production material which falls within the
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3AQ5a
specification limits cited. Information on production batches should be provided, if

necessary on an on-going basis.
7.1 Batch analysis results

The results should include:
- date of manufacture,
- batch size and number,
- place of manufacture,
- results of analytical determinations,
- use of batches.
As far as possible, the results should give actual figures for tests on, for example, impurity
levels. Results which merely state that the material "complies" with the test are not
sufficiently informative, especially where a relatively wide limit is allowed in the
specification.
The batch analyses should include all the tests set out in the specification. There may,
however, be cases where earlier batches of material were tested using a slightly different
specification. In these cases, a brief explanatory note should be included.
7.2 Discussion of results

Any apparently inconsistent or anomalous results in the batch analyses should be explained.
8. REFERENCE STANDARDS
The criteria for establishing the reference substances (primary and secondary) for routine
analysis should be given with full analytical profiles.
9. RADIOLABELLED PRODUCT
Information on radiolabelled material should be compatible with the above guidelines.
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3AQ6a
REQUIREMENTS IN RELATION TO ACTIVE

SUBSTANCES
Guideline Title R e q u i r e m e n t s in relation to Active S u b s t a n c e s

Date of entry into April 1992
force
P r e v i o u s titles/other None/ III/8315/89
references
Additional Notes This note for guidance clarifies the r e q u i r e m e n t s to b e
included in a m a r k e t i n g a u t h o r i s a t i o n for an active
substance d e p e n d i n g on the described classification.
CONTENTS
1. CLASSIFICATION OF ACITVE SUBSTANCES
2. NEW ACTP7E SUBSTANCES
3. EXISTING ACTRHE SUBSTANCES NOT INCLUDED IN THE P h . E u r . OR THE

PHARMACOPOEIA OF A MEMBER STATE
4. PHARMACOPOEIAL ACTP7E SUBSTANCES
DECISION TREE SHOWING SELECTION OF REGULATORY PROCEDURE FOR

ACTDTE SUBSTANCES
39
3AQ6a
REQUIREMENTS IN RELATION TO ACTIVE

SUBSTANCES
L CLASSIFICATION OF ACTIVE SUBSTANCES

Active substances may be classified into:
- new active substances;
- existing active substances not included in the Ph. Eur. or the pharmacopoeia of a
Member State;
- pharmacopoeial active substances included in the Ph. Eur. or the pharmacopoeia of a
Member State.
2. NEW ACTD7E SUBSTANCES

For new chemical active substances, the requirements are set out in the note for guidance
Chemistry of the Active Substance.
For biotechnologically derived new active substances, the requirements are described in the
specific "biotechnology" guidelines.
The information may be supplied either as part of the marketing authorisation (MA)
application or using the European Drug Master File procedure.
3. EXISTING ACTDVE SUBSTANCES NOT INCLUDED IN THE

PH. EUR. OR THE PHARMACOPOEIA OF A MEMBER
STATE
The requirements are as set out as above for new chemical active substances.
Where appropriate, information may be omitted in relation to proof of structure (e.g. where
this can be carried out by specific identification tests in relation to a reference substance
fully described in the dossier, where necessary).
Evidence of the stability of the active substance may be provided from the literature (see note
for guidance on Stability Tests on Active Substances and Finished Products).
The information may be supplied either as part of the MA application or using the European
Drug Master File procedure.
4. PHARMACOPOEIAL ACTIVE SUBSTANCES

Pharmacopoeial active substances may be divided into:
- inorganic substances;
- vegetable substances and vegetable substance preparations;
41
3AQ6a
- biotechnologically derived substances;

- organic substances (extracted from material of animal or human origin);
- organic substances (manufactured or extracted).
Each batch of substances must comply with the current requirements of the Ph. Eur. or
pharmacopoeia of a Member State.
In each case, evidence should be presented to the competent authorities to demonstrate the
suitability of the pharmacopoeial monograph for material from the named manufacturer.
In relation to solid-state properties, the official pharmacopoeial monographs are intended to
control the general suitability of an active substance for any of the likely intended uses. In
cases where it is necessary for the particular intended use to control the bulk substance with
respect to solid-state properties, a suitable specification must be proposed with details of test
methods, batch analyses, validation data, etc.
4.1 Inorganic substances

In the case of inorganic substances, it should be stated whether the manufacturer uses a
process which may leave impurities not adequately controlled in the monograph and, in that
case, details of the tests additional to those in the pharmacopoeial monograph should be
supplied to the competent authorities.
4.2 Vegetable substances and vegetable substance preparations

In the case of vegetable substances and vegetable substance preparations, it should be stated
whether the manufacturer uses a method of cultivation and preparation liable to leave
impurities not adequately controlled in the monograph (e.g. pesticide residues and
fumigante). In that case, details of the tests additional to those in the pharmacopoeial
monograph should be supplied to the competent authorities.
4.3 Biotechnologically derived substances

In the case of biotechnologically derived substances, full information shall be supplied, and
in particular information on the manufacture of the substance, measures to ensure freedom
from potentially pathogenic agents and stability shall be provided to the competent authorities
as part of the MA application or using the European DMF procedure.
4.4 Organic substances (extracted from material of animal or human

origin)
In the case of organic substances extracted from material of animal or human origin, full
information shall be supplied; in particular information on the collection, treatment and
storage of the animal or human source material, isolation of the active substance,
specification and control methods for source materials, measures to ensure freedom from
potentially pathogenic agents and stability shall be provided to the competent authorities as
part of the MA application or using the European DMF procedure.
42
3AQ6a
4.5 Organic substances (manufactured or extracted)

In relation to organic active substances from any manufacturer, there may be impurities
present which are not adequately controlled by the official pharmacopoeial monograph. The
suitability of the monograph should, in all cases, be demonstrated to the competent
authorities. The suitability may be shown in one of the following four ways:
4.5.1 Certificate of suitability of the monograph established by the European

Pharmacopoeia
The manufacturer of the active substance may submit documentation to the Secretariat of the
Commission of the European Pharmacopoeia with a view to evaluating the suitability of the
Ph. Eur. monograph in relation to the manufacturing method used (in this context,
appropriate procedures are in place within the Commission of the European Pharmacopoeia).
The applicant should include a copy of the certificate of compliance with the quality defined
by the European Pharmacopoeia, together with a written assurance that no significant
changes in the manufacturing method has taken place since the date of certification.
4.5.2 Other evidence of suitability of the European Pharmacopoeia monograph

The applicant may supply other evidence obtained from the active substance manufacturer
(ASM). This might include the following evidence:
a) information as to the length of time that the particular named source has been on sale
in the European Community and elsewhere; and
b) a statement that, in the above period, there had been no significant change in the
method of manufacture leading to a change in the impurity profile of the substance;
and
c) a statement that samples from the named source had been supplied to the Ph. Eur.
Commission or national Commission and had been taken into account in the
development of their monograph; and
d) a statement that no additional tests were necessary arising from the use of the
manufacturing route to identify and limit additional impurities (particularly toxic
impurities) not specifically controlled by the pharmacopoeial monograph.
The above is one possible approach to providing reassurance to the authorities of the
suitability of a well-defined active substance from the named source with long and safe
patient exposure. It is noted, however, that even where a monograph has been in force for
many years, it will not necessarily be sufficient in relation to a new route of manufacture.
4.5.3 Full details of manufacture

The applicant may submit full details on the active substance, its manufacture, control etc.
as outlined in the guideline on "Chemistry of active substances" but omitting, where
appropriate, information on proof of structure (where this can be shown by specific
identification tests in relation to reference substances fully described in the dossier, where
necessary) and stability (where adequate literature evidence can be cited and summarised
as indicated in the note for guidance Stability Tests on Active Substances and Finished
Products).
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4.5.4 European Drug Master File

The documentation relating to details of manufacture (item 4.5.3) may be submitted as a
Drug Master File as outlined in the note for guidance European Drug Master File Procedure.
44
3AQ6a
Decision Tree Showing Selection of Regulatory Procedure for Active Substances
Note: Reference to Ph. Eur. means the current edition of the European Pharmacopoeia, to
Ph. Eur./EC means the current edition of the European Pharmacopoeia or the pharmacopoeia
of a Member State.
Type of active ingredient?
^ = L
Non-pharmacopeial Ph. Eur
New Active Substance (i.e. not in Ph. Eur.) Monograph
1
Type of active
Vegetable Inorganic Organic active Oganic,

Biotech derived
drug drug (human or animal synthetic or
drug
origin) semi-synthet
L J
Pharmacopeia monograph + Does active manufacturer wiwh
necessary additional tests e.g. for to participate in
pesticides Ph. Eur. Certification Scheme
^ZT
Active in long use,
No
'31
Apply using Future
NO changes in the manufacture, Ph. Eur Scheme
NO uncontrolled toxic impurities etc.
Yes No
Use transparent Information on manufacture

Ph. Eur./EC monograph confidential, made by another
firm?
-i*.
<- 1 .
Yes No
Information in EuroDMF Information in MA Application
45
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EUROPEAN DRUG MASTER FILE PROCEDURE

FOR ACTIVE SUBSTANCES
Guideline Title E u r o p e a n Drug Master File P r o c e d u r e for Active

Substances
Date of first adoption May 1993
Date of entry i n t o May 1993
force
Status Last revised J u n e 1993
Previous titles/other Use of the European DMF Procedure III/5370/93 w h i c h
references included a n d replaced III/3500/91 a n d III/3499/91 a n d t h e
previous guidance Drug Master Files on Active
Ingredients III/3836/89
Additional Notes This note for guidance describes t h e legal basis and
possible way of p r e s e n t i n g t h e d o c u m e n t a t i o n r e q u i r e d
in P a r t 2 C and F of t h e Annexes to Directive 75/318/EEC
as amended a n d Directive 81/852/EEC as amended.
CONTENTS
A. LEGAL BASIS OF THE PROCEDURE
B. EUROPEAN DRUG MASTER FILE PROCEDURE
1. INTRODUCTION
2. CONTENT OF THE EUROPEAN DRUG MASTER FH
3. INFORMATION ACCOMPANYING AN EDMF
4. CRITICAL APPRAISAL OF DOCUMENTATION OF THE EDMF
5. GLOSSARY OF TERMS
C. USE OF THE EUROPEAN DRUG MASTER FILE (EDMF) PROCEDURE
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EUROPEAN DRUG MASTER FILE PROCEDURE

FOR ACTIVE SUBSTANCES
This compilation has been prepared in order to help applicants for marketing authorisations
and manufacturers of active substances when using the European Drug Master File
procedure in the preparation of dossiers for application for marketing authorisations. It does
not have any legal force and in case of doubt, applicants should refer to the original texts of
the directives.
The words "medicinal products" cover both products for use in humans and veterinary
medicinal products
Contents:
1. Legal basis of the procedure: annexes to Directives 75/318/EEC as amended and
81/852/EEC as amended, Part I C I , "Control of starting materials".
2. Note for guidance Use of the European Drug Master File Procedure, adopted by the
Committee for Proprietary Medicinal Products (CPMP) on 12.5.1993 and revised on
16.6.1993, and by the Committee for Veterinary Medicinal Products (CVMP) on
23.3.1994, with immediate entry into operation.
A. LEGAL BASIS OF THE PROCEDURE: EXTRACT FROM THE

ANNEX TO DIRECTIVE 75/318/EEC AS AMENDED, o AND
TO DIRECTIVE 81/852/EEC AS AMENDED, (*> "CONTROL OF
STARTING MATERIALS"
Controls of starting materials

In the case of:
- an active substance not described in the European Pharmacopoeia or in the
pharmacopoeia of a Member State, or
- an active substance described in the European Pharmacopoeia or in the pharmacopoeia
of a Member State, when prepared by a method liable to leave impurities not mentioned
in the pharmacopoeial monograph and for which the monograph is inappropriate to
adequately control its quality, which is manufactured by a person different from the
applicant, the latter may arrange for the detailed description of the manufacturing
method, quality control during manufacture and process validation to be supplied
directly to the competent authorities by the manufacturer of the active substance. In this
case, the manufacturer shall however provide the applicant with all the data which may
be necessary for the latter to take responsibility for the medicinal product. The
( *) as modified by Directive 91/507/EEC of 19 July 1991, Official Journal n L 270 of 26.6.1991

(**> as modified by Directive 92/18/EEC of 20 March 1992, Official Journal N L 97/1 of 10.4.1992
49
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manufacturer shall confirm in writing to the applicant that he shall ensure batch to
batch consistency and not modify the manufacturing process or specifications without
informing the applicant. Documents and particulars supporting the application for such
a change shall be supplied to the competent authorities.
B. EUROPEAN DRUG MASTER FILE PROCEDURE

Note for guidance concerning a possible way of presenting the documentation required in
Part 2 C and F of the Annex to Directives 75/318/EEC as amended and 81/852/EEC as
amended. The documentation needed is described in detail in the "Notice to Applicants for
marketing authorisations for medicinal products for human use in the Member States of the
European Union" Part II, C 1 and F 1 and in the same document dealing with veterinary
medicinal products and in the note for guidance Chemistry of Active Substances.
L INTRODUCTION
The European Drug Master File (DMF) procedure may be used when the active substance
manufacturer (ASM) is not the applicant for a product marketing authorisation (applicant),
with a view to protecting valuable know-how on the manufacture of the active substance.
A DMF is a document containing the information required to demonstrate that the quality of
the active substance is adequately controlled by the specification proposed by the applicant.
The applicant must, therefore, collaborate with the person submitting a separate DMF to
ensure that all relevant information required is supplied. Furthermore it must be ensured
that the applicant's part of the DMF (cf. below) contains all information needed for the
applicant to take full responsibility for the preparation, including the suitability of the active
substance (as supplied) for the intended route of administration.
It is not a requirement to present information on the active substance in the form of a DMF.
The information may also form part of the application for authorisation to place a medicinal
product on the market.
Three types of active substances may be described in a European DMF.
A. New active substances still covered by a patent, not described in the European
Pharmacopoeia or in the pharmacopoeia of a Member State.
B. Active substances off-patent, not described in the European Pharmacopoeia or the
C. Active substances described in the European Pharmacopoeia or in the pharmacopoeia of
a Member State when prepared by a method liable to leave impurities not mentioned in
the pharmacopoeial monograph and for which the monograph is inappropriate to
adequately control their quality (Directives 75/318/EEC as amended and 81/852/EEC as
amended).
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2. CONTENT OF THE EUROPEAN DRUG MASTER FILE

2.1 Overall content
Detailed information should be provided as indicated under the various headings of the
relevant "Notice to Applicants for M a r k e t i n g Authorisations for Medicinal Products in the
Member States of the European Union" - Part II C, Control of starting materials - active
substances and Part II F, Stability - stability tests on active substances.
2.2. Applicant's part and ASM Restricted Part of a European DMF

The DMF contains information which includes valuable know-how which should be kept
confidential and submitted to the authorities only. Therefore, it should be divided into 2 parts
- an applicant's part and an ASM Restricted Part. The applicant's part of a DMF is provided
by the ASM to the applicant directly and becomes part of the application for m a r k e t i n g
authorisation. Both the applicant's part and the ASM Restricted P a r t of the DMF a r e
submitted to the competent authorities. The applicant's part of the DMF is still a confidential
document which cannot be submitted to third parties without the written agreement of the
ASM.
a) Applicant's part of a DMF

The applicant must be supplied by the ASM with sufficient information to be able to take
responsibility for an evaluation of the suitability of the active substance specification to
control the quality of the substance. This normally includes a brief outline of the
manufacturing method, information on potential impurities originating from the
manufacturing method, from the isolation procedure (natural products) or from degradation
and, where applicable, information on the toxicity of specific impurities.
b) ASM R e s t r i c t e d P a r t of DMF
Detailed information on the individual steps of the m a n u f a c t u r i n g method such as reaction
conditions, temperature, validation and evaluation data for certain critical steps of the
manufacturing method, etc. and on quality control during manufacture may contain
valuable know-how. Such information may therefore be supplied to the authorities only.
An example is provided in the Table below of the division of the information which should be
included in the applicant's part and the ASM Restricted Part, respectively. However, the type
of information should always be adapted to the m a n u f a c t u r i n g method and the
characteristics of the individual active substance. The figures in brackets refer to sections in
Part II C or II F in the Notice to Applicants.
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TABLE
ASM Restricted Part Applicant's p a r t

Name(s) a n d site(s) of manufacturer(s) + +
Specification and routine tests (C.l.l.) +
Nomenclature (C. 1.2.1.) +
Description (C. 1.2.2.) +
Manufacturing method (C. 1.2.3.)
- brief outline (flow chart) +
- detailed description +
QC during manufacture (C. 1.2.4.) +
Process validation and evaluation of data +
Development chemistry (C. 1.2.5.)
- evidence of structure (if needed) +
- potential isomerism +
- physico-chemical characterisation +
- analytical validation +
Impurities (C. 1.2.6.) +
Batch analysis (incl. impurities) (C. 1.2.7.) +
Stability (where necessary) (F.l.) +
2.3. Discussion of potential toxicology of impurities

The ASM should, where applicable, include in the applicant's part of the DMF available
information on the potential toxicology of impurities by reference to literature or by the
presentation of data to justify the proposed limits. If the information is incomplete, the
applicant for the product marketing authorisation needs to supply additional information.
3. INFORMATION ACCOMPANYING AN EDMF

The ASM should give permission to the competent authorities to assess the data in his DMF
on behalf of a specified applicant in the form of a 'Letter of access'. Such a letter should
include the following information:
- name and address of the applicant;
- name of product and, if possible, date of application;
- name and address of the ASM;
- name and address of the importer of the active substance, where applicable;
- name and address of the distributor (agent or broker), where applicable.
A written assurance that there is a formal agreement between the ASM and the applicant
which ensures that if there is a significant change in the manufacturing method (likely to
alter the quality or safety of the product) or in active substance specification, this
information will be communicated to the applicant and to the authorities. It is the
responsibility of the applicant to consider any necessary changes in his specification and to
inform the competent authorities accordingly.
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A 'letter of access' must be lodged by the ASM for every application for marketing
authorisation.
4. CRITICAL APPRAISAL OF DOCUMENTATION OF THE

EDMF
The applicant for the product marketing authorisation has the full responsibility for the
inclusion in the Expert Report of the required critical evaluation of the dossier. However, as
information in the ASM Restricted Part of the DMF is not available to the applicant, a
critical appraisal should be included in the ASM Restricted Part of the DMF on specific
items important for the quality of the active substance. In particular this section should
consider:
- the manufacturing method and discuss how it will consistently guarantee material of
the desired quality;
- batch analyses and whether these show consistency of manufacture;
- demonstration of how the specification and routine tests in the applicant's part is
justified in relation to the ASM Restricted Part.
5. GLOSSARY OF TERMS
European Drug Master File: EC procedure where information can be provided to the
authorities and the applicant, where the active substance manufacturer is not the applicant
for a product marketing authorisation, with a view to protecting valuable manufacturing
know-how.
ASM: Active Substance Manufacturer.
Applicant's part of a DMF: Section of the European DMF given to the applicant to include
in the application for a product marketing authorisation.
ASM Restricted Part of DMF: Section of the European DMF given by the ASM only to the
authorities.
Letter of Access: Letter of authority from the ASM to allow the competent authorities to
assess the European DMF on behalf of a specified applicant.
C. USE OF THE EUROPEAN DRUG MASTER FILE (EDMF)

PROCEDURE
A practical guide to implementation by the chemical industry (manufacturers of active
substances) and the pharmaceutical industry (applicants for marketing authorisations).
1. Where is the procedure described?

The possibility of submitting confidential data to the competent authorities directly from an
Active Substance Manufacturer (ASM) who is different from the applicant for the marketing
53
3AQ7a
authorisation, is provided for in Directives 75/318/EEC as amended and 81/852/EEC a s

amended. Details of the procedure are given in the note for guidance European Drug Master
File Procedure for Active Substances.
2. Is t h e p r o c e d u r e m a n d a t o r y ?
The EDMF procedure is a possibility offered to the industry, it is not a requirement.

If there is no problem of confidentiality between the ASM and the applicant for a m a r k e t i n g
authorisation or if the ASM and the applicant can come to an agreement s a f e g u a r d i n g
confidentiality, all of the information on the active substance should be given directly in the
application for the marketing authorisation.
3. W h e n t o s u b m i t a n EDMF?
An EDMF may only be submitted if it is referred to in a m a r k e t i n g application for a
medicinal product. It should be submitted shortly after the submission of the application, so
t h a t the reference number given by the competent authority to the application for m a r k e t i n g
authorisation can be given in the letter of access. The EDMF may be submitted at the s a m e
time as the application, provided an unambiguous reference to the application can be given.
4. Who c a n s u b m i t an EDMF ?
Only ASMs and their authorised representatives (e.g. importers) may submit an EDMF.
If an applicant for a m a r k e t i n g authorisation wants to rely on alternative suppliers of the
active substance, he should refer to this situation in the application.
5. Where a n d h o w to s u b m i t an EDMF?
5.1 One copy of the complete EDMF, comprising the 2 parts (applicant's part and A S M ' s
restricted part) should be sent by the ASM directly to each of the competent authorities
concerned.
5.2 A copy of the applicant's part should be supplied in advance by the ASM to the
applicant. This applicant's part should be included in the application for m a r k e t i n g
authorisation.
6. H o w t o i n c l u d e a d d i t i o n a l s p e c i f i c a t i o n s for c e r t a i n d o s a g e forms?
Specific quality requirements for certain dosage forms may be included in the application
for m a r k e t i n g authorisation. Such requirements will not form part of the EDMF. T h e y
should be described in a formal contract between the ASM and the applicant and included i n
the application for marketing authorisation.
7. If a DMF h a s b e e n p r e v i o u s l y a s s e s s e d by t h e c o m p e t e n t authority, w h a t
i n f o r m a t i o n s h o u l d b e g i v e n w i t h a n e w DMF o n t h e s a m e a c t i v e s u b s t a n c e ?
7.1 If an EDMF has been updated since the previous assessment, the changes should be
clearly identified (particularly those in the method of manufacture and in the specifications
of the active substance).
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3AQ7a
7.2 If a DMF was not presented in accordance with the current guideline on EDMFs
(prior to 1992 for human medicinal products and prior to May 1993 for veterinary medicinal
products), it should be re-arranged into the 2-part format, and the different parts sent to the
authorities and to the applicant as described above.
8. Are EDMFs to be approved by the authorities?

An EDMF will never be approved as such, it may only be accepted in relation to a specific
application for marketing authorisation. No claims should therefore be made by any ASM as
to his EDMF having been "approved".
9. If further questions need to be answered before the assessment on an EDMF

can be completed, how will this be done?
One of the major objectives of the EDMF procedure is to ensure that the applicant is always
fully aware of any aspects of the active substance which could impact on its safety, quality or
efficacy. Thus, all deficiencies in the EDMF will normally be addressed by the competent
authorities to the applicant for the marketing authorisation. When asking such questions,
rather general terms will be used to avoid any disclosure of confidential data from the ASM
restricted part. The applicant will then ask the ASM to supply the further data to the
concerned competent authorities and also to ensure that his part of the EDMF is completed.
10. Will an ASM be advised of the results of the assessment of his EDMF?
The competent authority will not inform the Active Substance Manufacturer that his EDMF
has been accepted in relation to a particular application for marketing authorisation. This
information should be sought by the ASM from the applicant.
11. Need for identical specifications between the application and the EDMF
The specification of the active substance should be identical in the application and in the
applicant's part of the EDMF. Any proposal from the applicant to use a different test
procedure for the purity tests must be fully validated in relation to the procedure used by the
active substance manufacturer, in particular as regards the precision and accuracy of the
results of the tests, including the limits of detection for all possible impurities.
12. How to inform the interested parties when an ASM wants to modify the ASM
restricted part or the applicant's part?
12.1 If the only change to be made is in relation to the contents of the ASM restricted part
of the file, this information should only be given to the authorities. A prerequisite for such
procedure is that there is no change in specification and no change in impurity profile.
12.2 If changes are to be made in the applicant's part of the EDMF, this information must
also be given to any other applicant or holder of a marketing authorisation referring to this
EDMF. All the applicants involved will then need to seek to change their marketing
authorisation dossier (using the appropriate variation procedures).
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13. Fees to be paid to the competent authorities

13.1 Fees, if any, for the EDMF assessment are to be paid by the applicant for a marketing
authorisation according to the instructions given by the relevant competent authority.
13.2 Variations to marketing authorisations because of changes to the EDMF attract the
relevant fee, to be paid by the holder of the authorisation.
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IMPURITIES IN NEW ACTIVE SUBSTANCES *)
Guideline Title Impurities in New Active Substances

Date of entry into For studies commencing after November 1995
force
Previous titles/other ICH Q3A: Impurities in New Drug
references Suosfances/III/5442/94, CPMP/ICH/142/95
Additional Notes This note for guidance concerns the application of
Part 2, section C of the Annex to Directive 75/318/EEC as
amended, with a view to the granting of a marketing
authorisation for a new medicinal product. It replaces
the section on impurities in the guideline entitled
Chemistry of Active Substances in relation to new active
substances.
CONTENTS
1. PREAMBLE
2. CLASSIFICATION OF IMPURITIES
3. RATIONALE FOR THE REPORTING AND CONTROL OF IMPURITHSS
4. ANALYTICAL PROCEDURES
5. REPORTING IMPURITY CONTENT OF BATCHES
6. SPECIFICATION LIMITS FOR IMPURITIES
7. QUALIFICATION OF IMPURITIES
8. NEW IMPURITIES
9. GLOSSARY
DECISION TREE FOR SAFETY STUDHiS
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IMPURITIES IN NEW ACTIVE SUBSTANCES *)
L PREAMBLE
This document is intended to provide guidance for applicants on the content and
qualification of impurities in new active substances produced by chemical syntheses and not
previously authorised in a Member State. It is not intended to apply to the regulation of new
substances used during the clinical research stage of development.
Biological/biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation and
semi-synthetic products derived therefrom, herbal products, and crude products of animal or
plant origin are not covered.
Impurities in new substances are addressed from two perspectives:
Chemistry Aspects includes classification and identification of impurities, report
generation, setting specifications, and a brief discussion of analytical procedures; and,
Safety Aspects includes specific guidance for qualifying impurities which were not present
in batches of new substance used in safety and clinical studies and/or impurity levels
substantially higher than in those batches. Threshold limits are defined, below which,
qualification is not needed.
2. CLASSIFICATION OF IMPURITIES
Impurities may be classified into the following categories:
Organic Impurities (Process and Substance Related)
Inorganic Impurities
Residual Solvents
Organic impurities may arise during the manufacturing process and/or storage of the new
substance. They may be identified or unidentified, volatile or non-volatile, and include:
Starting Materials
By-Products
Intermediates
Degradation Products
Reagents, Ligands and Catalysts
Inorganic impurities may derive from the manufacturing process. They are normally
known and identified and include:
Reagents, Ligands and Catalysts
Heavy Metals
Inorganic Salts
Other Materials (e.g., Filter Aids, Charcoal, etc.)
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Solvents are organic or inorganic liquids used during the manufacturing process. Since
these are generally of known toxicity, the selection of appropriate controls is easily
accomplished.
Excluded from this document are: extraneous contaminants which should not occur in new
substances and are more appropriately addressed as GMP issues; polymorphic forms, a solid
state property of the new substance; and, enantiomeric impurities.
3. RATIONALE FOR THE REPORTING AND CONTROL OF

IMPURmES
3.1 Organic Impurities
The applicant should summarise those actual and potential impurities most likely to arise
during the synthesis, purification, and storage of the new substance. This summary should
be based on sound scientific appraisal of the chemical reactions involved in the synthesis,
impurities associated with raw materials which could contribute to the impurity profile of the
new substance, and possible degradation products. This discussion may include only those
impurities that may reasonably be expected based on knowledge of the chemical reactions
and conditions involved.
In addition, the applicant should summarise the laboratory studies conducted to detect
impurities in the new substance. This summary should include test results of batches
manufactured during the development process and batches from the proposed commercial
process, as well as results of intentional degradation studies used to identify potential
impurities arising during storage. Assessment of the proposed commercial process may be
deferred until the first batch is produced for marketing. The impurity profile of the active
substance lots intended for marketing should be compared with those used in development
and any differences discussed.
The studies conducted to characterise the structure of actual impurities present in the new
substance at or above an apparent level of 0.1% (e.g., calculated using the response factor of
the substance) should be described. Note that all recurring impurities at or above the 0.1%
level in batches manufactured by the proposed commercial process should be identified.
Degradation products observed in stability studies at recommended storage conditions
should be similarly identified. When identification of an impurity is not feasible, a
summary of the laboratory studies demonstrating the unsuccessful effort should be included
in the application. Where attempts have been made to identify impurities below the 0.1%
level, it is useful to also report the results of these studies
Identification of impurities below apparent levels of 0.1% is generally not considered
necessary. However, identification should be attempted for those potential impurities that are
expected to be unusually potent, producing toxic or pharmacological effects at a level lower
than 0.1%. In all cases, impurities should be qualified as described later in this guide.
Although it is common practice to round analytical results of between 0.05 and 0.09% to the
nearest number (i.e., 0.1%), for the purpose of these guidelines, such values would not be
rounded to 0.1% and these impurities would not require identification.
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3.2 Inorganic Impurities

Inorganic impurities are normally detected and quantitated using pharmacopoeial or other
appropriate procedures. Carry-over of catalysts to the new substance should be evaluated
during development. The need for inclusion or exclusion of inorganic impurities in the new
substance specifications should be discussed. Limits should be based on pharmacopoeial
standards or known safety data.
3.3 Solvents
The control of residues of the solvents used in the manufacturing process for the new
substance should be discussed. Any solvents which may appear in the substance should be
quantified using analytical procedures with an appropriate level of sensitivity.
Pharmacopoeial or other appropriate procedures should be utilised. Limits should be based on
pharmacopoeial standards or known safety data taking into consideration dose, duration of
treatment, and route of administration. Particular attention should be given to quantitation
of toxic solvents used in the manufacturing process
4. ANALYTICAL PROCEDURES
The application should include documented evidence that the analytical procedures are
validated and suitable for the detection and quantitation of impurities. Differences in the
analytical procedures used during development and proposed for the commercial product
should be discussed in the marketing authorisation application.
Organic impurity levels can be measured by a variety of techniques, including those which
compare an analytical response for an impurity to that of an appropriate reference standard
or to the response of the new substance itself. Reference standards used in the analytical
procedures for control of impurities should be evaluated and characterised according to their
intended uses. It is considered acceptable to use the substance to estimate the levels of
impurities. In cases where the response factors are not close, this practice may still be
acceptable, provided a correction factor is applied or the impurities are, in fact, being
overestimated. Specifications and analytical procedures used to estimate identified or
unidentified impurities are often based on analytical assumptions (e.g., equivalent detector
response, etc.). These assumptions should b discussed in the marketing authorisation
application.
5. REPORTING IMPURITY CONTENT OF BATCHES

Analytical results should be provided for all batches of the new substance used for clinical,
safety and stability testing, as well as for batches representative of the proposed commercial
process. The content of individual identified and unidentified and total impurities, observed
in these batches of the new substance, should be reported with the analytical procedures
indicated. A tabulation (e.g., spreadsheet) of the data is recommended. Impurities should be
designated by code number or by an appropriate descriptor, e.g., retention time. Levels of
impurities which are present but are below the validated limit of quantitation need not be
reported. When analytical procedures change during development, reported results should be
linked with the procedure used, with appropriate validation information provided.
Representative chromatograms should be provided. Chromatograms of such representative
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batches, from method validation studies showing separation and detectability of impurities
(e.g., on spiked samples), along with any other impurity tests routinely performed, can serve
as the representative impurity profiles. The applicant should ensure that complete impurity
profiles (i.e., chromatograms) of individual batches are available if requested.
A tabulation should be provided which links the specific new substance batch to each safety
study and each clinical study in which it has been used.
For each batch of the new substance, the report should include:
Batch Identity and Size
Date of Manufacture
Site of Manufacture
Manufacturing Process
Impurity Content, Individual and Total
Use of Batches
Reference to Analytical Procedure Used
6. SPECIFICATION LIMITS FOR IMPURITIES

The specifications for a new substance should include limits for impurities. Stability
studies, chemical development studies, and routine batch analyses can be used to predict
those impurities likely to occur in the commercial product. The selection of impurities to
include in the new substance specifications should be based on the impurities found in
batches manufactured by the proposed commercial process. Those impurities selected for
inclusion in the specifications for the new substance are referred to as "specified
impurities" in these guidelines. Specified impurities may be identified or unidentified and
should be individually listed in the new substance specifications.
A rationale for the inclusion or exclusion of impurities in the specifications should be
presented. This rationale should include a discussion of the impurity profiles observed in the
safety and clinical development batches, together with a consideration of the impurity profile
of material manufactured by the proposed commercial process. Specific identified impurities
should be included along with recurring unidentified impurities estimated to be at or above
0.1%. For impurities known to be unusually potent or to produce toxic or unexpected
pharmacological effects, the quantitation/detection limit of the analytical methods should be
commensurate with the level at which the impurities must be controlled. For unidentified
impurities, the procedure used and assumptions made in establishing the level of the
impurity should be clearly stated. Unidentified impurities included in the specifications
should be referred to by some appropriate qualitative analytical descriptive label (e.g.,
"unidentified A" unidentified with relative retention of 0.9", etc.). Finally, a general
specification limit of not more than 0.1% for any unspecified impurity should be included.
Limits should be set no higher than the level which can be justified by safety data, and,
unless safety data indicate otherwise, no lower than the level achievable by the
manufacturing process and the analytical capability. In other words, where there is no
safety concern, impurity specifications should be based on data generated on actual batches
of the new substance allowing sufficient latitude to deal with normal manufacturing and
analytical variation and, the stability characteristics of the new substance. Although
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normal manufacturing variations are expected, significant variation in batch to batch

impurity levels may indicate that the manufacturing process of the new substance is not
adequately controlled and validated.
In summary, the new substance specifications should include, where applicable, limits for:
Organic Impurities
Each Specified Identified Impurity
Each Specified Unidentified Impurity at or above 0.1%
Any Unspecified Impurity, with a limit of not more than 0.1%
Total Impurities
Residual Solvents
Inorganic Impurities
A summation of assay value and impurity levels generally may be used to obtain mass
balance for the test sample. The mass balance need not add to exactly 100% because of the
analytical error associated with each analytical procedure. The summation of impurity
levels plus the assay value may be misleading, e.g., when the assay procedure is non-
specific (e.g., Potentiometrie titrimetry) and the impurity level is relatively high.
7. QUALIFICATION OF IMPURITIES
Qualification is the process of acquiring and evaluating data which establishes the
biological safety of an individual impurity or a given impurity profile at the level(s)
specified. The applicant should provide a rationale for selecting impurity limits based on
safety considerations. The level of any impurity present in a new substance which has been
adequately tested in safety and/or clinical studies is considered qualified. Impurities which
are also significant metabolites present in animal and/or human studies do not need further
qualification. A level of a qualified impurity higher than that present in a new substance
can also be justified based on an analysis of the actual amount of impurity administered i n
previous safety studies.
If data are not available to qualify the proposed specification level of an impurity, studies to
obtain such data may be needed when the usual qualification threshold limits given below
are exceeded:
Maximum Daily Dose Qualification Threshold
< 2g/day 0.1% or 1 mg per day intake (whichever is lower)
> 2g/day 0.05%

Higher or lower threshold limits for qualification of impurities may be appropriate for some
individual substances based on scientific rationale and level of concern, including product
class effects and clinical experience. For example, qualification may be especially
important when there is evidence that such impurities in certain substances or therapeutic
classes have previously been associated with adverse reactions in patients. In these
instances, a lower qualification threshold limit may be appropriate. Conversely, a higher
qualification threshold limit may be appropriate for individual substances when the level of
concern for safety is less than usual based on similar considerations (patient population,
substance class effects, clinical considerations, etc.). Technical factors (manufacturing
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capability and control methodology) may be considered as part of the justification for
selection of alternative threshold limits. Proposals for alternative threshold limits are
considered on a case by case basis.
The "Decision Tree for Safety Studies" (Attachment I) describes considerations for the
qualification of impurities when thresholds are exceeded. In some cases, decreasing the
level of impurity below the threshold may be simpler than providing safety data.
Alternatively, adequate data may be available in the scientific literature to qualify an
impurity. If neither is the case, additional safety testing should be considered. The studies
desired to qualify an impurity will depend on a number of factors, including the patient
population, daily dose, route and duration of medicinal product administration. Such studies
are normally conducted on the new substance containing the impurities to be controlled,
although studies using isolated impurities are acceptable.
8. NEW IMPURITIES
During the course of a substance development program, the qualitative impurity profile of the
new substance may change, or a new impurity may appear as a result of synthetic route
changes, process optimisation, scale-up, etc. New impurities may be identified or
unidentified. Such changes call for consideration of the need for qualification of the level of
the impurity unless it is below the threshold values as noted above. When a new impurity
exceeds the threshold, the "Decision Tree for Safety Studies" should be consulted. Safety
studies should compare the new substance containing a representative level of the new
impurity with previously qualified material, although studies using the isolated impurity
are also acceptable (these studies may not always have clinical relevance).
9. GLOSSARY
Chemical Development Studies: Studies conducted to scale-up, optimise, and validate
the manufacturing process for a new substance.
Enantiomers: Compounds with the same molecular formula as the substance, which differ
in the spatial arrangement of atoms within the molecule and are non- superimposable
mirror images.
Extraneous Substance: An impurity arising from any source extraneous to the
manufacturing process.
Herbal Products: Medicinal products containing, exclusively, plant material and/or
vegetable preparations as active substances. In some traditions, materials of inorganic or
animal origin may also be present.
Identified Impurity: An impurity for which a structural characterisation has been
achieved.
Impurity: Any component of the new substance which is not the chemical entity defined as
the new substance.
Impurity Profile: A description of the identified and unidentified impurities present in a
new substance.
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Intermediate: A material produced during steps of the synthesis of a new substance which
must undergo further molecular change before it becomes a new substance.
Ligand: An agent with a strong affinity to a metal ion.
New Substance: The designated therapeutic moiety which has not been previously
registered in a region or member state (also referred to as a new molecular entity or new
chemical entity). It may be a complex, simple ester, or salt of a previously approved
substance.
Polymorphism: The occurrence of different crystalline forms of the same substance.
Potential Impurity: An impurity which, from theoretical considerations, may arise from
or during manufacture. It may or may not actually appear in the new substance.
Qualification: The process of acquiring and evaluating data which establishes the
biological safety of an individual impurity or a given impurity profile at the level(s)
specified.
Reagent: A substance, other than a starting material or solvent, which is used in the
manufacture of a new substance.
Safety Information: The body of information that establishes the biological safety of an
individual impurity or a given impurity profile at the level(s) specified.
Solvent: An inorganic or an organic liquid used as a vehicle for the preparation of
solutions or suspensions in the synthesis of a new substance.
Specified Impurity: Identified or unidentified impurity that is selected for inclusion i n
the new substance specifications and is individually listed and limited in order to assure
the safety and quality of the new substance.
Starting Material: A material used in the synthesis of a new substance which is
incorporated as an element into the structure of an intermediate and/or of the new
substance. Starting materials are normally commercially available and of defined
chemical and physical properties and structure.
Toxic Impurity: Impurities having significant undesirable biological activity.
Unidentified Impurity: An impurity which is defined solely by qualitative analytical
properties (e.g., chromatographic retention time).
Validated Limit of Quantitation: For impurities at a level of 0.1%, the validated limit of
quantitation should be less than or equal to 0.05%. Impurities limited at higher levels may
have higher limits of quantitation.
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Decision Tree for Safety Studies
Decrease impurity YES Above threshold? NO Qualified

level below
threshold
4, YES
Structure elucidated
J, YES
YES
Toxicity documented and
sufficient?
J, NO
NO Related to others with known Acceptable
toxicity? justification?
i NO VES
Consider patient population and Qualified

duration of use
I
Consider need for:
1. Genotoxicity studies (point mutation, chromosonal abberation)
2. General toxicity studies (one species, min. 14 days, max90 days)

3. Other specific toxicity endpoints as appropiate
Adverse Effects
YES NO
Consider additional testing Qualified

or removal of impurity
If considered desirable, a minimum screen for genotoxic potential should be conducted. A study to detect point mutations and
one to detect chromosomal aberrations, both in vitro, are seen as an acceptable minimum screen.
If general toxicity studies are desirable, study(ies) should be designed to allow comparison of unqualified to qualified
material. The study duration should be based on available relevant information and performed in the species most likely to
maximise the potential to detect the toxicity of an impurity. In general, a minimum duration of 14 days and a maximum
duration of 90 days will be acceptable.
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EXCIPIENTS IN THE DOSSIER FOR APPLICATION

FOR MARKETING AUTHORISATION OF A
MEDICINAL PRODUCT
Guideline Title Excipients in the Dossier for Application for Marketing

Authorisation of a Medicinal Product
Date of first adoption February 1994
Date of entry into August 1994
force
references
Additional Notes This note for guidance concerns the application to
excipients of Part 2, sections A, C, E and F of the Annex to
Directive 75/318/EEC as amended, with a view to the
granting of a marketing authorisation for a new m e d i c i n a l
product.
CONTENTS
INTRODUCTION
2 COMPOSITION OF THE MEDICINAL PRODUCT
2.A.4 DEVELOPMENT PHARMACEUTICS
2.C.2 EXCIPIENTS
2.E THE FINISHED PRODUCT
ANNEX
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EXCIPIENTS IN THE DOSSIER FOR APPLICATION

FOR MARKETING AUTHORISATION OF A
MEDICINAL PRODUCT
INTRODUCTION
This note for guidance is concerned with the application to excipients of Part 2, sections A, C,
E, F of the annex to Directive 75/318/EEC as amended with a view to the granting of a
marketing authorisation for a new medicinal product.
The data should be presented according to the standard format described in the Notice to
Applicants (Volume II of "The Rules Governing Medicinal Products in the European Union"
series), parts II A, C, E and F.
PART 2.A.1 COMPOSITION OF THE MEDICINAL PRODUCT

Excipients must be listed, specifying their common name, their quantity and the use and
reference to any relevant standard. When the common name is not sufficient to indicate
functional specifications, the brand name with commercial grade should be specified. In the
case of excipients presented as a mixture of compounds, details as to the composition should
be provided in qualitative and quantitative terms. However, for flavouring agents and
aromatic substances, it is permitted to give the qualitative composition only.
PART 2.A.4 DEVELOPMENT PHARMACEUTICS

This section should comprise an explanation of the choice of the excipient (and grade where
necessary) according to the note for guidance Development Pharmaceutics and Process
Validation.
PART 2.C.2 EXCIPIENTS

Examples of different kinds of excipients are given in the annex.
2.1 Specifications and routine tests

2.1.1 Excipients described in the European Pharmacopoeia or, failing this, in the
pharmacopoeia of a Member State
The routine tests which are to be carried out on each batch of starting materials must be
stated in the application for marketing authorisation. If tests other than those mentioned i n
the pharmacopoeia are used, proof must be supplied that the test methods used are suitable to
establish that the starting materials meet the quality requirements of that pharmacopoeia.
When the monograph covers a family of related products, the particular specifications
chosen for the excipients must be submitted. In addition and when necessary, the test used to
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determine the quality of the excipient should be shown to be in relation to the function that it
fulfils in the medicinal product.
Data on microbiological contamination of the excipients used in the manufacture of sterile
products should always be given where membrane filtration is used to achieve sterility.
2.1.2 Excipients not described in the European Pharmacopoeia or in the pharmacopoeia of a
Member State
The entire routine test procedure and storage conditions must follow on from the scientific
data (2.2.2): an appropriate specification of the excipient must be established, based on the
following types of tests:
Physical characteristics
Identification tests
' Purity tests, including limits for total or individual impurities, which should be
named. Purity tests may be physical, chemical, biological and, if appropriate,
immunological.
Where sterile filtration is used in the manufacture of a parenteral medicinal product, data
and routine tests on microbiological contamination of excipients should always be given.
Other relevant tests including, e.g. the tests on parameters which may influence the
performance of the dosage form.
Assay or limit tests if necessary.
2.2 Scientific data

This documentation has an important role to play in justifying the choice and use of an
excipient which is used for a particular purpose: it will determine the properties which must
be checked during the routine tests and which will be the subject of certain specifications i n
connection with the bioavailability of the product (see note for guidance: Specifications and
Control Tests on the Finished Product).
Nevertheless, scientific data are not systematically required for well-known excipients. For
example, they are not required for excipients which have been used in similar medicinal
products for a long period of time and when their characteristics and properties have not
changed significantly.
For solid and semi-solid dosage forms, the scientific data should, if necessary, provide
information on the relevant characteristics of the excipient. Special tests are often necessary
(e.g. to verify the capacity of the excipient to emulsify and disperse, or to measure the
viscosity...).
Appropriate data are needed for excipients used in a new route of administration.
2.2.1 Scientific data on excipients already included in the European Pharmacopoeia, or
failing this, in the pharmacopoeia of a Member State and other well-known excipients
already used in a medicinal product
For these excipients, scientific data will normally not be required. However, any particular
specification concerning the characteristics, as defined in Part 2.A.4, should be justified (e.g.
sieve analysis, in relation to microcry stallini ty).
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2.2.2 Other excipients: a dossier should be established containing the same data as
required for new active substances:
a) A strict definition of the excipient, its function and its conditions of use. If the excipient
is complex or is made of a mixture of compounds, the composition must be specified i n
qualitative and quantitative terms.
b) For new excipients and for excipients presented as a mixture of compounds the
following should be taken into consideration:
i. Any bibliographical data on the chemistry and on the toxicology and the field i n
which the product is already used.
ii. The Community provisions concerning additives in foodstuffs: any criteria
which are based on the toxicological data, with cross-references to these data.
The quality specifications which have been laid down in the directives are satisfactory
as long as the routine control tests used are validated.
iii. The international specifications (FAO/WHO/JECFA), and other publications
such as the Food Chemical Codex.
iv. For medicinal products for topical use, data on the starting material in cosmetic
products (Directive 76/768/EEC).
v. Data concerning the toxicology of the new excipient should be presented
according to the dosage form and the route of administration of the medicinal
product (if applicable).
c) Documentation on chemistry of excipients is required for all new excipients, taking as
its basis the note for guidance Chemistry of Active Substances.
The origin of the excipient, including the name and address of manufacturer.
A general outline of the synthesis (manufacture and purification).
Structure.
Physical, chemical properties, identification and purity tests.
Validated methods of analysis with a presentation of batch results.
Miscellaneous information (microbiological tests, etc.).
Contamination, presence of foreign substances, residual solvents, etc.
In the case of an excipient obtained from a mixture of several components, the
quality of each component and the physico-chemical tests for the mixture should
be described.
The routine test procedures and limits should be established on the basis of the
documentation given in the dossier.
PART 2.E THE FINISHED PRODUCT

Apart from those situations envisaged in the note for guidance Specifications and Control tests
on the Finished Product it is not usually necessary to carry out identity testing and an assay
of the excipients in the finished product at release.
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1. Stability tests on starting materials

For new excipients, stability data should be provided as required for new active substances.
2. Stability tests on the finished product

The maintenance of the physico-chemical properties of the finished product are dependent
upon the properties and the stability of the excipients (see note for guidance Specifications and
Control Tests on the Finished Product).
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ANNEX
Examples of requirements concerning different kinds of excipients
1. Excipients which are a single chemical entity include, for example, organic and
inorganic acids and their salts, sugars and alcohols.
They may have undergone physical treatments which gave them special technological
characteristics (e.g. micronisation).
2. Chemically transformed excipients include excipients which have undergone a special

chemical treatment in order to confer certain technological characteristics (e.g.
modified starch).
The name and quality of such excipients should be defined in such a way as to avoid
confusion with an unmodified excipient.
3. Mixtures of chemically related components include, for example, polyol esters

(mixture of mono, di and tri esters), hydrogenated glucose syrup, maltitol syrup.
For these products the dossier should specify the following characteristics of the
excipient:
the nature and content of each component with a statement of its acceptable
limits;
technological criteria (appropriate criteria to the performance of dosage form);
any additives which may be present.
4. Mixed excipients are ready-for-use preparations, for example for direct compression or
film coating.
The qualitative and quantitative composition of the mixed excipient should be
submitted, the specifications of the product as a whole and of each component
must be stated.
5. Excipients of natural origin, so called "natural" products have often undergone some
kind of chemical treatment.
In general and if relevant for the quality control of the product, data should give a n
outline of the operations carried out to obtain and to purify the product, and any special
characteristics: decomposition products, specific impurities, chemical substances used
during the treatment with residual limits, methods of sterilisation or decontamination,
with a description of the effect of these processes on the excipient (e.g. modification of
the physical structure).
6. For biological excipients of animal or human origin, the risk of transmitting

adventitious agents should be considered and appropriate documentation submitted
(e.g.; method of preparation and control of tissues and body fluids used as starting
materials). In addition, the name of the manufacturer and site of manufacture should
be specified.
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7. Flavouring agents (flavours and aromatic substances) are either natural products
and/or products obtained by chemical synthesis. Because of the complexity of their
composition, it is only necessary to describe the general qualitative composition
mentioning the main constituents with an appropriate process of identification to
ensure the consistency of the composition (in particular, identification of the m a i n
constituents and if necessary carriers).
Most constituents of artificial flavours have internationally accepted purity criteria i n
food use (FAO/WHO). Reference to these standards is acceptable for medicinal
products.
8. Colouring matters: Community legislation on colouring matters in foodstuffs and

medicinal products is applicable.
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PLASTIC PRIMARY PACKAGING MATERIALS
Guideline Title Plastic P r i m a r y P a c k a g i n g Materials

Legislative basis Directive 75/318/EEC a s a m e n d e d
Date of first adoption F e b r u a r y 1994
force
Status Last revised F e b r u a r y 1994
P r e v i o u s titles/ o t h e r Plastic Containers and Packaging Material (Immediate
references Packaging) /9090/90
Additional Notes This note for guidance concerns the application to p l a s t i c
p r i m a r y packaging materials of P a r t 2, sections A, C, and F
of t h e Annex to Directive 75/318/EEC as amended, w i t h a
view to t h e granting of a m a r k e t i n g authorisation for a
new m e d i c i n a l p r o d u c t . The p r o v i s i o n s of C o m m u n i t y
legislation r e l a t i n g to plastic materials i n t e n d e d to c o m e
into contact w i t h foodstuffs, in p a r t i c u l a r D i r e c t i v e
90/218/EEC should be t a k e n into account.
CONTENTS
INTRODUCTION
2.A.2 IMMEDIATE PACKAGING
2.A.4 DEVELOPMENT PHARMACEUTICS
2.C.3 PACKAGING MATERIAL (PRIMARY OR IMMEDIATE PACKAGING)
2.F.2 STABILITY - STUDIES OF MIGRATIONS AND INTERACTIONS
SUMMARY PRESENTATION
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PLASTIC PRIMARY PACKAGING MATERIALS
INTRODUCTION
This note for guidance is concerned with the application to plastic primary packaging
materials of Part 2, sections A, C, F of the Annex to Directive 75/318/EEC as amended, with a
view to the granting of a marketing authorisation for a new medicinal product.
The present note is limited to plastic primary (or immediate) packaging materials i.e.
packaging materials intended to be in direct contact with the medicinal product. They
comprise containers, closures, seals and other parts which come into contact with the
medicinal product.
The data should be presented according to the standard format described in the Notice to
Applicants (Volume II of The Rules governing Medicinal Products in the European Union),
parts 2 A2, A4, C3 and F2.
Two other notes for guidance refer to containers: development pharmaceutics and process
validation and stability tests on active substances and finished products.
The importance and characteristics of data to be given are related to the pharmaceutical
dosage form (see appended summary).
When establishing the specifications for packaging materials for non-parenteral
preparations, the provisions of Community legislation relating to plastic materials intended
to come into contact with foodstuffs in particular Directive 90/128/EEC as amended, should be
taken into account.
For packaging materials used for ophthalmic and parenteral products, appropriate
development studies should be carried out if they are not described in the European
Pharmacopoeia or in the pharmacopoeia of a Member State.
Reference should be made to monographs of the European Pharmacopoeia or of the national
PART 2 A.2 IMMEDIATE PACKAGING

This part of the dossier should contain a brief description of the container. It should include:
- the nature of the packaging material, indicating in particular the qualitative
composition of its different parts and the non-plastic parts;
a description of the closure (nature and method) including, if necessary, its watertight
and airtight seal;
- a description of the method of opening and, if necessary, safety devices;
- information on the container (single or multidose) and dosing devices;
- a description of any tamper-evident closure and child resistant closure.
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PART 2 A.4 DEVELOPMENT PHARMACEUTICS

A justification of the choice of the container should be given in relation to data on stability of
the active substance(s) and of the finished product, to the method of administration and to
any sterilisation procedures.
The data collected during the development should be presented to justify the choice of the
plastic material(s) and of the container(s). These data should include details on:
- tightness of closure
- protection of the contents against external factors.
- container/contents interaction (e.g. sorption, leaching)
- influence of the manufacturing process on the container (e.g. sterilisation conditions).
PART 2 C.3 PACKAGING MATERIAL (PRIMARY OR

IMMEDIATE PACKAGING)
3.1 Specifications and routine test
Information should be provided on:
- The construction of the container, with a list of the different components.
- The type of materials used in the different parts, and nature of polymers
- The specifications: the nature, the extent and the frequency of routine tests of
packaging materials vary significantly according to the type of material, the type of
immediate packaging and the use of the product. Due account should be taken of the
route of administration, e.g. for parenteral and ophthalmic products.
For example, the following tests may be performed:
- identification of plastic material
- visual inspection
- dimensional tests
- physical tests (as appropriate: e.g. tensile strength...)
- microbiological tests (as appropriate).
If the plastic material is not described in the European Pharmacopoeia or in the
pharmacopoeia of a Member State it is advisable to use another pharmacopoeia monograph as
a guide to establish the routine tests and the general methods of the pharmacopoeia to
establish the specifications. The tests and limits applied should be justified.
3.2 Scientific data

Scientific data collected during the development of the medicinal product concern the
following points: information as to the plastic itself and the plastic packaging material,
uniformity of the material used for packaging and interaction studies.
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3.2.1.Information concerning the plastic material used for packaging

pharmaceutical products
A. General information
The following information should be provided for plastic materials used in the container,
including those already described in the pharmacopoeia where the monographs authorise the
use of several additives from which the manufacturer may choose one or several (within
certain limits).
- The name and grade given by the manufacturer of the material.
- For ophthalmic and parenteral preparations, the name of the plastic manufacturer.
- The chemical name of the material.
- The chemical name(s) of any monomer used.
- The complete qualitative composition of the plastic material is required where a n
interaction between the container and the contents occurs. The qualitative composition
covers all substances, including additives such as antioxidants, stabilisers, catalysts,
plasticisers, lubricants, solvents and/or dyes (comprising the colour index number
and/or the EC number).
If the material has not been approved for use for packaging of food, toxicological data
should be provided. In addition, toxicological information is required for plastics
normally approved for use in food packaging, if they are used for parenteral or
ophthalmic medicinal products.
B. Technical information
- Characteristics
Description of the material, its solubility in various solvents.
- Identification of the material
generally by infrared absorption spectrophotometry, with indication of the position of
characteristic absorption bands. The infrared spectrum of the reference material
should be provided: other methods of identification may be appropriate.
- Identification of the main additives
in particular those which are likely to migrate into the contents (such as antioxidants,
plasticisers, catalysts, initiators, etc.... and, for PVC, phthalates, adipates and organic
tin compounds).
- Identification of dyes
by using chromatographic or any other appropriate method.
Tests
General tests
Mechanical tests
Physical tests: an extraction test should be performed where the plastic material
is used as primary packaging material for liquid and semi-solid preparations.
The choice of solvent for this test depends on the composition of the product. The
test should investigate the level of extractives (antioxidants, plasticisers...).
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3.2.2 Container
The name of the manufacturer (also called converter) of the container should be specified for
ophthalmic and parental preparations.
The converter should ensure that the manufacturing process is reproducible and that there i s
no change in the composition of the material as defined for the type-sample.
Any significant change in the composition of the plastic material needs new specifications
and verification by the pharmaceutical manufacturer that the new packaging is suitable for
the intended use.
PART 2 F.2 STABILITY - STUDIES OF MIGRATIONS AND

INTERACTIONS
The choice of a packaging material is determined from studies concerning the protective
effect of the packaging against various external influences and compatibility studies
between the dosage form and the packaging.
The compatibility study forms part of the stability tests on the finished products and includes
compatibility with synthetic elastomer closures.
A study scheme should be proposed, designed for each dosage form: solid forms, semi-solid
forms and liquid forms.
Particular attention should be given to ophthalmic and parenteral preparations.
1. Dosage forms
1.1 Solid forms
For oral or topical solid dosage forms, the risk of migration is low and generally does not
require a content/container interaction study. Solid forms intended for parenteral use may
need interaction studies between the elastomer closure and the components of the
formulation.
1.2 Semi-solid forms

The study should consist mainly of technological controls of the container and study of the
eventual migration of additives or dyes. The risk of migration into aqueous or non-aqueous
semi-solid requires suitable specific studies for each formulation.
The study should be performed under normal and accelerated conditions.
1.3 Liquid forms

The risks of migration require suitable specific studies for each formulation.
In the case of parenteral and ophthalmic products, determination of the active substance and
preservative contents should be performed under simulated conditions of use.
Levels of extractives (e.g. antioxidants, plasticisers, catalysts processing aids etc..) should
be investigated mainly for parenteral and ophthalmic products.
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2. General scheme
2.1 Samples
During the development stage, migration studies on initial formulations often allow the
choice of a suitable packaging material for the finished product to be chosen.
The study should be performed on at least one batch of finished product.
2.2 Study condition

Studies should be performed under normal and accelerated conditions according to the
current notes for guidance on stability.
2.3 Study methods

Simulation studies performed with extraction solvents (as in the case of food) can only be
considered as predictive tests and do not preclude the need to perform a study on the finished
product.
- Migration and interaction studies should include:
- the control of technological characteristics for each pharmaceutical form.
- a study on the leaching of antioxidants, mono- and oligomers, plasticisers, mineral
compounds likely to migrate (e.g. calcium, barium, tin for PVC) and other additives
according to the composition of the packaging material. Maximum limits may need to
be proposed.
- a study of the sorption of the formulation components to the packaging material.
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SUMMARY PRESENTATION OF THE DOCUMENTATION OF PLASTIC CONTAINERS

AND PACKAGING MATERIAL
Solid Semi-solid/liquid Ophthalmic and

preparations preparations Parenteral preparations
Plastic material
Name of material + + +
Chemical name + + +
Manufacturer +
Complete composition if necessary +
including additives
Container/closure
Manufacturer +
(converter)
Description + + +
Identification* polymer + + +
Dimensional + + +
properties
4=
if necessary if necessary
Technical properties
Suitability** + + +
. . ** if necessary if necessary
Toxicity
Compatibility, + +
migration
* Routine tests
** The studies of suitability toxicity and compatibility content-container and migrations are carried out during
the development and not for routine test.
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SPECIFICATIONS AND CONTROL TESTS ON THE

FINISHED PRODUCT
G u i d e l i n e Title Specifications a n d Control Tests on t h e Finished P r o d u c t

Date of entry into J u n e 1992
force
P r e v i o u s titles/other Control of the Finished Product! III/3324/89
references
Additional Notes This note for guidance concerns t h e application of P a r t 2,
section E of t h e Annex to Directive 75/318/EEC as
amended, with a view to t h e g r a n t i n g of a m a r k e t i n g
a u t h o r i s a t i o n for a n e w medicinal product.
CONTENTS
1. SPECIFICATIONS
2. TEST PROCEDURES
3. BATCH ANALYSIS
APPENDIX
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SPECIFICATIONS AND CONTROL TESTS ON THE

FINISHED PRODUCT
Note for guidance concerning the application of Part 2, section E of the Annex to Directive
75/318/EEC as amended, for the purposes of granting a marketing authorisation.
A glossary of terms used is included in an appendix to this note.
1 SPECIFICATIONS
LI Quality characteristics covered by the specifications
Quality characteristics cover the following area:
- general characteristics of the pharmaceutical form, particularly pharmacotechnical;
that is to say those characteristics, determined in general by physical tests with limits
of acceptance, relating to the product performance or handling (e.g. hardness, friability
of a conventional tablet);
- identification of the active substance(s);
- assay of active substances (and also for herbal medicines, quantitative determination
of the constituents with known therapeutic activity);
- if necessary, identification and assay of the excipients such as:
- identification of colorants used,
- identification and assay of antimicrobial agents or antioxidant preservatives (with
acceptance limits);
- purity tests (if necessary, the investigation of breakdown products, residual solvents or
other process related impurities, microbial contamination);
- pharmaceutical tests (e.g. dissolution);
- safety tests including abnormal or specific toxicity tests, where applicable, in
particular for biological products.
In order to determine the specifications of the finished product, the quality characteristics
related to the manufacturing process should be taken into account.
An appropriate specification for each aspect of quality studied during the phase of
development and during the validation of the manufacturing process should be determined.
At least those aspects considered to be critical should be the object of specifications routinely
verified.
1.2 Relationship between dossier specifications and the

Pharmacopoeia
The general provisions of the European Pharmacopoeia describe and define the content and
legal weight of the various sections of its monographs and general methods.
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Directive 75/318/EEC as amended recapitulates those aspects of quality which must be the
object of appropriate specifications by referring to the general monographs of the European
Pharmacopoeia or, failing this, to pharmacopoeias of Member States.
Concerning monographs on medicinal products, the European or national pharmacopoeiae
define the reference quality level. In the marketing authorisation application, the applicant
should determine the most appropriate means for reaching the stated objective, and supply the
appropriate analytical validation data. In all cases, routine tests on the finished product at
release follow the stipulations of section 1.4.
In addition, as a result of the fact that these monographs correspond to regulatory
specifications, they represent the limit values of medicinal product at the end of their shelf
life (except for those provisions referring to production). The specifications for release of the
finished product must comply with the criteria defined by Directive 75/318/EEC as amended,
i.e. 5% for the assay of active substance(s) except when otherwise justified.
1.3 Relationships between the specification of a finished product at

the end of shelf life and at manufacture (at release)
a) The aim of the application dossier for a marketing authorisation is to set the quality
level of the medicinal product as intended for marketing. It establishes specifications,
i.e. qualitative and quantitative characteristics, with test procedures and acceptance
limits, with which the medicinal product must comply during its intended shelf life.
In Part II F of the dossier, the applicant proposes a shelf life for the medicinal product
mainly on the basis of the level of active constituents (efficacy) and the admissible
level of any breakdown products or impurities (safety) and consistent
pharmacotechnical properties. From the behaviour of the medicinal product the
applicant deduces the appropriate storage conditions which will maintain compliance
with the specifications of the medicinal product.
b) The specification limits of the finished product at the time of batch release are set by the
marketing authorisation applicant such that the specifications proposed at the end of
shelf life are guaranteed; they are established on the basis of a critical detailed review
of the data gathered from the batches analysed. Nevertheless, acquired experience is a
factor recognised to be very important in terms of good manufacturing practice. One of
the basic requirements of GMP (see the Guide to GMP) is the systematic review of all
manufacturing processes in the light of experience. Thus, the applicant, in compliance
with Directive 65/65/EEC as amended, Article 9. a, shall adapt or refine the
specifications at release as a function of experience acquired by the manufacturer(s) of
the medicinal product.
c) The specifications of the finished product at manufacture may be different from those
of the medicinal product at expiry.
In certain cases, for characteristics of the medicinal product which may change during
storage under the approved conditions, the quality required at the end of shelf life
should be taken into account in determining appropriate specifications at the time of
manufacture, for example in the case of overages for reasons of stability.
It is desirable that all specifications (characteristics and acceptance limits) of the
medicinal product and the finished product at the time of release be presented in the
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form f a summary table. In this table, the limits of any likely breakdown products
which may form under the approved conditions of storage should be stated.
The summary table could be presented as follows:
SUMMARY PRESENTATION OF THE SPECIFICATIONS OF THE MEDICINAL

PRODUCT AND THE FINISHED PRODUCT (AT MANUFACTURE)
Methods and Acceptance Limits

Characteristics of the medicinal product up of the finished product at
to the end of shelf life release
1. Characteristics of the Indicate with an asterisk the specification limits which
pharmaceutical form may require updating in the light of experience acquired
after the first "n" production batches
2. Identification and assay of
active constituents
3. Purity tests
4. Excipient: Identification
for example of colorants,
preservatives, limit values
of preservatives etc.
Details should be given of specification reference number and signature and date of
approval.
This presentation of the complete series of specifications does not affect the choice and the
frequency of the testing which will effectively be carried out on the finished product at
manufacture (or possibly on the bulk product or intermediate product) (see 1.4).
1.4 Specifications a n d r o u t i n e t e s t s for t h e r e l e a s e of b a t c h e s of

finished p r o d u c t at t h e t i m e of m a n u f a c t u r e (at r e l e a s e )
1.4.1 Relationship between validation of the manufacturing process, GMP and
establishment of specifications
The compliance of each batch of finished product with its specifications at manufacture
should be guaranteed by GMP (see Guide to GMP). Nevertheless, those features of the batch
documentation required by GMP may not be included in the marketing authorisation
dossier.
In the marketing authorisation dossier, it must be shown that the manufacturing process
used in compliance with GMP is capable of producing the finished product consistently in
compliance with the specifications chosen; these specifications take into account:
- development studies described in Part II A 4 of the marketing authorisation dossier
(see note for guidance Development Pharmaceutics and Process Validation ) which will
have led to the identification of the features of the formulation and the manufacturing
process, which are essential for the quality of product;
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- the validation of critical steps in the manufacturing process described in Part III 3 of
the marketing authorisation dossier which enable scale up and batch reproducibility to
be monitored.
1.4.2 Routine tests and periodic tests

Different types of tests may exist:
a) tests to be carried out batch by batch on the finished product or, possibly, on the bulk
product;
b) tests whose performance during a manufacturing step (intermediate products or in-
process controls) will contribute a greater guarantee of finished product compliance
than their performance on the finished product or on the bulk product;
c) periodic tests (not carried out batch by batch on the finished product or, possibly on the
intermediate product or bulk product), where the frequency of performance is highly
dependent on other parameters of GMP (e.g. microbiological quality);
d) tests whose performance on the finished product or possibly on the bulk product at
manufacture can be replaced by the verification of another highly dependent
specification (for example replacement of the test for uniformity of mass with the test
for uniformity of content);
e) tests which are not carried out routinely once the guarantees of compliance are
furnished by the manufacturer; these specific cases are exceptional (e.g. identification
of colorants);
f) tests corresponding to critical points in the manufacturing process to be monitored
particularly during the first V production batches and temporarily in the course of
any substantial modification (for example changing the manufacturing site,
materials, etc.). Subsequently, as a function of acquired experience and especially
validation of the production process, their batch by batch performance can be omitted
(e.g. residual solvents).
There may exist situations other than those described above.
1.4.3 Scheme for verifying specifications

As a function of the conclusions of parts II A 4 and II 3, the dossier must clearly indicate
the individual tests to be carried out for the release of the finished product and state whether
they are done routinely on each batch of finished product, or indicate the periodicity of testing
(batch by batch, every V batches, the first "n" batches etc.).
Two cases are possible depending upon whether or not the products are known and for which
the manufacturer has available substantial production experience.
In the first case, for example a new dosage strength of an already authorised product, or a
new conventional formulation (solution, compressed tablet etc.) of a well known
pharmacopoeial active substance, tests carried out routinely and periodically can be stated at
the time of application.
In the second case, for example with formulations of a new active substance or new dose
delivery system such as modified release tablets, adjustments as a result of product
experience should also be described, where possible, and the regulatory authorities informed
of appropriate developments based on the results obtained.
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The tests proposed in both cases should be presented in the form of summary tables as
indicated below (Tables 1 and 2). Whenever necessary, the tests proposed during the first "n"
batches (Table 1) should be distinguished from those envisaged in a routine industrial
situation (Table 2).
Table 1
Proposed scheme for testing during the initial period of industrial production
Frequency of Testing
Tests
(Surveillance of the first "n" batches)
The applicant should state the number ("n") of batches involved where applicable.
Table 2
Testing scheme confirmed following stabilised industrial production
Tests Frequency of Testing
Each batch of medicinal product marketed must comply with all the specifications defining
its expected quality level, regardless of the testing plans envisaged, or confirmed after
experience.
Testing may be chemical, physical, pharmaceutical, microbiological or biological.
1.5 Acceptance limits

The acceptance limits for specifications of the different quality characteristics (refer to
section 1.1) are established by taking into account all significant elements related to the
quality of the medicinal product (constancy of its characteristics), its activity (level of active
constituent) and, if necessary, its safety (risk of microbial contamination, breakdown
products, etc.).
1.5.1 Acceptance limits of pharmacotechnical parameters

For most pharmacotechnical specifications, the European Pharmacopoeia or failing this the
national pharmacopoeias of the Member States, describes general test procedures with, in
some cases, standards or maximal limits.
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In the context of these specifications, it is necessary to establish minimal and/or maximal

limits, specific and adapted to the medicinal product which is the object of the marketing
authorisation in order to guarantee the reproducibility of the finished product at
manufacture.
1.5.2 Maximum acceptable deviation in the content of active substances

Directive 75/318/EEC as amended states "Unless there is appropriate justification, the
maximum acceptable deviation in the active substance content of the finished products shall
not exceed 5% at the time of manufacture. On the basis of the stability tests the
manufacturer must propose and justify maximum acceptable tolerance limits in the active
substance content of the finished product up to the end of the proposed shelf life".
- Release limits of 5% are acceptable without further justification.
- Release limits wider than 5% would need to be justified in the part "Development
Pharmaceutics" with experimental results which are normally based on a confidence
level of 95%. The wider limits also include both, the variation of the production and of
the test procedure for the assay.
- Use of inadequate manufacturing procedures or inadequate test procedures (low
precision) will not be accepted as a justification for wider release limits.
- It is left to the responsibility of the manufacturer if -to satisfy the 5% requirements- he
has to apply an adjustment of the amount of active substance in the production of the
finished product (factorisation). In such a case, the overage must be clearly stated in
Part II 1. The release limit will remain within 5% of the stated content.
- References to pharmacopoeias cannot normally be accepted as a justification for wider
limits as monographs do not describe a defined composition of the medicinal product,
and do not pertain to release, but to recontrol by official laboratories over the whole
shelf life of the product.
- Exceptionally, for certain products with a well known degradation process and which
pose no safety problems, an overage at release can be tolerated (vitamins, etc.). This
overage, the aim of which is to guarantee a sufficient level at the end of shelf life, must
not cause an excessive level at release, and release limits must be adapted
accordingly.
- Release limits 5% primarily concern the manufacturers of the product and not any
outside laboratory. At recontrol by official laboratories, the release limits of the
manufacturer will be taken into consideration despite the fact that the limits used at
recontrol may not be identical to the release limits of the manufacturer (due to
interlaboratory variability). Where recontrol tests are carried out by another
laboratory, the test procedures must be validated in their hands.
- Overfilling to guarantee the delivery of the theoretical unit dose must be justified i
Part II A 4 "Development Pharmaceutics". The acceptance limits for determination of
unit content are adapted in consequence.
1.5.3 Acceptance limits for excipients

- Excipients which affect the bioavailability of an active substance must be the object of a
quantitative determination in each batch, unless bioavailability is guaranteed by other
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appropriate tests, established on a case by case basis as a function of development

studies.
In the case of preservatives, content limits of 90-110% at release should be acceptable
without further justification except in special cases. On expiry the lower limit of
antimicrobial preservatives may be reduced subject to the results of satisfactory
preservative efficacy testing. For chemical preservatives (antioxidants), the lower
limit may be considerably lower than 90% during the shelf life, because of the
preferential degradation of these agents.
2. TEST PROCEDURES
Test procedures must be described in a sufficiently detailed manner to enable any official
laboratory to verify the compliance of the medicinal product up to the end of shelf life.
Control methods must be validated in accordance with the note for guidance Validation of
Analytical Procedures: Methodology. The results of such analytical validation must be
included in the dossier.
Thus, the description of the test procedure(s) should, if necessary, include the description of
the reference substance, including its specifications and the description of the calculation
formula or an example of calculation (whether or not the calculations are performed by an
automatic instrument), chromatograms and spectra (etc.) furnishing the proof of the results
obtained.
Except for those officially included in the European Pharmacopoeia, e.g. sterility tests, it is
not necessary to describe the sampling procedures, which are the domain of GMP and depend
on inspection services.
A test procedure may use either an official reference substance (European Pharmacopoeia,
national pharmacopoeias, WHO) or a working standard, providing the latter is
standardised against the official reference substance (see note for guidance Validation of
Analytical Procedures: Methodology ).
It is to be remembered that an analytical result cannot be dissociated from the method used.
Thus in a pharmacopoeial monograph, it is assumed that the specifications adopted for the
tests and assay are established from the methods described in the Pharmacopoeia.
Methods other than the methods described in the Pharmacopoeia may be used for control
purposes providing that these methods are validated with reference to the official method and
providing that these methods used enable an unequivocal decision to be made as to whether
compliance with the standards of the monograph would be achieved if the official methods
were used (see general provisions of the European Pharmacopoeia).
In addition, the general methods described in the Pharmacopoeia may be used for products
not described in the Pharmacopoeia or for specifications not described in a monograph.
Utilisation of these methods requires validation for the specific case envisaged.
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3. BATCH ANALYSIS
The data which must be produced are summarised in the Notice to Applicants. The section
"batch analysis" must include the results obtained for all specifications at release, whether
or not they are intended to be verified batch to batch.
Where possible, the consecutive batches should correspond to production scale batches
manufactured by all manufacturers and at all manufacturing sites declared in the
marketing authorisation, according to the process described in Part 1 A of the dossier. If
these data are not available for industrial scale batches, they should be supplied to the
competent authorities as soon as possible after the marketing authorisation is granted.
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APPENDIX
Glossary
The following definitions are applicable to the terms used in this note for guidance.
Analytical Validation (Refer to note for guidance Validation of Analytical Procedures:

Methodology
Batch (Refer to Directive 75/318/EEC as amended, Annex, Part 2 E)

For the control of the finished product, a batch of a proprietary medicinal product comprises
all the units of a pharmaceutical form which are made from the same initial mass of
material and have undergone a single series of manufacturing operations or a single
sterilisation operation or, in the case of a continuous production process, all the units
manufactured in a defined period of time.
Bulk Product (Refer to Guide to GMP)

Any product which has completed all processing stages up to, but not including final
packaging.
Factorisation
Adjustment by calculation of the mass of an substance (usually the active substance) added
for the manufacture of a batch of a product on the actual potency as determined by assay of
that particular batch of substance. For example, if the theoretical batch quantity of an active
substance in a batch of product is 100 gram, and the assay "as is" of the batch of substance
being used is 92.0% m/m, then the factorised mass to be added would be 100 g 100/92.0 =
108.7 g.
Finished Product
A medicinal product which has undergone all stages of production including packaging
(refer to Guide to GMP).
In-Process Control (Refer to Guide to GMP)

Controls performed during production in order to monitor and if necessary to adjust the
process to ensure that the finished product conforms to its specifications.
Intermediate Product (Refer to Guide to GMP)

Partly processed material which must undergo further manufacturing steps before it becomes
a bulk product.
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Overage
An additional amount of an substance (usually the active substance) over and above the
amount stated in the unit formula, added to prepare a batch of product in order to compensate
for losses incurred during manufacture and/or storage in the finished pack. It is usually
expressed as a percentage.
Quality Assurance (Refer to Guide to GMP)

For the medicinal product, it is the sum total of the organised arrangements made with the
object of ensuring that manufactured medicinal products are of the quality required for their
intended use.
Routine Tests and Periodic Tests

Routine tests are carried out on each industrial batch of the intermediate product, the bulk
product or the finished product.
If necessary, these tests are extended by special tests called "periodic tests" which are carried
out according to a certain periodicity defined in each case; they are mentioned separately.
Specification
Qualitative and/or quantitative characteristics with test procedures and acceptance limits,
with which a given product must comply.
Specifications of the Finished Product (At Release)

Monograph defining qualitative and quantitative characteristics with test procedures and
their acceptance limits, with which the finished product must comply at the time of the
manufacture (at its release).
Specifications of the Finished Product (Up to the End of Shelf life)

Monograph defining qualitative and quantitative characteristics with test procedures and
their acceptance limits, with which a medicinal product (on the market) must comply
throughout its valid shelf life.
Validation of Manufacturing Process (Refer to Guide to GMP)

Action of proving, in accordance with the principles of Good Manufacturing Practice, that
any procedure, process, equipment, material, activity or system actually leads to the expected
results.
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IMPURITIES IN NEW MEDICINAL PRODUCTS *)
G u i d e l i n e Title I m p u r i t i e s in New Medicinal Products*)

Legislative basis Directive 75/318/EEC
Date of e n t r y into For studies commencing after J u n e 1997
force
Previous titles/other ICH Q3B: Impurities in New Drug Products,
references CPMP/ICH/282/96
section E of t h e Annex to Directive 75/318/EEC as
authorisation for a n e w medicinal product.
It is an annex to t h e Impurities in New Active Substances
guideline w h i c h should be consulted for basic
principles.
CONTENTS
1. INTRODUCTION
2. GUIDELINES
3. GLOSSARY
ATTACHMENT I
ATTACHMENT II
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IMPURITIES IN NEW MEDICINAL PRODUCTS *)
L INTRODUCTION
LI Objective of the Guideline
This guideline provides guidance recommendations for applications for marketing
authorisation on the content and qualification of impurities in new medicinal products
produced from chemically synthesised new active substances not previously registered in a
member state.
1.2 Background
This Guideline is an annex to the Impurities in New Active Substances Guideline which
should be consulted for basic principles.
1.3 Scope of t h e Guideline

This Guideline addresses only those impurities in medicinal products classified as
degradation products of the active substance or reaction products of the active substance with
an excipient and/or immediate container/closure system (collectively referred to as
"degradation products" in this Guideline). Impurities arising from excipients present in the
product are not covered by this Guideline. This Guideline also does not address the
regulation of products used during the clinical research stages of development.
Biological/biotechnological products, peptides, oligonucleotides, radiopharmaceuticals,
fermentation and semi-synthetic products derived therefrom, herbal products, and crude
products of animal or plant origin are not covered. Also excluded from this Guideline are:
extraneous contaminants which should not occur in medicinal products and are more
appropriately addressed as good manufacturing practice issues, polymorphic form, a solid
state property of the new active substance, and enantiomeric impurities. Impurities present
in the new active substance need not be monitored in medicinal products unless they are also
degradation products.
2. GUIDELINES
2.1 Analytical Procedures
The application for a marketing authorisation should include documented evidence that the
analytical procedures have been validated and suitable for the detection and quantitation of
degradation products. Analytical methods should be validated to demonstrate that impurities
unique to the new active substance do not interfere with or are separated from specified and
unspecified degradation products in the product.
Degradation product levels can be measured by a variety of techniques, including those
which compare an analytical response for a degradation product to that of an appropriate
reference standard or to the response of the new active substance itself. Reference standards
used in the analytical procedures for control of degradation products should be evaluated and
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characterised according to their intended uses. The active substance may be used to estimate
the levels of degradation products. In cases where the response factors are not close, this
practice may still be used if a correction factor is applied or the degradation products are, in
fact, being overestimated. Specifications and analytical procedures used to estimate
identified or unidentified degradation products are often based on analytical assumptions
(e.g., equivalent detector response). These assumptions should be discussed in the application
for marketing authorisation. Differences in the analytical procedures used during
development and those proposed for the commercial product should be discussed.
2.2 Rationale for the Reporting and Control of Impurities

The applicant should summarise those degradation products observed during stability
studies of the medicinal product. This summary should be based on sound scientific
appraisal of potential degradation pathways in the medicinal product and impurities arising
from the interaction with excipients and/or the immediate container/closure system. In
addition, the applicant should summarise any laboratory studies conducted to detect
degradation products in the medicinal product. This summary should include test results of
batches manufactured during the development process and batches representative of the
proposed commercial process. A rationale should be provided for exclusion of those
impurities which are not degradation products, e.g., process impurities from the active
substance and excipients and their related impurities. The impurity profile of the batches
representative of the proposed commercial process should be compared with the profiles of
batches used in development and any differences discussed.
Degradation products observed in stability studies conducted at recommended storage

conditions should be identified when the identification thresholds given in Attachment I are
equalled or exceeded (although it is common practice to round analytical results of between
0.05 and 0.09 percent to the nearest number, i.e., 0.1 percent, for the purpose of these
guidelines such values would not be rounded to 0.1 percent). When identification of a
degradation product is not feasible, a summary of the laboratory studies demonstrating the
unsuccessful effort should be included in the application for marketing authorisation.
Degradation products below the indicated levels generally would not need to be identified.
However, identification should be attempted for those degradation products that are suspected
to be unusually potent, producing toxic or significant pharmacologic effects at levels lower
than indicated.
2.3 Reporting Impurity Content of Batches

Analytical results should be provided in tabular format for all relevant batches of new
medicinal product used for clinical, safety and stability testing, as well as batches which are
representative of the proposed commercial process. Because the degradation test procedure
can be an important support tool for monitoring the manufacturing quality as well as for
deciding the expiration dating period of the product, the reporting level should be set below the
identification threshold. The recommended target value for the reporting threshold
(expressed as a percentage of the active substance) is found in Attachment I. A higher
reporting threshold should only be proposed, with justification, if the target reporting
threshold cannot be achieved.
In addition, where an analytical method reveals the presence of impurities in addition to the
degradation products (e.g., impurities arising from the synthesis of the active substance) the
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origin of these impurities should be discussed. Chromatograms, or equivalent data (if other
methods are used), from representative batches including long-term and accelerated stability
conditions should be provided. The procedure should be capable of quantifying at least at the
reporting threshold and the chromatograms should show the location of the observed
degradation products and impurities from the new active substance.
The following information should be provided:
Batch identity, strength and size
Date of manufacture
Site of manufacture
Manufacturing process, where applicable
Immediate container/closure
Degradation product content, individual and total
Use of batch
Reference to analytical procedure(s) used
Batch number of the drug substance used in the drug product
Storage conditions
2.4 Specification Limits for Impurities

The specifications for a new medicinal product should include limits for degradation
products expected to occur under recommended storage conditions. Stability studies,
knowledge of degradation pathways, product development studies and laboratory studies
should be used to characterise the degradation profile. Specifications should be set taking into
account the qualification of the degradation products, the stability data, the expected expiry
period, and the recommended storage conditions for the product, allowing sufficient latitude
to deal with normal manufacturing, analytical and stability profile variation. The
specification for the product should include, where applicable, limits for:
Each specified degradation product
Any unspecified degradation product
Total degradation products.
Although some variation is expected, significant variation in batch to batch degradation
profiles may indicate that the manufacturing process of the new medicinal product is not
adequately controlled and validated. A rationale for the inclusion or exclusion of impurities
in the specifications should be presented. This rationale should include a discussion of the
impurity profiles observed in the safety and clinical studies, together with a consideration of
the impurity profile of the product manufactured by the proposed commercial process.
2.5 Q u a l i f i c a t i o n of I m p u r i t i e s
Qualification is the process of acquiring and evaluating data which establishes the
biological safety of an individual degradation product or a given degradation profile at the
level(s) specified. The applicant should provide a rationale for selecting degradation product
limits based on safety considerations. The level of any degradation product present in a new
medicinal product which has been adequately tested and found safe in safety and/or clinical
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studies is considered qualified. Therefore, it is useful to include any available information

on the actual content of degradation products in the relevant batches at the time of use i n
safety and/or clinical studies. Degradation products which are also significant metabolites,
present in animal and/or human studies, do not need further qualification. It may be
possible to justify a higher level of a degradation product than the level administered i n
safety studies. The justification should include consideration of factors such as: the amount
of degradation product administered in previous safety and/or clinical studies and found to
be safe; the percentage change in the degradation product; and, other safety factors as
appropriate.
If data is not available to qualify the proposed specification level of a degradation product,
studies to obtain such data may be needed (see ATTACHMENT II) when the usual
qualification thresholds set out in ATTACHMENT I are equalled or exceeded. Higher or
lower thresholds for qualification of degradation products may be appropriate for some
individual products based on scientific rationale and level of concern, including drug class
effects and clinical experience. For example, qualification may be especially important
when there is evidence that such degradation products in certain medicinal products or
therapeutic classes have previously been associated with adverse reactions in patients. In
these instances, a lower qualification threshold may be appropriate. Conversely, a higher
qualification threshold may be appropriate for individual products when the level of concern
for safety is less than usual based on similar considerations (e.g., patient population, drug
class effects, and clinical considerations). In unusual circumstances, technical factors (e.g.,
manufacturing capability, a low active substance to excipient ratio, or the use of excipients
that are also crude products of animal or plant origin) may be considered as part of the
justification for selection of alternative thresholds. Proposals for alternative thresholds
would be considered on a case by case basis.
The "Decision Tree for Safety Studies" (see guideline for Impurities in New Medicinal
Products and ATTACHMENT II) describes considerations for the qualification of impurities
when thresholds are equalled or exceeded. Alternatively, if data are available in the
scientific literature then such data may be submitted for consideration to qualify a
degradation product. If neither is the case, additional safety testing should be considered.
The studies desired to qualify a degradation product will depend on a number of factors,
including the patient population, daily dose, route and duration of product administration.
Such studies should normally be conducted on the product or substance containing the
degradation products to be controlled, although studies using isolated degradation products
are considered acceptable.
2.6 New Impurities

During the course of drug development studies, the qualitative degradation profile of a new
medicinal product may change resulting in new degradation products which exceed the
identification and/or qualification threshold. In this event, these new degradation products
should be identified and/or qualified. Such changes call for consideration of the need for
qualification of the level of the impurity unless it is below the threshold values as set out i n
ATTACHMENT I.
When a new degradation product equals or exceeds the threshold (for rounding, see section
2.2), the "Decision Tree for Safety Studies" should be consulted. Safety studies should provide
a comparison of results of safety testing of the product or substance containing a
representative level of the degradation product with previously qualified material, although
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studies using the isolated degradation products are also considered acceptable (these studies
may not always have clinical significance).
3. GLOSSARY
Degradation Product
A molecule resulting from a chemical change in the substance brought about over time
and/or by the action of, e.g., light, temperature, pH, or water or by reaction with an excipient
and/or the immediate container/closure system (also called decomposition product).
Degradation Profile
A description of the degradation products observed in the active substance or medicinal
product.
Development Studies
Studies conducted to scale-up, optimise and validate the manufacturing process for a
medicinal product.
Identified Impurity
An impurity for which a structural characterisation has been achieved.
Impurity
Any component of the medicinal product which is not the chemical entity defined as the
active substance or an excipient in the product.
Impurity Profile
A description of the identified and unidentified impurities present in a medicinal product.
New Active Substance

The designated therapeutic moiety which has not been previously registered in a member
state (also referred to as a new molecular entity or new chemical entity). It may be a
complex, simple ester, or salt of a previously approved substance.
Potential Degradation Product

An impurity which, from theoretical considerations, may arise during or after manufacture
or storage of the medicinal product. It may or may not actually appear in the active substance
or medicinal product.
Qualification
The process of acquiring and evaluating data which establishes the biological safety of an
individual impurity or a given impurity profile at the level(s) specified.
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Reaction Product
Product arising from the reaction of a substance with an excipient in the medicinal product
or immediate container/closure system.
Safety Information
The body of information that establishes the biological safety of an individual impurity or a
given impurity profile at the level(s) specified.
Specified Degradation Product

Identified or unidentified degradation product that is selected for inclusion in the new
medicinal product specifications and is individually listed and limited in order to assure
the safety and quality of the new medicinal product.
Toxic Impurity
An impurity having significant undesirable biological activity.
Unidentified Degradation Product

An impurity which is defined solely by qualitative analytical properties, e.g.,
chromatographic retention time.
Unspecified Degradation Product

A degradation product which is not recurring from batch to batch.
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ATTACHMENT I
THRESHOLDS FOR REPORTING OF DEGRADATION PRODUCTS IN NEW

MEDICINAL PRODUCTS
Maximum Daily Dose1 Threshold

0.1%
>lg 0.05%
THRESHOLDS FOR IDENTIFICATION OF DEGRADATION PRODUCTS IN NEW

MEDICINAL PRODUCTS
Maximum Daily Dose1 Threshold 2

< 1 mg 1.0% or 5 ug TDI3 whichever is lower
1 mg - 10 mg 0.5% or 20 ug TDI whichever is lower
> 10 mg - 2 g 0.2% or 2 mg TDI whichever is lower
>2g 0.1%
THRESHOLDS FOR QUALIFICATION OF DEGRADATION PRODUCTS IN NEW

MEDICINAL PRODUCTS
Maximum Daily Dose1 Threshold 2

< 10 mg 1.0% or 50 ug TDI whichever is lower
10 mg - 100 mg 0.5% or 200 ug TDI whichever is lower
>100mg-2g 0.2% or 2 mg TDI whichever is lower
>2g 0.1%
The amount of substance administered per day

Threshold is based on percent of the substance
Total Daily Intake
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1 Thresholds for Identification, Qualification and Reporting of Degradation

Products in New Medicinal Products
OS

I de i t fica tio
""" Qualification
Reporting
2000
Maximum LbflyDose expressed in mg of Aclive Substance
Expanded scale:
mm mm ^ mm -Thresholds for Identification, Qualification and Reporting
o f Degradation Products in New Medicinal Products
- I deri:fica tio
Qualification
le porting
+ + +
0 5 10 15 20
Maximum Eafl y Dos e expres d in mg of Active Substance
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ATTACHMENT II: DECISION TREE FOR SAFETY STUDIES
Decrease degradation product level YES Above threshold? NO Qualified

below threshold ^ *
YES
Structure elucidated?
YES
YES Toxicity documented

and sufficient?
NO NO

Related to others YES Acceptable
with known toxicity? justification?
J, NO NO \ YES
Consider patient population and Qualified

duration of use
t
Consider need for:
1. Genotoxicity studies (point mutation, chromosomal aberration)11
2General toxicity studies (one species, min. 14 days, max. 90 days)1*c
3. Other specific toxicity endpoints, as appropriate
i
Adverse Effects
YES NO
Consider additional testing or removal/ Qualified

lowering level of degradation products
If considered desirable, a minimum screen e.g., genotoxic potential, should be conducted. A study to detect
point mutations and one to detect chromosomal aberrations, both in vitro, are seen as an acceptable
minimum screen, as discussed in the ICH-Guidelines: Specific Aspects of Regulatory Genotoxicity Tests
(included in Volume 3B ) and Genotoxicity: Definition of Core Battery Tests.
If general toxicity studies are desirable, study(ies) should be designed to allow comparison of unqualified to
qualified material. The study duration should be based on available relevant information and performed in
the species most likely to maximise the potential to detect the toxicity of an impurity. In general, a minimum
duration of 14 days and a maximum duration of 90 days would be acceptable.
On a case by case basis, single dose studies may be acceptable, especially for single dose drugs, and when
such studies are conducted using an isolated impurity. If repeat-dose studies are desirable, a maximum
duration of 90 days would be acceptable.
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VALIDATION OF ANALYTICAL PROCEDURES:

METHODOLOGY *)
Guideline Title Validation of Analytical Procedures: Methodology

Date of entry into For studies commencing after June 1997
force
Previous titles/other ICH Q2B/CPMP/ICH/281/95
references
Additional Notes This note for guidance concerns test procedures used in
documentation submitted in accordance with Part 2,
sections -F of the Annex to Directive 75/318/EEC as
authorisation for a medicinal product.
This guideline is complementary to, and should be read
in conjunction with, the guideline on Validation of
Analytical procedures: Definitions and Terminology.
CONTENTS
INTRODUCTION
1. SPECIFICITY
2. LINEARITY
3. RANGE
4. ACCURACY
5. PRECISION
6. DETECTION LIMIT
7. QUANTITATION LIMIT
8. ROBUSTNESS
9. SYSTEM SUITABILITY TESTING
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METHODOLOGY *)
INTRODUCTION
This guideline is complementary to the parent guideline* which presents a discussion of the
characteristics that should be considered during the validation of analytical procedures. Its
purpose is to provide some guidance and recommendations on how to consider the various
validation characteristics for each analytical procedure. In some cases (for example,
demonstration of specificity), the overall capabilities of a number of analytical procedures in
combination may be investigated in order to ensure the quality of the active substance or
medicinal product. In addition, the document provides an indication of the data which should
be presented in an application for marketing authorisation.
All relevant data collected during validation and formulae used for calculating validation
characteristics should be submitted and discussed as appropriate.
Approaches other than those set forth in this guideline may be applicable and acceptable. It i s
the responsibility of the applicant to choose the validation procedure and protocol most
suitable for the product. However it is important to remember that the main objective of
validation of an analytical procedure is to demonstrate that the procedure is suitable for its
intended purpose. Due to their complex nature, analytical procedures for biological and
biotechnological products in some cases may be approached differently than in this
document.
Well-characterised reference materials, with documented purity, should be used throughout
the validation study. The degree of purity necessary depends on the intended use.
In accordance with the parent guideline*, and for the sake of clarity, this document
considers the various validation characteristics in distinct sections. The arrangement of
these sections reflects the process by which an analytical procedure may be developed and
evaluated.
In practice, it is usually possible to design the experimental work so that the appropriate
validation characteristics can be considered simultaneously to provide a sound, overall
knowledge of the capabilities of the analytical procedure, for instance: specificity, linearity,
range, accuracy and precision.
L SPECIFICITY
An investigation of specificity should be conducted during the validation of identification
tests, the determination of impurities and the assay. The procedures used to demonstrate
specificity will depend on the intended objective of the analytical procedure.
It is not always possible to demonstrate that an analytical procedure is specific for a
particular analyte (complete discrimination). In this case a combination of two or more
analytical procedures is recommended to achieve the necessary level of discrimination.
Note for guidance on Validation of Analytical Procedures: Definitions and Terminology
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LI Identification
Suitable identification tests should be able to discriminate between compounds of closely
related structures which are likely to be present. The discrimination of a procedure may be
confirmed by obtaining positive results (perhaps by comparison with a known reference
material) from samples containing the analyte, coupled with negative results from samples
which do not contain the analyte. In addition, the identification test may be applied to
materials structurally similar to or closely related to the analyte to confirm that a positive
response is not obtained. The choice of such potentially interfering materials should be based
on sound scientific judgement with a consideration of the interferences that could occur.
1.2 Assay a n d I m p u r i t y Test(s)

For chromatographic procedures, representative chromatograms should be used to
demonstrate specificity and individual components should be appropriately labelled. Similar
considerations should be given to other separation techniques.
Critical separations in chromatography should be investigated at an appropriate level. For
critical separations, specificity can be demonstrated by the resolution of the two components
which elute closest to each other.
In cases where a non-specific assay is used, other supporting analytical procedures should be
used to demonstrate overall specificity. For example, where a titration is adopted to assay the
active substance for release, the combination of the assay and a suitable test for impurities
can be used.
The approach is similar for both assay and impurity tests.
1.2.1 Discrimination ofanalytes where impurities are available

For the assay, this should involve demonstration of the discrimination of the analyte in the
presence of impurities and/or excipients; practically, this can be done by spiking pure
substances (active substance or product) with appropriate levels of impurities and/or
excipients and demonstrating that the assay result is unaffected by the presence of these
materials (by comparison with the assay result obtained on unspiked samples).
For the impurity test, the discrimination may be established by spiking active substance or
product with appropriate levels of impurities and demonstrating the separation of these
impurities individually and/or from other components in the sample matrix.
1.2.2 Discrimination of the analyte where impurities are not available

If impurity or degradation product standards are unavailable, specificity may be
demonstrated by comparing the test results of samples containing impurities or degradation
products to a second well-characterised procedure e.g.: pharmacopoeial method or other
validated analytical procedure (independent procedure). As appropriate, this should include
samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis
and oxidation.
for the assay, the two results should be compared.
for the impurity tests, the impurity profiles should be compared.
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Peak purity tests may be useful to show that the analyte chromatographic peak is not
attributable to more than one component (e.g., diode array, mass spectrometry).
2. LINEARITY
A linear relationship should be evaluated across the range (see section 3) of the analytical
procedure. It may be demonstrated directly on the active substance (by dilution of a standard
stock solution) and/or on separate weighings of synthetic mixtures of the product
components, using the proposed procedure. The latter aspect can be studied during
investigation of the range.
Linearity should be evaluated by visual inspection of a plot of signals as a function' of
analyte concentration or content. If there is a linear relationship, test results should be
evaluated by appropriate statistical methods, for example, by calculation of a regression line
by the method of least squares. In some cases, to obtain linearity between assays and sample
concentrations, the test data may need to be subjected to a mathematical transformation prior
to the regression analysis. Data from the regression line itself may be helpful to provide
mathematical estimates of the degree of linearity.
The correlation coefficient, y-intercept, slope of the regression line and residual sum of
squares should be submitted. A plot of the data should be included. In addition, an analysis
of the deviation of the actual data points from the regression line may also be helpful for
evaluating linearity.
Some analytical procedures, such as immunoassays, do not demonstrate linearity after any
transformation. In this case, the analytical response should be described by an appropriate
function of the concentration (amount) of an analyte in a sample.
For the establishment of linearity, a minimum of 5 concentrations is recommended. Other
approaches should be justified.
3. RANGE
The specified range is normally derived from linearity studies and depends on the intended
application of the procedure. It is established by confirming that the analytical procedure
provides an acceptable degree of linearity, accuracy and precision when applied to samples
containing amounts of analyte within or at the extremes of the specified range of the
analytical procedure.
The following minimum specified ranges should be considered:
for the assay of an active substance or a finished product: normally from 80 to 120
percent of the test concentration;
for content uniformity, covering a minimum of 70 to 130 percent of the test
concentration, unless a wider more appropriate range, based on the nature of the
dosage form (e.g., metered dose inhalers), is justified;
for dissolution testing: +/-20 % over the specified range; e.g., if the specifications for a
controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24
hours, the validated range would be 0-110% of the label claim.
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for the determination of an impurity: from the reporting level of an impurity* to 120%
of the specification; for impurities known to be unusually potent or to produce toxic or
unexpected pharmacological effects, the detection/ quantitation limit should be
commensurate with the level at which the impurities must be controlled.
Note: for validation of impurity test procedures carried out during development, it may
be necessary to consider the range around a suggested (probable) limit;
if assay and purity are performed together as one test and only a 100% standard is
used, linearity should cover the range from the reporting level of the impurities 1 to
120% of the assay specification.
4. ACCURACY
Accuracy should be established across the specified range of the analytical procedure.
4.1 Assay
4.1.1 Active Substance
Several methods of determining accuracy are available:
a) application of an analytical procedure to an analyte of known purity (e.g. reference
material);
b) comparison of the results of the proposed analytical procedure with those of a second
well-characterised procedure, the accuracy of which is stated and/or defined
(independent procedure, see 1.2.2);
c) accuracy may be inferred once precision, linearity and specificity have been
established.
4.1.2 Medicinal Product

Several methods for determining accuracy are available:
a) application of the analytical procedure to synthetic mixtures of the product components
to which known quantities of the substance to be analysed have been added;
b) in cases where it is impossible to obtain samples of all product components , it may be
acceptable either to add known quantities of the analyte to the product or to compare
the results obtained from a second, well characterised procedure, the accuracy of
which is stated and/or defined (independent procedure, see 1.2.2).
c) accuracy may be inferred once precision, linearity and specificity have been
established.
4.2 I m p u r i t i e s (Quantitation)
Accuracy should be assessed on samples (substance/ product) spiked with known amounts of
impurities.
see chapters "Reporting Impurity Content of Batches" of the corresponding Guidelines: "Impurities in New
Active Substances" and "Impurities in New Medicinal Products"
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In cases where it is impossible to obtain samples of certain impurities and/or degradation

products, it is considered acceptable to compare results obtained by an independent procedure
(see 1.2.2). The response factor of the drug substance can be used.
It should be clear how the individual or total impurities are to be determined e.g.,
weight/weight or area percent, in all cases with respect to the major analyte.
4.3 R e c o m m e n d e d D a t a
Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3
concentration levels covering the specified range (e.g. 3 concentrations/ 3 replicates each of
the total analytical procedure).
Accuracy should be reported as percent recovery by the assay of known added amount of
analyte in the sample or as the difference between the mean and the accepted true value
together with the confidence intervals.
5. PRECISION
Validation of tests for assay and for quantitative determination of impurities includes an
investigation of precision.
5.1 Repeatability
Repeatability should be assessed using:
a) a minimum of 9 determinations covering the specified range for the procedure (e.g.
3 concentrations/3 replicates each)
or
b) a minimum of 6 determinations at 100% of the test concentration.
5.2 I n t e r m e d i a t e P r e c i s i o n
The extent to which intermediate precision should be established depends on the
circumstances under which the procedure is intended to be used. The applicant should
establish the effects of random events on the precision of the analytical procedure. Typical
variations to be studied include days, analysts, equipment, etc. It is not considered necessary
to study these effects individually. The use of an experimental design (matrix) is
encouraged.
5.3 Reproducibility
Reproducibility is assessed by means of an inter-laboratory trial. Reproducibility should be
considered in case of the standardisation of an analytical procedure, for instance, for
inclusion of procedures in pharmacopoeias. This data is not part of the marketing
authorisation dossier.
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The standard deviation, relative standard deviation (coefficient of variation) and
confidence interval should be reported for each type of precision investigated.
6. DETECTION LIMIT
Several approaches for determining the detection limit are possible, depending on whether
the procedure is a non-instrumental or instrumental. Approaches other than those listed
below may be acceptable.
6.1 Based on Visual Evaluation

Visual evaluation may be used for non-instrumental methods but may also be used with
instrumental methods.
The detection limit is determined by the analysis of samples with known concentrations of
analyte and by establishing the minimum level at which the analyte can be reliably
detected.
6.2 Based on Signal-to-Noise

This approach can only be applied to analytical procedures which exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by comparing measured signals
from samples with known low concentrations of analyte with those of blank samples and
establishing the minimum concentration at which the analyte can be reliably detected. A
signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the
detection limit.
6.3 Based on t h e S t a n d a r d Deviation of t h e Response a n d t h e Slope

The detection limit (DL) may be expressed as:
3.3
DL
S
where = the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of
may be carried out in a variety of ways, for example:
6.3.1 Based on the Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by
analysing an appropriate number of blank samples and calculating the standard deviation
of these responses.
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6.3.2 Based on the Calibration Curve

A specific calibration curve should be studied using samples containing an analyte in the
range of DL. The residual standard deviation of a regression line or the standard deviation
of y-intercepts of regression lines may be used as the standard deviation.
The detection limit and the method used for determining the detection limit should be
presented. If DL is determined based on visual evaluation or based on signal to noise ratio,
the presentation of the relevant chromatograms is considered acceptable for justification.
In cases where an estimated value for the detection limit is obtained by calculation or
extrapolation, this estimate may subsequently be validated by the independent analysis of a
suitable number of samples known to be near or prepared at the detection limit.
Several approaches for determining the quantitation limit are possible, depending on
whether the procedure is a non-instrumental or instrumental. Approaches other than those
listed below may be acceptable.
7.1 B a s e d on Visual E v a l u a t i o n
Visual evaluation may be used for non-instrumental methods but may also be used with
instrumental methods.
The quantitation limit is generally determined by the analysis of samples with known
concentrations of analyte and by establishing the minimum level at which the analyte can
be quantified with acceptable accuracy and precision.
7.2 Based on Signal-to-Noise A p p r o a c h

This approach can only be applied to analytical procedures that exhibit baseline noise.
Determination of the signal-to-noise ratio is performed by comparing measured signals
from samples with known low concentrations of analyte with those of blank samples and by
establishing the minimum concentration at which the analyte can be reliably quantified. A
typical signal-to-noise ratio is 10:1.
7.3 Based on t h e S t a n d a r d Deviation of t h e Response a n d t h e Slope

The quantitation limit (QL) may be expressed as:
QL
S
where = the standard deviation of the response

S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte. The estimate of
may be carried out in a variety of ways including:
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7.3.1 Based on Standard Deviation of the Blank

Measurement of the magnitude of analytical background response is performed by
analysing an appropriate number of blank samples and calculating the standard deviation
of these responses.
7.3J Based on the Calibration Curve

A specific calibration curve should be studied using samples, containing an analyte in the
range of QL. The residual standard deviation of a regression line or the standard deviation
of y-intercepts of regression lines may be used as the standard deviation.
7.4 Recommended Data

The quantitation limit and the method used for determining the quantitation limit should be
presented.
The limit should be subsequently validated by the analysis of a suitable number of samples
known to be near or prepared at the quantitation limit.
8. ROBUSTNESS
The evaluation of robustness should be considered during the development phase and
depends on the type of procedure under study. It should show the reliability of an analysis
with respect to deliberate variations in method parameters.
If measurements are susceptible to variations in analytical conditions, the analytical
conditions should be suitably controlled or a precautionary statement should be included in
the procedure. One consequence of the evaluation of robustness should be that a series of
system suitability parameters (e.g., resolution test) is established to ensure that the validity
of the analytical procedure is maintained whenever used.
Examples of typical variations are:
stability of analytical solutions,
extraction time
In the case of liquid chromatography, examples of typical variations are
influence of variations of pH in a mobile phase,
influence of variations in mobile phase composition,
different columns (different lots and/or suppliers),
temperature,
flow rate.
In the case of gas-chromatography, examples of typical variations are
different columns (different lots and/or suppliers),
temperature,
flow rate.
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9. SYSTEM SUITABILITY TESTING

System suitability testing is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, electronics, analytical operations and samples to be
analysed constitute an integral system that can be evaluated as such. System suitability test
parameters to be established for a particular procedure depend on the type of procedure being
validated. See Pharmacopoeias for additional information.
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DEFINITIONS AND TERMINOLOGY *)
Guideline Title Validation of Analytical Procedures: Definitions and

Terminology*)
Date of first adoption November 1994
Date of entry into For studies commencing after June 1994
force
Previous titles/other ICH Q2A: Validation of Analytical Methods: Definitions
references and Terminology/III/5626/94
Additional Notes This note for guidance concerns the definitions and
terminology used in the application of the guideline on
Validation of Analytical Procedures: Methodology which
is applicable to test procedures used in documentation
submitted in accordance with Part 2, sections -F of the
Annex to Directive 75/318/EEC as amended, with a view to
the granting of a marketing authorisation for a medicinal
product.
This guideline is complementary to, and should be read
in conjunction with the guideline on Validation of
Analytical Procedures: Methodology.
It replaces and expands the 1989 CPMP guideline
Analytical Validation.
CONTENTS
1. INTRODUCTION
2. TYPES OF ANALYTICAL PROCEDURE TO BE VALIDATED
GLOSSARY
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DEFINITIONS AND TERMINOLOGY *)
L INTRODUCTION
This document presents a discussion of the characteristics for consideration during the
validation of the analytical procedures included as part of applications submitted within the
EC, Japan and USA. This document does not necessarily seek to cover the testing that may be
required for registration in, or export to, other areas of the world. Furthermore, this text
serves as a collection of terms, and their definitions, and is not intended to provide direction
on how to accomplish validation. These terms and definitions are meant to bridge the
differences that often exist between various compendia and regulators of the EC, Japan and
USA.
The objective of validation of an analytical procedure is to demonstrate that it is suitable for
its intended purpose. A tabular summation of the characteristics applicable to identification,
control of impurities and assay procedures is included. Other analytical procedures may be
considered in future additions to this document.
2. TYPES OF ANALYTICAL PROCEDURES TO BE VALIDATED

The discussion of the validation of analytical procedures is directed to the four most common
types of analytical procedures:
- Identification tests.
- Quantitative tests for impurities' content.
- Limit tests for the control of impurities.
- Quantitative tests of the active moiety in samples of substance or medicinal product or
other selected component(s) in the medicinal product.
Although there are many other analytical procedures, such as dissolution testing for
medicinal products or particle size determination for substance, these have not been
addressed in the initial text on validation of analytical procedures. Validation of these
additional analytical procedures is equally important to those listed herein and may be
addressed in subsequent documents.
A brief description of the types of tests considered in this document is provided below.
- Identification tests are intended to ensure the identity of an analyte in a sample. This
is normally achieved by comparison of a property of the sample (e.g., spectrum,
chromatographic behaviour, chemical reactivity, etc.) to that of a reference standard.
- Testing for impurities can be either a quantitative test or a limit test for the impurity
in a sample. Either test is intended to accurately reflect the purity characteristics of the
sample. Different validation characteristics are required for a quantitative test than
for a limit test.
- Assay procedures are intended to measure the analyte present in a given sample. In
the context of this document, the assay represents a quantitative measurement of the
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major component(s) in the substance. For the medicinal product, similar validation
characteristics also apply when assaying for the active or other selected component(s).
The same validation characteristics may also apply to assays associated with other
analytical procedures (e.g., dissolution).
The objective of the analytical procedure should be clearly understood since this will govern
the validation characteristics which need to be evaluated. Typical validation characteristics
which should be considered are listed below:
- Accuracy
- Precision
- Repeatability
- Intermediate Precision
- Specificity
- Detection Limit
- Quantitation Limit
- Linearity
- Range
Each of these validation characteristics is defined in the attached Glossary. The table lists
those validation characteristics regarded as the most important for the validation of different
types of analytical procedures. This list should be considered typical for the analytical
procedures cited but occasional exceptions should be dealt with on a case by case basis. It
should be noted that robustness is not listed in the table but should be considered at an
appropriate stage in the development of the analytical procedure.
Furthermore revalidation may be necessary in the following circumstances:
- changes in the synthesis of the active substance;
- changes in the composition of the medicinal product;
- changes in the analytical procedure.
The degree of revalidation required depends on the nature of the changes. Certain other
changes may require validation as well.
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TABLE
ASSAY
Type of analytical Testing for - dissolution
Identification
procedure impurities (measurement only)
- content/potency
characteristics quantitat. limit
Accuracy - + +
Precision
Repeatability - + +
Interra. Precision - + (1) - + (D
Specificity (2) + + + +
Detection Limit - -(3) + -
Quantitation Limit - + -
Linearity - + +
Range - + +
- signifies that this characteristic is not normally evaluated

+ signifies that this characteristic is normally evaluated
(1) in cases where reproducibility (see glossary) has been performed, intermediate
precision is not needed
(2) lack of specificity of one analytical procedure could be compensated by other supporting
analytical procedure(s)
(3) may be needed in some cases
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GLOSSARY
1. ANALYTICAL PROCEDURE
The analytical procedure refers to the way of performing the analysis. It should describe i n
detail the steps necessary to perform each analytical test. This may include but is not limited
to: the sample, the reference standard and the reagents preparations, use of the apparatus,
generation of the calibration curve, use of the formulae for the calculation, etc.
2. SPECIFICITY
Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, dgradants,
matrix, etc.
Lack of specificity of an individual analytical procedure may be compensated by other
supporting analytical procedure(s).
This definition has the following implications:
Identification: to ensure the identity of an analyte.
Purity Tests: to ensure that all the analytical procedures performed allow an accurate
statement of the content of impurities of an analyte, i.e. related substances
test, heavy metals, residual solvents content, etc.
Assay (content to provide an exact result which allows an accurate statement on the
or potency): content or potency of the analyte in a sample.
3. ACCURACY
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value
and the value found.
This is sometimes termed trueness.
4. PRECISION
The precision of an analytical procedure expresses the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions. Precision may be considered at three
levels: repeatability, intermediate precision and reproducibility.
Precision should be investigated using homogeneous, authentic samples. However, if it is
not possible to obtain a homogeneous sample it may be investigated using artificially
prepared samples or a sample solution.
The precision of an analytical procedure is usually expressed as the variance, standard
deviation or coefficient of variation of a series of measurements.
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4.1. Repeatability
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision.
4.2 Intermediate precision
Intermediate precision expresses within laboratory variations: different days, different
analysts, different equipment, etc.
4.3. Reproducibility
Reproducibility expresses the precision between laboratories (collaborative studies, usually
applied to standardisation of methodology).
5. DETECTION LIMIT
The detection limit of an individual analytical procedure is the lowest amount of analyte i n
a sample which can be detected but not necessarily quantitated as an exact value.
The quantitation limit of an individual analytical procedure is the lowest amount of analyte
in a sample which can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays for low levels of compounds i n
sample matrices, and is used particularly for the determination of impurities and/or
degradation products.
7. LINEARITY
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the
sample.
8. RANGE
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which
it has been demonstrated that the analytical procedure has a suitable level of precision,
accuracy and linearity.
9. ROBUSTNESS
The robustness of an analytical procedure is a measure of its capacity to remain unaffected
by small, but deliberate variations in method parameters and provides an indication of its
reliability during normal usage.
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STABILITY TESTING OF NEW ACTIVE SUBSTANCES

AND MEDICINAL PRODUCTS *)
G u i d e l i n e Title Stability Testing of New Active Substances a n d Medicinal

Products*)
Date of e n t r y into Compulsory for applications s u b m i t t e d after 1/1/1998
force
Previous titles/other ICH Q1A: Stability Testing of New Drug Substances and
references Products, IH/3352/92, CPMP/ICH/380/95
sections C a n d F of t h e Annex to Directive 75/318/EEC as
a u t h o r i s a t i o n for a n e w medicinal p r o d u c t .
This guideline replaces those sections of t h e CPMP
guideline entitled Stability tests on Active Ingredients and
Finished Products which refer to n e w active substances
and medicinal p r o d u c t s containing new active substances.
CONTENTS
PREAMBLE
OBJECTIVE
SCOPE
ACTrVE SUBSTANCE
PRODUCT
ANNEX I
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STABILITY TESTING OF NEW ACTIVE SUBSTANCES

AND MEDICINAL PRODUCTS *)
PREAMBLE
The following guideline sets out the stability testing requirement for a marketing
authorisation within the three areas of the EC, Japan and the USA. It does not seek
necessarily to cover the testing that may be required for registration in or export to other
areas of the world.
The guideline seeks to exemplify the core stability data package required for new active
substances and medicinal products. It is not always necessary to follow this when there are
scientifically justifiable reasons for using alternative approaches.
The guideline provides a general indication on the requirements for stability testing, but
leaves sufficient flexibility to encompass the variety of different practical situations
required for specific scientific situations and characteristics of the materials being
evaluated.
The principle that information on stability generated in any one of the three areas of the EC,
Japan and the USA would be mutually acceptable in both of the other two areas has been
established, provided it meets the appropriate requirements of this guideline and the
labelling is in accord with national/regional requirements.
Details of the specific requirements for sampling, test requirements for particular dosage
forms/packaging etc., are not covered in this guideline.
OBJECTRHE
The purpose of stability testing is to provide evidence on how the quality of a an active
substance or medicinal product varies with time under the influence of a variety of
environmental factors such as temperature, humidity and light, and enables recommended
storage conditions, re-test periods and shelf lives to be established.
SCOPE
The guideline primarily addresses the information required in marketing authorisations
for new active substances and associated medicinal products.
This guideline does not currently seek to cover the information required for abbreviated or
abridged applications, variations, clinical trial applications, etc.
The choice of test conditions defined in this guideline is based on an analysis of the effects
of climatic conditions in the three areas of the EC, Japan and the USA. The mean kinetic
temperature in any region of the world can be derived from climatic data (Grimm, W.
Drugs Made in Germany, 28, 196-202, 1985 and 29, 39-47, 1986).
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ACTrVE SUBSTANCE
General
Information on the stability of the active substance is an integral part of the systematic
approach to stability evaluation.
Stress Testing
Stress testing helps to determine the intrinsic stability of the molecule by establishing
degradation pathways in order to identify the likely degradation products and to validate the
stability indicating power of the analytical procedures used.
Formal Studies
Primary stability studies are intended to show that the active substance will remain within
specification during the retest period if stored under recommended storage conditions.
Selection of Batches
Stability information from accelerated and long term testing is to be provided on at least
three batches. The long term testing should cover a minimum of 12 months duration on at
least three batches at the time of submission.
The batches manufactured to the minimum of pilot plant scale should be by the same
synthetic route and use a method of manufacture and procedure that simulates the final
process to be used on a manufacturing scale.
The overall quality of the batches of active substance placed on stability should be
representative of both the quality of the material used in preclinical and clinical studies
and the quality of material to be made on a manufacturing scale.
Supporting information may be provided using stability data on batches of active substance
made on a laboratory scale.
The first three production batches of active substance manufactured post approval, if not
submitted in the original marketing authorisation application, should be placed on long term
stability studies using the same stability protocol as in the approved marketing authorisation
application.
Test Procedures and Test Criteria

The testing should cover those features susceptible to change during storage and likely to
influence quality, safety and/or efficacy. Stability information should cover as necessary
the physical, chemical and microbiological test characteristics. Validated stability
indicating testing methods must be applied. The need for the extent of replication will depend
on the results of validation studies.
Specification
Limits of acceptability should be derived from the profile of the material as used in the pre
clinical and degradation products, the justification for which should be influenced by the
levels observed in material used in preclinical studies and clinical trials.
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Storage Conditions
The length of the studies and the storage conditions should be sufficient to cover storage
shipment and subsequent use. Application of the same storage conditions as applied to the
medicinal product will facilitate comparative review and assessment. Other storage
conditions are allowable if justified. In particular, temperature sensitive active substances
should be stored under an alternative, lower temperature condition which will then become
the designated long term testing storage temperature. The six months accelerated testing
should then be carried out at a temperature at least 15C above this designated long term
storage temperature (together with the appropriate relative humidity conditions for that
temperature). The designated long term testing conditions will be reflected in the labelling
and re-test date.
Conditions Minimum Time Period at Submission

Long term testing 25C2C/ 60% RH5% 12 months
Accelerated Testing 40C2C/ 75% RH+5% 6 months
Where 'significant change' occurs during six months storage under conditions of
accelerated testing at 40C2C/75 percent RH5 percent, additional testing at an
intermediate condition (such as 30C2C/60 percent RH5 percent) should be conducted for
active substances to be used in the manufacture of dosage forms tested long term at 25C/60
percent RH and this information included in the marketing authorisation application. The
initial marketing authorisation application should include a minimum of 6 months data
from a 12 months study.
'Significant change' at 40C/75 percent RH or 30C/60 percent RH is defined as failure to
meet the specification.
The long term testing will be continued for a sufficient period of time beyond 12 months to
cover all appropriate re-test periods, and the further accumulated data can be submitted to the
Authorities during the assessment period of the marketing authorisation application.
The data (from accelerated testing at an intermediate condition) may be used to evaluate the
impact of short term excursions outside the label storage conditions such as might occur
during shipping.
Testing Frequency-
Frequency of testing should be sufficient to establish the stability characteristics of the active
substance. Testing under the defined long term conditions will normally be every three
months, over the first year, every six months over the second year and then annually.
Packaging/Containers
The containers to be used in the long term, real time stability evaluation should be the same
as or simulate the actual packaging used for storage and distribution
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Evaluation
The design of the stability study is to establish, based on testing a minimum of three batches
of the active substance and evaluating the stability information (covering as necessary the
physical, chemical and microbiological test characteristics), a retest period applicable to all
future batches of the bulk substance manufactured under similar circumstances. The degree
of variability of individual batches affects the confidence that a future production batch will
remain within specification until the retest date.
An acceptable approach for quantitative characteristics that are expected to decrease with
time is to determine the time at which the 95% one-sided confidence limit for the mean
degradation curve intersects the acceptable lower specification limit. If analysis shows that
the batch to batch variability is small, it is advantageous to combine the data into one overall
estimate and this can be done by first applying appropriate statistical tests (for example,
values for level of significance of rejection of more than 0.25) to the slopes of the regression
lines and zero time intercepts for the individual batches. If it is inappropriate to combine
data from several batches, the overall retest period may depend on the minimum time a
batch may be expected to remain within acceptable and justified limits.
The nature of any degradation relationship will determine the need for transformation of
the data for linear regression analysis. Usually the relationship can be represented by a
linear, quadratic or cubic function on an arithmetic or logarithmic scale. Statistical methods
should be employed to test the goodness of fit of the data on all batches and combined batches
(where appropriate) to the assumed degradation line or curve.
The data may show so little degradation and so little variability that it is apparent from
looking at the data that the requested retest period will be granted. Under the circumstances,
it is normally unnecessary to go through the formal statistical analysis but merely to
provide a full justification for the omission.
Limited extrapolation of the real time data beyond the observed range to extend expiration
dating at approval time, particularly where the accelerated data supports this, may be
undertaken. However, this assumes that the same degradation relationship will continue to
apply beyond the observed data and hence the use of extrapolation must be justified in each
application in terms of what is known about the mechanism of degradation, the goodness of
fit of any mathematical model, batch size, existence of supportive data etc.
Any evaluation should cover not only the assay, but the levels of degradation products and
other appropriate attributes.
Statements/Labelling
A storage temperature range may be used in accordance with relevant national/regional
requirements. The range should be based on the stability evaluation of the active substance.
Where applicable, specific requirements should be stated, particularly for active substances
that cannot tolerate freezing. The use of terms such as 'ambient conditions' or 'room
temperature' is unacceptable.
A re-test period should be derived from the stability information.
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PRODUCT
General
The design of the stability program for the finished product should be based on the knowledge
of the behaviour and properties of the active substance and the experience gained from
clinical formulation studies and from the stability studies on the active substance. The
likely changes on storage and the rationale for the selection of product variables to include
in the testing programme should be stated.
Selection of Batches
Stability information from accelerated and long term testing is to be provided on three
batches of the same formulation and dosage form in the containers and closure proposed for
marketing. Two of these three batches should be at least pilot scale. The third one may be
smaller (e.g., 25,000 to 50,000 tablets or capsules for solid oral dosage forms). The long term
testing should cover at least 12 months duration at the time of submission. The
manufacturing process to be used should meaningfully simulate that which would be applied
to large scale batches for marketing. The process should provide medicinal product of the
same quality intended for marketing, and meeting the same quality specification as to be
applied for release of material. Where possible, batches of the finished product should be
manufactured using identifiably different batches of active substance.
Data on the laboratory scale batches is not acceptable as primary stability information. Data
on associated formulations or packaging may be submitted as supportive information. The
first three production batches manufactured post approval, if not submitted in the original
marketing authorisation application, should be placed on accelerated and long term stability
studies using the same stability protocols as in the approved marketing authorisation.
Test procedures and Test Criteria

The testing should cover those features susceptible to change during storage and likely to
influence quality, safety and/or efficacy. Analytical test procedures should be fully
validated and the assays should be stability-indicating. The need for the extent of replication
will depend on the results of validation studies.
The range of testing should cover not only chemical and biological stability but also loss of
preservative, physical properties and characteristics, organoleptic properties and where
required, microbiological attributes. Preservative efficacy testing and assays on stored
samples should be carried out to determine the content and efficacy of antimicrobial
preservatives.
Specifications
Limits of acceptance should relate to the release limits (where applicable), to be derived from
consideration of all the available stability information. The shelf life specification could
allow acceptable and justifiable derivations from the release specification based on the
stability evaluation and the changes observed on storage. It will need to include specific
upper limits for degradation products, the justification for which should be influenced by the
levels observed in material used in pre-clinical studies and clinical trials. The justification
for the limits proposed for certain other tests such as particle size and/or dissolution rate will
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require reference to the results observed for batch(es) used in bioavailability and/or clinical
studies. Any differences between the release and shelf life specifications for antimicrobial
preservatives should be supported by preservative efficacy testing.
Storage Test Conditions

The length of the studies and the storage conditions should be sufficient to cover storage,
shipment and subsequent use (e.g., reconstitution or dilution as recommended in the
labelling).
See Table below for accelerated and long term storage conditions and minimum times. An
assurance that long term testing will continue to cover the expected shelf life should be
provided.
Other storage conditions are allowable if justified. Heat sensitive finished products should be
stored under an alternative lower temperature condition which will eventually become the
designated long term storage temperature. Special consideration may need to be given to
products which change physically or even chemically at lower storage conditions e.g.,
suspensions or emulsions which may sediment or cream, oils and semi-solid preparations
which may show an increased viscosity. Where a lower temperature condition is used, the
six months accelerated testing should be carried out at a temperature at least 15C above its
designated long term storage temperature (together with appropriate relative humidity
conditions for that temperature). For example, for a product to be stored long term under
refrigerated conditions, accelerated testing should be conducted at 25C2C/60 percent RH5
percent RH. The designated long term testing conditions will be reflected in the labelling
and expiration date.
Storage under conditions of high relative humidities applies particularly to solid dosage
forms. For products such as solutions, suspensions etc., contained in packs designed to
provide a permanent barrier to water loss, specific storage under conditions of high relative
humidity is not necessary but the same range of temperatures should be applied. Low relative
humidity (e.g., 10-20 percent RH) can adversely affect medicinal products packed in semi-
permeable containers (e.g. solutions in plastic bags, nose drops in small plastic containers
etc.) and consideration should be given to appropriate testing under such conditions.
Conditions Minimum Time Period at Submission

Long term testing 25C2C/60% RH5% 12 months
Accelerated Testing 40C2C/75% RH5% 6 months
Where 'significant change' occurs due to accelerated testing, additional testing at an

intermediate condition e.g. 30C2C/60 percent 5 percent RH should be conducted.
'Significant change' at the accelerated condition is defined as:
1. A 5 percent potency loss from the initial assay value of a batch;
2. Any specified degradant exceeding its specification limit;
3. The product exceeding its pH limits;
4. Dissolution exceeding the specification limits for 12 capsules or tablets.
5. Failure to meet specifications for appearance and physical properties e.g., colour, phase
separation, resuspendibility, delivery per actuation, caking, hardness, etc.
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Should significant change occur at 40C/75 percent RH then the initial marketing
authorisation application should include a minimum of 6 months data from an ongoing one
year study at 30C/60 percent RH; the same significant change criteria shall then apply.
The long term testing will be continued for a sufficient time beyond 12 months to cover shelf
life at appropriate test periods. The further accumulated data should be submitted to the
authorities during the assessment period of the marketing authorisation application.
The first three production batches manufactured post approval, if not submitted in the
original marketing authorisation application, should be placed on accelerated and long term
stability studies using the same stability protocol as in the approved marketing
authorisation.
Testing Frequency
Frequency of testing should be sufficient to establish the stability characteristics of the
medicinal product. Testing will normally be every three months over the first year, every
six months over the second year and then annually.
The use of matrixing or bracketing can be applied, if justified.(See Glossary).
Packaging Materials
The testing should be carried out in the final packaging proposed for marketing. Additional
testing of unprotected finished product can form a useful part of the stress testing and pack
evaluation, as can studies carried out in other related packaging materials in supporting the
definitive pack(s).
Evaluation
A systematic approach should be adopted in the presentation and evaluation of the stability
information which should cover as necessary physical, chemical, biological, microbiological
quality characteristics, including particular properties of the dosage form (for example
dissolution rate for oral solid dose forms).
The design of the stability study is to establish, based on testing a minimum of three batches
of the finished product, a shelf life and label storage instructions applicable to all future
batches of the dosage form manufactured and packed under similar circumstances. The
degree of variability of individual batches affects the confidence that a future production
batch will remain within specification until the expiration date.
An acceptable approach for quantitative characteristics that are expected to decrease with
time is to determine the time at which the 95% one-sided confidence limit for the mean
degradation curve intersects the acceptable lower specification limit. If analysis shows that
the batch to batch variability is small, it is advantageous to combine the data into one overall
estimate and this can be done by first applying appropriate statistical tests (for example,
values for level of significance of rejection of more than 0.25) to the slopes of the regression
lines and zero time intercepts for the individual batches. If it is inappropriate to combine
data from several batches, the overall shelf life may depend on the minimum time a batch
may be expected to remain within acceptable and justified limits.
The nature of the degradation relationship will determine the need for transformation of the
data for linear regression analysis. Usually the relationship can be represented by a linear,
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quadratic or cubic function on an arithmetic or logarithmic scale. Statistical methods should

be employed to test the goodness of fit on all batches and combined batches (where
appropriate) to the assumed degradation line or curve.
Where the data shows so little degradation and so little variability that it is apparent from
looking at the data that the requested shelf life will be granted, it is normally unnecessary to
go through the formal statistical analysis but only to provide a justification for the omission.
Limited extrapolation of the real time data beyond the observed range to extend expiration
dating at approval time, particularly where the accelerated data supports this, may be
undertaken. However, this assumes that the same degradation relationship will continue to
apply beyond the observed data and hence the use of extrapolation must be justified in each
application in terms of what is known about the mechanisms of degradation, the goodness of
fit of any mathematical model, batch size, existence of supportive data, etc.
Any evaluation should consider not only the assay, but the levels of degradation products
and appropriate attributes. Where appropriate, attention should be paid to reviewing the
adequacy of the mass balance, different stability and degradation performance.
The stability of the medicinal products after reconstituting or diluting according to
labelling, should be addressed to provide appropriate and supportive information.
Statements/Labelling
A storage temperature range may be used in accordance with relevant national/regional
requirements. The range should be based on the stability evaluation of the medicinal
product. Where applicable, specific requirements should be stated particularly for medicinal
products that cannot tolerate freezing.
The use of terms such as 'ambient conditions' or 'room temperature' is unacceptable.
There should be a direct linkage between the label statement and the demonstrated stability
of the medicinal product.
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ANNEX 1
Glossary and Information
The following terms have been in general use and the following definitions are provided to
facilitate interpretation of the guideline.
Accelerated Testing
Studies designed to increase the rate of chemical degradation or physical change of an active
substance or medicinal product by using exaggerated storage conditions as part of the
formal, definitive, storage programme.
These data, in addition to long term stability studies, may also be used to assess longer term
chemical effects at non-accelerated conditions and to evaluate the impact of short term
excursions outside the label storage conditions such as might occur during shipping. Results
from accelerated testing studies are not always predictive of physical changes.
Bracketing
The design of a stability schedule so that at any time point only the samples on the extremes,
for example of container size and/or dosage strengths, are tested. The design assumes that
the stability of the intermediate condition samples are represented by those at the extremes.
Where a range of dosage strengths is to be tested, bracketing designs may be particularly
applicable if the strengths are very closely related in composition (e.g., for a tablet range
made with different compression weights of a similar basic granulation, or a capsule range
made by filling different plug fill weights of the same basic composition into different size
capsule shells). Where a range of sizes of immediate containers are to be evaluated,
bracketing designs may be applicable if the material of composition of the container and the
type of closure are the same throughout the range.
Climatic Zones
The concept of dividing the world into four zones based on defining the prevalent annual
climatic conditions.
Dosage Form; Preparation

A pharmaceutical product type, for example tablet, capsule, solution, cream etc. that contains
an active substance generally, but not necessarily, in association with excipients.
Medicinal Product; Finished Product

The dosage form in the final immediate packaging intended for marketing.
Excipient
Anything other than the active substance in the dosage form.
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Expiry/Expiration Date
The date placed on the container/labels of a product designating the time during which a
batch of the product is expected to remain within the approved shelf life specification if stored
under defined conditions, and after which it must not be used.
Formal (Systematic) Studies

Formal studies are those undertaken to a pre-approval stability protocol which embraces the
principles of these guidelines.
Long Term (Real Time) Testing

Stability evaluation of the physical, chemical, biological and microbiological characteristics
of a product and an active substance, covering the expected duration of the shelf life and re-
test period, which are claimed in the submission and will appear on the labelling.
Mass Balance; Material Balance

The process of adding together the assay value and levels of degradation products to see how
closely these add up to 100 per cent of the initial value, with due consideration of the margin
of analytical precision.
This concept is a useful scientific guide for evaluating data but it is not achievable in all
circumstances. The focus may instead be on assuring the specificity of the assay, the
completeness of the investigation of routes of degradation, and the use, if necessary, of
identified dgradants as indicators of the extent of degradation via particular mechanisms.
Matrixing
The statistical design of a stability schedule so that only a fraction of the total number of
samples are tested at any specified sampling point. At a subsequent sampling point, different
sets of samples of the total number would be tested. The design assumes that the stability of
the samples tested represents the stability of all samples. The differences in the samples for
the same product should be identified as, for example, covering different batches, different
strengths, different sizes of the same container and closure and possibly in some cases
different container/closure systems.
Matrixing can cover reduced testing when more than one variable is being evaluated. Thus
the design of the matrix will be dictated by the factors needing to be covered and evaluated.
This potential complexity precludes inclusion of specific details and examples, and it may
be desirable to discuss design in advance with the Regulatory Authority, where this is
possible. In every case it is essential that all batches are tested initially and at the end of the
long term testing.
Mean Kinetic Temperature

When establishing the mean value of the temperature, the formula of J. D. Haynes (J.
Pharm. Sci. 60, 927-929, 1971) can be used to calculate the mean kinetic temperature. It is
higher than the arithmetic mean temperature and takes into account the Arrhenius equation
from which Haynes derived his formula.
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New Molecular Entity; New Active Substance

An active substance which has not previously been registered as a new active substance with
the national or regional authority concerned.
Pilot Plant Scale

The manufacture of either active substance or product by a procedure fully representative of
and simulating that to be applied on a full manufacturing scale.
For oral solid dosage forms this is generally taken to be at a minimum scale of one tenth
that of full production or 100,000 tablets or capsules, whichever is the larger.
Primary Stability Data

Data on the active substance stored in the proposed packaging under storage conditions that
support the proposed re-test date.
Data on the product stored in the proposed container-closure for marketing under storage
conditions that support the proposed shelf life.
Re-Test Date
The date when samples of the active substance should be re-examined to ensure that
material is still suitable for use.
Re-Test Period
The period of time during which the active substance can be considered to remain within the
specification and therefore acceptable for use in the manufacture of a given product,
provided that it has been stored under the defined conditions; after this period, the batch
should be retested for compliance with specification and then used immediately.
Shelf life; Expiration Dating Period

The time interval that a product is expected to remain within the approved shelf life
specification provided that it is stored under the conditions defined on the label in the
proposed containers and closure.
Specification - Release
The combination of physical, chemical, biological and microbiological test requirements
that determine a product is suitable for release at the time of its manufacture.
Specification - Check/Shelf life

The combination of physical, chemical, biological and microbiological test requirements
that a active substance must meet up to its re-test date or a product must meet throughout its
shelf life.
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Storage Conditions Tolerances

The acceptable variation in temperature and relative humidity of storage facilities.
The equipment must be capable of controlling temperature to a range of 2C and Relative
Humidity to 5% RH. The actual temperatures and humidities should be monitored during
stability storage. Short term spikes due to opening of doors of the storage facility are accepted
as unavoidable. The effect of excursions due to equipment failure should be addressed by the
applicant and reported if judged to impact stability results. Excursions that exceed these
ranges (i.e., 2C and/or 5 percent RH) for more than 24 hours should be described in the
study report and their impact assessed.
Stress Testing (Active substance)

These studies are undertaken to elucidate intrinsic stability characteristics. Such testing is
part of the development strategy and is normally carried out under more severe conditions
than those used for accelerated tests.
Stress testing is conducted to provide data on forced decomposition products and
decomposition mechanisms for the active substance. The severe conditions that may be
encountered during distribution can be covered by stress testing of definitive batches of
active substance.
These studies should establish the inherent stability characteristics of the molecule, such as
the degradation pathways, and lead to identification of degradation products and hence
support the suitability of the proposed analytical procedures. The detailed nature of the studies
will depend on the individual active substance and type of product.
This testing is likely to be carried out on a single batch of material and to include the effect
of temperatures in 10C increments above the accelerated temperature test condition (e.g.,
50C, 60C, etc.) humidity where appropriate (e.g., 75 per cent or greater); oxidation and
photolysis on the active substance plus its susceptibility to hydrolysis across a wide range of
pH values when in solution or suspension.
Results from these studies will form an integral part of the information provided to
regulatory authorities.
Light testing should be an integral part of stress testing. [The standard conditions for light
testing are discussed in the note for guidance Photostability Testing of New Active Substances
and Medicinal Products.]
It is recognised that some degradation pathways can be complex and that under forcing
conditions decomposition products may be observed which are unlikely to be formed under
accelerated or long term testing. This information may be useful in developing and
validating suitable analytical methods, but it may not always be necessary to examine
specifically for all degradation products, if it has been demonstrated that in practice these
are not formed.
Stress Testing (Product)

Light testing should be an integral part of stress testing (see note for guidance on
Photostability Testing of New Active Substances and Medicinal Products).
Special test conditions for specific products (e.g., metered dose inhalations and creams and
emulsions) may require additional stress studies.
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Supporting Stability Data

Data other than primary stability data, such as stability data on early synthetic route batches
of active substance, small scale batches of materials, investigational formulations not
proposed for marketing, related formulations, product presented in containers and/or
closures other than those proposed for marketing, information regarding test results on
containers, and other scientific rationale that support the analytical procedures, the proposed
re-test period or shelf life and storage conditions.
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STABILITY TESTING ON ACTIVE INGREDIENTS AND

FINISHED PRODUCTS
Guideline Title Stability Testing on Active Substances a n d Finished

Products
Date of first adoption J u l y 1988
Date of entry into J a n u a r y 1989
force
Previous titles/other None
references
section F of t h e Annex to Directive 75/318/EEC as
a u t h o r i s a t i o n for a medicinal p r o d u c t .
This note for guidance is c u r r e n t l y (1997) being revised
by t h e CPMP, b u t until a new version is adopted, it
applies to applications for medicinal p r o d u c t s c o n t a i n i n g
k n o w n active substances. Applications for medicinal
p r o d u c t s containing new active substances should be
based on the preceding note for guidance: Stability Testing
on New Active Substances and Medicinal Products which
refers to new active substances a n d medicinal p r o d u c t s
containing new active substances.
CONTENTS
1. INTRODUCTION
2. INTRINSIC PROPERTD3S OF THE ACTRÊ INGREDIENT
3. STABILITY TESTS ON THE FINISHED PRODUCT
ANNEX
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STABILITY TESTS ON ACTIVE INGREDIENTS AND

FINISHED PRODUCTS
Note for guidance concerning the application of Part 2, section F of the Annex to Directive
75/318/EEC, as amended, with a view to the granting of a marketing authorisation for a new
medicinal product.
L INTRODUCTION
This note concerns research enabling the applicant to determine what shelf-life to propose.
The purpose of stability tests is to obtain information which enables proposals to be made for
the shelf-life of the medicinal product and to recommend storage conditions.
The quality of a medicinal product is determined by its content of active substance(s), its
purity (limitation or absence of decomposition products of the active substance(s)) and its
organoleptic, physico-chemical and microbiological properties.
The purpose of the stability studies is to ascertain how the quality of a medicinal product
varies as a function of time and under the influence of a variety of environmental factors.
On the basis of the information thus obtained, storage conditions are recommended (the
purpose of these studies is to produce recommendations) which will guarantee maintenance
of the quality of the medicinal product, in relation to its safety, efficacy and acceptability,
throughout the proposed shelf-life (i.e. during storage, distribution, dispensing and use).
The design of the finished product stability studies for a medicinal product is based on the
knowledge obtained from the studies on the active substance and from the Development
Pharmaceutics studies.
2. INTRINSIC PROPERTIES OF THE ACTrVE INGREDIENT

Information on the stability of the active ingredient can be ascertained:
- for new active ingredient: by experimental studies;
for known active ingredients: as a rule, evidence from the scientific literature is
acceptable, but comparative accelerated stability studies may need to be considered in
certain cases, such as when there is a significant change in the route of synthesis from
that approved by the competent authorities, or when there is a significant change in the
production method.
Where there are several possible active ingredient manufacturers and/or methods of
obtaining the active ingredient, consideration may need to be given to the examination of
studies on material from each source.
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2.1 Batches examined

- the number of batches (minimum of two), together with the batch identification number,
date of manufacture and size of each batch and the name of the manufacturer.
2.2 General methodology of the study

- details of the accelerated and proposed storage conditions used, taking into account the
physical and chemical properties of the substance: temperature, humidity and light;
exposure to air and chemical agents (particularly in solution in water and/or other
solvents);
- duration of exposure under various conditions;
- materials and containers used.
2.3 Analytical methods

- the assay techniques used, normally with evidence from the analytical validation
studies of their specificity (i.e. that they separate the active ingredient from its
degradation products);
- method of determination (wherever possible) of the degradation products.
2.4 Results
- details of results obtained, as notes or numerical data, in table form.
2.5 Interpretation
- the conclusions as to the most appropriate storage conditions for the active ingredient,
and the duration of storage before the substance needs retesting to check for compliance
with specification;
- the discussion of the significance of the decomposition products, particularly as
regards their potential toxicity.
2.6 Conclusions
The stability data on the active ingredient and the development studies enable a preliminary
choice of the formulation and packing material to be made. They also enable some
consideration to be given to the choice of analytical methods and test conditions for the
studies on the pharmaceutical form.
3 STABILITY TESTS ON THE FINISHED PRODUCT

3.1 Objective
The analysis of the results of the stability studies on the finished product should allow the
determination of the shelf-life, the recommendations for storage conditions (where relevant
before and after opening the container), and the justification of any overage of the active
ingredient applied to guarantee the potency at the end of the shelf-life.
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The design of the stability tests is based on the known properties of the active ingredient (see
section 2), the results of the Development Pharmaceutics studies, the properties of the chosen
formulation and the recommendations for use of the product.
The specifications proposed at the time of manufacture and to the end of the proposed shelf-
life must reflect, as far as possible, the results of the stability studies, particularly in relation
to any parameters which could have a bearing on efficacy and safety and product
acceptability.
Specific in-use stability tests must be carried out where the product is labile once the
container is opened, or where the product is to be diluted or reconstituted before use.
3.2 Study methods

3.2.1 Real-time studies
These studies should be carried out under a range of controlled test conditions which will
enable the shelf-life and the product container label/package insert storage requirements to
be defined. This will normally include studies which will allow the properties of the product
at temperatures between 20C and 30C to be evaluated. However, 25C should be used as the
mean kinetic testing temperature for products in the European Union.
For each study, the mean temperature, the ranges of temperature and the mean humidity
should be stated.
3.2.2 Studies under varied conditions

Such studies should be carried out to provide essential additional information. They can
fulfil a number of objectives such as:
- to support the initial shelf-life request, by complementing the limited results of the
early real-time studies as the decomposition, if it is occurring, is likely to be
accelerated,
- to produce useful data at an early stage of development,
- to demonstrate the effects of adverse storage on the packaging and product, and to
enable storage conditions and suitable labelling to be provided,
- to help to define suitable conditions for storage of the product,
- to support a request to extend the shelf-life,
- to support major changes in formulation, packaging materials or method of
preparation.
The various test conditions should be stated. Depending on the nature and objectives of the
stability study, the following may need to be considered:
- various test temperatures: three or more (see 3.7) particularly if long term real-time
data is unavailable. The effect of low temperatures may, in addition, need to be
considered such as below -15C (freezer), 2-8C (refrigerator) and freeze-thaw recycling,
- high humidity: relative humidity not less than 75%,
- elevated temperature and humidity in combination: e.g. temperature of 40C associated
with a relative humidity of 75%, possibly the effects of cycling between different
temperatures and humidities,
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- light: either natural day light or defined artificial illumination.
3.3 Definition of the product under study

3.3.1 Number and nature of the batches tested
The number of batches tested must be stated, with the batch number, details of the
composition, date of manufacture, the size of the batch and the batch number and name of the
manufacturer of the active ingredient(s) used. These should include where possible
production scale batches of the product.
Normally at least 3 batches of the dosage form must be studied; however in circumstances
where the active substance is an existing one (already used in licensed products), and it is
found that the active substance is stable in the product and no significant decomposition
products are formed, the stability study can be limited to 2 batches.
3.3.2 Immediate packaging

The batches of product must be packaged in the way proposed for marketing. However,
supplementary evidence from batches of product in related packs must be used to augment
this data.
Details of the packaging should be stated as:
- type(s) of container and closure, and nature of the constituent materials,
- nature of any dessicant used.
3.4 Characteristics
The characteristics studied should be:
- those in the finished product specification that are likely to be affected by storage, and
- those not monitored routinely at the time of manufacture, but which may be indicative
of the stability/instability of the particular dosage form, e.g. dissolution of tablets.
3.4.1 Physical characteristics and microbiological aspects of the finished

product
- organoleptic properties,
- physical properties specific to the dosage form, such as tablet hardness,
- important quality parameters such as the in vitro dissolution test, moisture content
(e.g. in relation to any dessicant used in the packaging and particle size,
- efficacy of preservatives at the end of the reported storage test period or at the end of the
shelf-life, except where otherwise justified,
- any other physical characteristics of the finished product that must be known in order
to assess the product stability.
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3.4.2 Chemical characteristics of the finished product

- assay of the active principle(s),
content of decomposition products,
- content of other agents (such as antimicrobial preservatives and antioxidants),
any other chemical characteristics that must be known in order to assess the quality of
the product.
3.4.3 Characteristics of the packaging to be considered

- study of the container and closure interaction with the contents in any case where this
is a risk.
3.5 E v a l u a t i o n m e t h o d s
The test methods used must be fully described. It must be shown that they are capable of
detecting decomposition of the active ingredient in the medicinal product, and unless
justified, of quantifying any decomposition products. If possible, the assay method for the
active substance in the finished product should be stability indicating.
The test procedures applied to the stability tests on the finished product must be validated.
3.6 P r e s e n t a t i o n of r e s u l t s
The results should summarised (e.g. as tables and graphs). For each batch of product, the
initial results (at the time of manufacture), the results during storage and at the end of the
proposed shelf -line should be given. However, results of real time data should be supplied as
they become available.
3.7 D i s c u s s i o n , i n t e r p r e t a t i o n a n d c o n c l u s i o n s
The discussion in the Expert Report should provide a critical evaluation of the suitability of
the test methods used, the results obtained and proposed shelf-life specification.
If necessary to carry out any further studies due to significant changes in physical
properties, an explanation should be given, together with the results of these studies.
Studies under accelerated test conditions will increase the decomposition and may permit
some extrapolation of the room temperature shelf-life from that which would otherwise be
acceptable. However, such studies would always need to be supplemented by long-term real
time studies, and normally at least 6 month real time data should be presented in the
application for marketing authorisation.
If batches of the product demonstrate a different stability profile, the shelf-life proposed and
any overage should be based on the stability of the least stable, unless an explanation can be
given.
The shelf-life should be proposed for the product as packaged for sale. If necessary, a
recommendation should also be given regarding the shelf-life before and after opening the
container, and after dilution or reconstitution, and storage during marketing and use.
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If there is evidence that batches of the stored product as packed for sale are stable at
temperatures up to 30C, the product need bear no special temperature storage instructions.
However, if there is evidence that the product must be stored under defined conditions of
storage, this must be stated on the container label and the package insert (if included). The
maximum (or minimum) storage temperature should be stated in Celsius (e.g. store below
25C, store in a refrigerator at 2-8C, do not refrigerate - store above 8C). These
label/package insert storage recommendations must fully reflect conditions found in the
Member State for which marketing authorisation is sought (see Annex), and those in any
other Member State to which the product is likely to be supplied (whether by the person
responsible for placing the product on the market or another person).
3.8 Ongoing stability

Where data on routine production batches is not provided in the application for marketing
authorisation, ongoing stability studies should be carried out on 2 or 3 of the first production
batches and the result provided to the competent authorities on an ongoing basis.
3.9 Variation to the marketing authorisation

Significant changes to the particulars and documents on which the marketing authorisation
was approved, such as:
- major changes in composition,
- changes in packaging materials,
- major changes in the method of preparation for the product,
would normally necessitate the results of comparative accelerated or long term stability
studies being provided to the competent authorities before such changes are introduced.
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ANNEX
International Climatic Zones and Climatic Conditions
Zone II Zone III

Climatic Zone I Zone rV
Mediterranean Hot/dry or
Condition Temperate Vey hot/humid
(sub-tropical) Hot/moderate RH
Mean Annual
<20C 20.5-24C >24C >24C
Temperature
Kinetic Mean
Temperature
21C 26C 31C 31C
(Virtual
temperature)
Mean Annual
45% 60% 40% 70%
Relative
Humidity
Zones I and II are those in which EEC countries are situated.
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STABILITY TESTING: REQUIREMENTS FOR NEW

DOSAGE FORMS *)
Guideline Title Stability Testing: Requirements for New Dosage Forms *)

Date of entry into For studies commencing after January 1998
force
Previous titles/other ICH QIC /CPMP/ICH/28095
references
Additional Notes This note for guidance concerns the application of Part 2,
section F of the Annex to Directive 75/318/EEC as
authorisation for a new dosage form of an already
authorised medicinal product.
It is an annex to the Stability Testing of New Active
Substances and Medicinal Products guideline which
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STABILITY TESTING: REQUIREMENTS FOR NEW

DOSAGE FORMS *)
GENERAL
This document is an annex to the note for guidance: Stability Testing of New Active
Substances and Medicinal Products and addresses the recommendations on the data which
should be submitted regarding stability of new dosage forms by the owner of the original
application, after the original submission for new active substances and medicinal products.
NEW DOSAGE FORMS

A new dosage form is defined as a medicinal product which is a different pharmaceutical
product type, but containing the same active substance as included in an existing product
approved by the pertinent regulatory authority.
Such pharmaceutical product types include products of a different route of administration
(e.g., oral to parenteral), new specific functionality/delivery systems (e.g., immediate
release tablet to modified release tablet) and different dosage forms of the same route of
administration (e.g., capsule to tablet, solution to suspension).
Stability protocols for new dosage forms should follow the guidance in the parent stability
guideline in principle. However, a reduced stability database at submission time (e.g., 6
months accelerated and 6 months long term data from ongoing studies) may be acceptable in
certain justified cases.
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PHOTOSTABILITY TESTING OF NEW ACTIVE

SUBSTANCES AND MEDICINAL PRODUCTS *)
Guideline Title Photostability Testing of New Active Substances and Medicinal

Products *)
Date of entry into For studies commencing after January 1998
force
Previous titles/other ICH QIB: Photostability Testing of New Drug Substances
references and Products, CPMP/ICH/279/95
sections C and F of the Annex to Directive 75/318/EEC as
authorisation for a new medicinal product.
This guideline is an annex to the Stability Testing of New
Active Substances and Medicinal Products guideline
which should be consulted for basic principles.
CONTENTS
1. GENERAL
2. SUBSTANCES
3. MEDICINAL PRODUCT
4. ANNEX
5. GLOSSARY
6. REFERENCES
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PHOTOSTABILITY TESTING OF NEW ACTIVE

SUBSTANCES AND MEDICINAL PRODUCTS
I GENERAL
The note for guidance on Stability Testing of New Active Substances and Medicinal Products,
originally published as an ICH Harmonised Tripartite Guideline (hereafter referred to as
the Parent Guideline) notes that light testing should be an integral part of stress testing. This
document is an annex to the Parent Guideline and addresses the recommendations for
photostability testing.
II Preamble
The intrinsic photostability characteristics of new active substances and medicinal products
should be evaluated to demonstrate that, as appropriate, light exposure does not result i n
unacceptable change. Normally, photostability testing is carried out on a single batch of
material selected as described under Selection of Batches in the Parent Guideline. Under
some circumstances these studies should be repeated if certain changes are made to the
product (e.g., formulation, packaging). Whether these studies should be repeated depends on
the photostability characteristics determined at the time of first submission of an application
and the type of change made.
The guideline primarily addresses the generation of photostability information for
submission in applications for marketing authorisations for new active substances and
associated medicinal products. The guideline does not cover the photostability of medicinal
products after administration (i.e. under conditions of use) and those applications not
covered by the Parent Guideline. Alternative approaches may be used if they are
scientifically sound and justification is provided.
A systematic approach to photostability testing is recommended covering, as appropriate,
studies such as:
i) Tests on the active substance;
ii) Tests on the exposed product outside of the immediate pack,
and if necessary;
iii) Tests on the product in the immediate pack;
and if necessary;
iv) Tests on the product in the marketing pack.
The extent of product testing should be established by assessing whether or not acceptable
change has occurred at the end of the light exposure testing as described in the Decision Flow
Chart for Photostability Testing of Medicinal Products. Acceptable change is change within
limits justified by the applicant.
The formal labelling requirements for photolabile active substances and products are
established by national requirements.
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DECISION FLOWCHART FOR PHOTOSTABILITY TESTING OF MEDICINAL

PRODUCTS
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1.2 Light sources

The light sources described below may be used for photostability testing. The applicant should
either maintain an appropriate control of temperature to minimise the effect of localised
temperature changes or include a dark control in the same environment unless otherwise
justified. For both options 1 and 2, a pharmaceutical manufacturer/applicant may rely on the
spectral distribution specification of the light source manufacturer.
Option 1
Any light source that is designed to produce an output similar to the D65/ID65 emission
standard such as an artificial daylight fluorescent lamp combining visible and ultraviolet
(UV) outputs, xenon, or metal halide lamp. D65 is the internationally recognised standard
for outdoor daylight as defined in ISO 10977 (1993). ID65 is the equivalent indoor indirect
daylight standard. For a light source emitting significant radiation below 320 nm, an
appropriate filter(s) may be fitted to eliminate such radiation.
Option 2
For option 2 the same sample should be exposed to both the cool white fluorescent and near
ultraviolet lamp.
1. A cool white fluorescent lamp designed to produce an output similar to that specified i n
ISO 10977(1993); and
2. A near UV fluorescent lamp having a spectral distribution from 320 nm to 400 nm with
a maximum energy emission between 350 nm and 370 nm; a significant proportion of
UV should be in both bands of 320 to 360 nm and 360 to 400 nm.
1.3 Procedure
For confirmatory studies, samples should be exposed to light providing an overall
illumination of not less than 1.2 million lux hours and an integrated near ultraviolet
energy of not less than 200 watt hours/square meter to allow direct comparisons to be made
between the substance and product.
Samples may be exposed side-by-side with a validated chemical actinometric system to
ensure the specified light exposure is obtained, or for the appropriate duration of time when
conditions have been monitored using calibrated radiometers/lux meters. An example of an
actinometric procedure is provided in the Annex.
If protected samples (e.g., wrapped in aluminium foil) are used as dark controls to evaluate
the contribution of thermally induced change to the total observed change, these should be
placed alongside the authentic sample.
2. ACTIVE SUBSTANCE
For active substances, photostability testing should consist of two parts: forced degradation
testing and confirmatory testing.
The purpose of forced degradation testing studies is to evaluate the overall photosensitivity of
the material for method development purposes and/or degradation pathway elucidation. This
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testing may involve the active substance alone and/or in simple solutions/suspensions to
validate the analytical procedures. In these studies, the samples should be in chemically
inert and transparent containers. In these forced degradation studies, a variety of exposure
conditions may be used, depending on the photosensitivity of the active substance involved
and the intensity of the light sources used. For development and validation purposes it is
appropriate to limit exposure and end the studies if extensive decomposition occurs. For
photostable materials, studies may be terminated after an appropriate exposure level has been
used. The design of these experiments is left to the applicant's discretion although the
exposure levels used should be justified.
Under forcing conditions, decomposition products may be observed that are unlikely to be
formed under the conditions used for confirmatory studies. This information may be useful
in developing and validating suitable analytical methods. If in practice it has been
demonstrated they are not formed in the confirmatory studies, these degradation products
need not be further examined.
Confirmatory studies should then be undertaken to provide the information necessary for
handling, packaging, and labelling (see section 1.3, Procedure, and 2.1, Presentation, for
information on the design of these studies).
Normally, only one batch of active substance is tested during the development phase, and
then the photostability characteristics should be confirmed on a single batch selected as
described in the Parent Guideline if the active substance is clearly photostable or photolabile.
If the results of the confirmatory study are equivocal, testing of up to two additional batches
should be conducted. Samples should be selected as described in the Parent Guideline.
2.1 Presentation of samples

Care should be taken to ensure that the physical characteristics of the samples under test are
taken into account and efforts should be made, such as cooling and/or placing the samples
in sealed containers, to ensure that the effects of the changes in physical states such as
sublimation, evaporation or melting are minimised. All such precautions should be chosen
to provide minimal interference with the exposure of samples under test. Possible
interactions between the samples and any material used for containers or for general
protection of the sample, should also be considered and eliminated wherever not relevant to
the test being carried out.
As a direct challenge for samples of solid active substances, an appropriate amount of
sample should be taken and placed in a suitable glass or plastic dish and protected with a
suitable transparent cover if considered necessary. Solid active substances should be spread
across the container to give a thickness of typically not more than 3 millimetres. Active
substances that are liquids should be exposed in chemically inert and transparent
containers.
2.2 Analysis of samples

At the end of the exposure period, the samples should be examined for any changes in
physical properties (e.g., appearance, clarity, or colour of solution) and for assay and
dgradants by a method suitably validated for products likely to arise from photochemical
degradation processes.
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Where solid active substance samples are involved, sampling should ensure that a
representative portion is used in individual tests. Similar sampling considerations, such as
homogenisation of the entire sample, apply to other materials that may not be homogeneous
after exposure. The analysis of the exposed sample should be performed concomitantly with
that of any protected samples used as dark controls if these are used in the test.
2.3 E v a l u a t i o n of r e s u l t s
The forced degradation studies should be designed to provide suitable information to develop
and validate test methods for the confirmatory studies. These test methods should be capable
of resolving and detecting photolytic dgradants that appear during the confirmatory studies.
When evaluating the results of these studies, it is important to recognise that they form part
of the stress testing and are not therefore designed to establish qualitative or quantitative
limits for change.
The confirmatory studies should identify precautionary measures needed in manufacturing
or in formulation of the product, and if light resistant packaging is needed. When
evaluating the results of confirmatory studies to determine whether change due to exposure to
light is acceptable, it is important to consider the results from other formal stability studies
in order to assure that the active substance will be within justified limits at time of use (see
the relevant Stability and Impurity Guidelines).
3. MEDICINAL PRODUCT
Normally, the studies on products should be carried out in a sequential manner starting with
testing the fully exposed product then progressing as necessary to the product in the
immediate pack and then in the marketing pack. Testing should progress until the results
demonstrate that the product is adequately protected from exposure to light. The product
should be exposed to the light conditions described under the procedure in section 1.3.
Normally, only one batch of product is tested during the development phase, and then the
photostability characteristics should be confirmed on a single batch selected as described i n
the Parent Guideline if the product is clearly photostable or photolabile. If the results of the
confirmatory study are equivocal, testing of up to two additional batches should be conducted.
For some products where it has been demonstrated that the immediate pack is completely
impenetrable to light, such as aluminium tubes or cans, testing should normally only be
conducted on directly exposed product.
It may be appropriate to test certain products such as infusion liquids, dermal creams, etc., to
support their photostability in-use. The extent of this testing should depend on and relate to the
directions for use, and is left to the applicant's discretion.
The analytical procedures used should be suitably validated.
3.1 Presentation of samples

Care should be taken to ensure that the physical characteristics of the samples under test are
taken into account and efforts, such as cooling and/or placing the samples in sealed
containers, should be made to ensure that the effects of the changes in physical states are
minimised, such as sublimation, evaporation, or melting. All such precautions should be
chosen to provide a minimal interference with the irradiation of samples under test. Possible
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interactions between the samples and any material used for containers or for general
protection of the sample should also be considered and eliminated wherever not relevant to
the test being carried out.
Where practicable when testing samples of the product outside of the primary pack, these
should be presented in a way similar to the conditions mentioned for the active substance.
The samples should be positioned to provide maximum area of exposure to the light source.
For example, tablets, capsules, etc., should be spread in a single layer.
If direct exposure is not practical (e.g., due to oxidation of a product), the sample should be
placed in a suitable protective inert transparent container (e.g., quartz).
If testing of the product in the immediate container or as marketed is needed, the samples
should be placed horizontally or transversely with respect to the light source, whichever
provides for the most uniform exposure of the samples. Some adjustment of testing conditions
may have to be made when testing large volume containers (e.g., dispensing packs).
3.2 A n a l y s i s of s a m p l e s
At the end of the exposure period, the samples should be examined for any changes in
physical properties (e.g., appearance, clarity or colour of solution, dissolution/disintegration
for dosage forms such as capsules, etc.) and for assay and dgradants by a method suitably
validated for products likely to arise from photochemical degradation processes.
When powder samples are involved, sampling should ensure that a representative portion is
used in individual tests. For solid oral dosage form products, testing should be conducted on
an appropriately sized composite of, for example, 20 tablets or capsules. Similar sampling
considerations, such as homogenisation or solubilisation of the entire sample, apply to other
materials that may not be homogeneous after exposure (e.g., creams, ointments, suspensions,
etc.). The analysis of the exposed sample should be performed concomitantly with that of any
protected samples used as dark controls if these are used in the test.
3.3 Evaluation of results

Depending on the extent of change special labelling or packaging may be needed to mitigate
exposure to light. When evaluating the results of photostability studies to determine whether
change due to exposure to light is acceptable, it is important to consider the results obtained
from other formal stability studies in order to assure that the product will be within proposed
specifications during the shelf life (see the relevant Stability and Impurity Guidelines).
4. ANNEX
4.1 Quinine chemical actinometry
The following provides details of an actinometric procedure for monitoring exposure to a
near UV fluorescent lamp (based on FDA/National Institute of Standards and Technology
study). For other light sources/actinometric systems, the same approach may be used, but
each actinometric system should be calibrated for the light source used.
Prepare a sufficient quantity of a 2 per cent weight/volume aqueous solution of quinine
monohydrochloride dihydrate (if necessary, dissolve by heating).
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Option 1
Put 10 millilitres (ml) of the solution into a 20 ml colourless ampoule seal it hermetically,
and use this as the sample. Separately, put 10 ml of the solution into a 20 ml colourless
ampoule (see note 1), seal it hermetically, wrap in aluminium foil to protect completely from
light, and use this as the control. Expose the sample and control to the light source for a n
appropriate number of hours. After exposure determine the absorbances of the sample (AT)
and the control (A) at 400 nm. Calculate the change in absorbance, A = Ap - A0. The length
of exposure should be sufficient to ensure a change in absorbance of at least 0.9.
Option 2
Fill a 1 cm quartz cell and use this as the sample. Separately fill a 1 cm quartz cell, wrap in
aluminium foil to protect completely from light, and use this as the control. Expose the
sample and control to the light source for an appropriate number of hours. After exposure
determine the absorbances of the sample (AT) and the control (A0) at 400 nm. Calculate the
change in absorbance, A = AT - AQ. The length of exposure should be sufficient to ensure a
change in absorbance of at least 0.5.
Alternative packaging configurations may be used if appropriately validated. Alternative
validated chemical actinometers may be used.
Note 1: Shape and Dimensions (See Japanese Industry Standard (JIS) R3512
(1974) for ampoule specifications)
Stem diameter: 2L8 0.40
Bore (at cutting position)
Bore: 7.0 0.7 mm

Stem length: 80.0 1.2 mm
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5. GLOSSARY
Immediate (primary) pack is that constituent of the packaging that is in direct contact with
the active substance or finished product, and includes any appropriate label.
Marketing pack is the combination of immediate packaging and other secondary packaging
such as a carton.
Forced degradation testing studies are those undertaken to degrade the sample deliberately.
These studies, which may be undertaken in the development phase normally on the active
substances, are used to evaluate the overall photosensitivity of the material for method
development purposes and/or degradation pathway elucidation.
Confirmatory studies are those undertaken to establish photostability characteristics under
standardised conditions. These studies are used to identify precautionary measures needed
in manufacturing or formulation and whether light resistant packaging and/or special
labelling is needed to mitigate exposure to light. For the confirmatory studies, the batch(es)
should be selected according to batch selection for long-term and accelerated testing which is
described in the Parent Guideline.
6. REFERENCES
Quinine Actinometry as a method for calibrating ultraviolet radiation intensity in light-
stability testing of pharmaceuticals.
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QUALITY OF PROLONGED RELEASE ORAL SOLID

DOSAGE FORMS
Guideline Title Quality of Prolonged Release Oral Solid Dosage Forms

Date of entry into November 1992
force
Previous titles/other None/ III/3172/92
references
prolonged release oral solid dosage forms of Part 2,
sections B, D, E and F of the Annex to Directive
75/318/EEC as amended, with a view to the granting of a
CONTENTS
1. INTRODUCTION
2. DEFINITIONS
3. DEVELOPMENT PHARMACEUTICS
4. MANUFACTURING PROCESS VALIDATION
5. CONTROL TESTS
6. STABILITY
7. CHANGES TO PRODUCTS
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QUALITY OF PROLONGED RELEASE ORAL SOLID

DOSAGE FORMS
L INTRODUCTION
Pharmaceutical dosage forms may be developed in which the rate of release of active
substance(s) has in some way been modified compared with conventional formulations. Such
modification in release of active substances may have a number of objectives, but the
intention of this note for guidance is to cover those formulations in which the release of the
active substance is prolonged in some way in order to maintain therapeutic activity, to
reduce toxic effects or for some other therapeutic purpose.
The details required in the application for marketing authorisation will reflect:
- the therapeutic intention
- the nature of the active substance
- the nature of the formulation
- the route of administration
and data must be provided in the various sections of the dossier in support of the application
taking into account these various requirements. The note for guidance will cover the various
parts 2.A to 2.F of the application for marketing authorisation and highlight areas which
need to be addressed. It is clear therefore that this note for guidance will cross-refer to other
quality guidelines, to the Notice to Applicants and in particular to the note for guidance
Clinical Testing of Prolonged Action Forms with Special Reference to Extended Release Forms.
The note for guidance concerns quality aspects, including in vitro testing, of oral solid
dosage forms in which release of active substance forms the rate-limiting step in absorption.
While the note for guidance is intended to be specific for prolonged release oral solid dosage
forms, many of the principles discussed will be relevant to other prolonged action dosage
forms intended for administration via other routes.
2. DEFINITIONS
It is important to clearly define the terminology used to describe the different types of release
models to which the note for guidance relates. The definitions of various types of release
characteristics should be considered in relation to the pharmacokinetic properties and to the
therapeutic intention of the formulation (see note for guidance Clinical Testing of Prolonged
Action Forms with Special Reference to Extended Release Forms), and are correlated as
closely as possible with pharmacopoeial definitions. The more general terms "prolonged
release" can be applied to a range of different release models; terms such as "controlled
release" and "sustained release" are looser than those defined and should be avoided.
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2.1 Modified release

This term is defined in the European Pharmacopoeia as a modification of the rate or place at
which the active substance is released. Modified release products cover a wide range of
release models, the principal types of which would include "delayed release" and "prolonged
release" products. It is the intention of this note for guidance to focus only on pharmaceutical
aspects of prolonged release oral solid dosage products.
2.1.1 Delayed release

A modified release product in which the release of active substance is delayed for a finite
"lag time", after which release is unhindered [e.g. enteric coated or "Gastro resistant" (Ph.
Eur.) oral tablets or capsules which remain intact in the stomach and only disintegrate in
the higher pH of the small intestine]. Delayed release results in a longer Tmax but with
Tmax and elimination half life unchanged.
2.1.2 Prolonged release

A product in which the rate of release of active substance from the formulation after
administration has been reduced, in order to maintain therapeutic activity, to reduce toxic
effects, or for some other therapeutic purpose.
3. DEVELOPMENT PHARMACEUTICS
3.1 Therapeutic objectives
The therapeutic objectives and rationale for developing the prolonged release product should
be provided. Pharmacokinetic and physico-chemical characteristics of the active substance
relevant to the development of the product should be given.
3.2 Principle of t h e p r o l o n g e d release s y s t e m

The prolonged release system should be described:
- the manner in which prolonged release is intended to be achieved (membrane type,
matrix, etc.);
- single non-disintegrating unit or multi-unit pelletised preparation etc.;
- release mechanism and kinetics if known (diffusion, erosion, osmosis, etc. or
combinations of these).
3.3 T e s t i n g of t h e prolonged-release s y s t e m
3.3.1 In vitro testing
The release rate should be tested in vitro by a dissolution test method which has been shown
to discriminate between batches with acceptable and unacceptable in vivo performance. Test
conditions providing the most suitable discrimination should be chosen.
The dissolution apparatus should be one of those described in the European Pharmacopoeia.
The continuous flow-through method of the European Pharmacopoeia monograph may be of
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particular value in testing poorly soluble substances. The use of methods other than the
official methods in the European Pharmacopoeia should be justified.
The choice of rotation speed should be justified by carrying out the test at different speeds and
the speed giving appropriate discrimination between batches with acceptable and
unacceptable bioavailability should be chosen.
The test medium should preferably be aqueous-based; organic or aqueous-organic media
should be avoided. For poorly soluble substances, a minimal content of an appropriate
surfactant may be added. Buffer solutions at a number of pH values spanning the
physiological rate (pH 0.8-2, stomach; pH 5-6.5, jejunum; pH 6-7.5, ileum; Davis et al 1989)
should be used to determine the relationship between dissolution and pH. The data obtained
could usefully be represented using three-dimensional dissolution profiles (i.e. % dissolved
as a function of time and pH).
In order to achieve adequate discrimination, it may be necessary to limit the solubility of the
medicinal product and still achieve sink conditions in the dissolution medium. It may also
be necessary to consider the ionic strength and surface tension of the medium. The volume
of medium used should preferably ensure sink conditions which may be assumed if the
amount of substance in solution does not exceed 30% of the saturation concentration. The
solubility of the substance in the chosen dissolution medium should be stated.
Identical test conditions should be used for different strengths of the same product.
The robustness of the dissolution test should be determined by examining the effect on the
dissolution rate of variations in temperature, pH and speed of rotation.
Dissolution profiles should be determined for:
- each strength of the prolonged release product if more than one strength is to be
marketed;
- halved tablets where the release mechanism permits tablets to be broken in half for
dosage purposes;
- any changes in the composition of the product during development.
At each time point individual dosage unit results (n _ 6), the mean value and a measure of
variability should be presented.
The definitive dissolution profile and the corresponding specification will be based on i n
vitro results of batches used in in vivo testing and will provide an assurance that batches
will routinely give the desired in vivo behaviour. It may be necessary to validate the
specification for any variations in the substance or excipients, e.g. the particle size or
polymorphic form of the active substance, the gelling properties or particle size of the
release-controlling excipients.
The content of any key excipient which has a determining effect on the release of the active
substance should not vary outside validated limits. These limits should be established on a
case by case basis during the development of each product.
3.3.2 In vivo testing

A summary of the bioavailability testing should be given. The data should include batch
numbers, formulations (if different from the proposed marketing formulation) and
dissolution results of batches used in in vivo studies.
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Bioavailability studies should preferably be performed on at least pilot production scale

batches. Where these are not available, studies could be performed on non-production scale
batches provided it is demonstrated by valid in vitro testing that subsequent production
batches comply with the same model.
3.3.3 In vitro-in vivo comparison

To justify the specification limits of the in vitro dissolution test, an attempt should be made to
establish a meaningful correlation between in vitro release characteristics and In vivo
bioavailability parameters. In order to accomplish this, a number of techniques may be
employed. These include, In order of decreasing predictive power:
a) comparison of the in vitro dissolution curve of the product with the in vivo dissolution
curves generated by deconvolution of plasma level data or by other appropriate
methods;
b) comparison of the mean in vitro dissolution time of the product to either the mean i n
vivo residence time or the mean in vivo dissolution time of the product derived by
using the principles of statistical moment analysis;
c) comparison of the mean in vitro dissolution time to one mean pharmacokinetic
parameter, e.g. Tmax.
Other approaches are acceptable especially if the above methods fail to demonstrate a
correlation. Examples of other approaches include demonstrating bioequivalence of the
proposed formulation to formulations with dissolution profiles at the upper and lower limits
of the specification, or alternatively, the specification limits may be derived from the spread
of in vitro dissolution results of batches used in bioavailability testing the choice of approach
should be justified by the applicant.
4. MANUFACTURING PROCESS VALIDATION

Precise details of the manufacturing process should be given. Critical process parameters
which can significantly affect the release of the substance (e.g. tablet hardness, coating
procedure, moisture content) should be identified. If an in vitro-in vivo correlation has been
established, limits for the critical parameters should be validated by dissolution testing of the
product made under different processing conditions to demonstrate that allowable variations
in these parameters do not result in unacceptable changes to the dissolution profile.
If the manufacturing process has been validated using small-sized batches the effect of scale
up on the dissolution characteristics of the product should be established.
5. CONTROL TESTS
5.1 In-process (if necessary)
A dissolution specification which may be applied to Intermediate products (e.g. cores, pellets)
may be the same or different from that to be applied to the finished product. If different, an
explanation for the limits chosen should be provided.
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5.2 Finished product

The finished product specification normally includes a dissolution test. The dissolution
specification is of importance not only to ensure consistent substance release from batch to
batch at time of manufacture but also to set acceptance limits for the dissolution of the product
during its shelf life. The dissolution specification should be deduced from the profile(s)
obtained during the development of the product and revalidated with at least pilot production
scale batches. Selection of specifications should take into account pharmacokinetics,
pharmacodynamics and in vitro assay precision.
The specification should clearly state the number of dosage units to be tested and whether or
not the limits apply to individual dosage unit results or to the mean value. In the latter case
the individual dosage unit results should be controlled so that they do not lie substantially
outside the specification limits. If retesting is proposed in the event of non compliance with
the specification the number of units to be retested and the limits to be applied should be
specified and justified.
A minimum of three points should be included in the specification: an early time point to
exclude dose dumping at least one point to ensure compliance with the shape of the
dissolution profile and one to ensure that the majority of the substance has been released.
Where both upper and lower limits are specified at any time point the difference between
them should not usually exceed 20% of the labelled content of active substance in the
formulation unless wider limits have been shown to provide reproducible and acceptable in
vivo performance.
Where an in vitro-in vivo correlation has not been satisfactorily established it may be
necessary to carry out additional control tests on the finished product. For example the
content of any release-determining excipients may need to be controlled within an upper and
lower limit on each batch of product (see note for guidance Specifications and Control tests on
the Finished Product).
5.3 Validation of the dissolution assay

Analytical validation is required for the dissolution assay procedure (see note for guidance
Validation of Analytical Procedures: Methodology). The assay method should be validated for
specificity and linearity over the expected concentration range; accuracy and precision
should be determined at the low as well as the high end of the expected concentration range.
The stability of the active substance in solution in the medium should be addressed.
5.4 B a t c h r e s u l t s
Batch analytical results should be provided for at least three batches one of which should be
production scale for each product strength. Individual dosage unit dissolution results should
be included.
6. STABILITY
It must be demonstrated that the dissolution profile of the active substance is maintained
within specification throughout the proposed shelf life of the product. The results of
dissolution testing should include mean values of individual dosage units together with
maximum and minimum values for all the batches undergoing the stability tests.
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7. CHANGES TO PRODUCTS
Where the dissolution specification has been correlated with in vivo results minor changes
to the data may be acceptable on the basis of in vitro testing. Minor changes include changes
to the composition (e.g. nature and/or quantity of excipients which do not influence the
release characteristics) method or site of manufacture or manufacturing equipment. Other
changes may however necessitate further in vitro-in vivo correlation studies or in vivo
bioavailability studies.
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RADIOPHARMACEUTICALS
Guideline Title Radiopharmaceuticals

Legislative basis Directives 65/65/EEC, 75/318/EEC as amended, Directive
89/343/EEC
force
references
radiopharmaceuticals of Directive 65/65/EEC and of parts
2, 3 and 4 of the Annex to Directive 75/318/EEC as
authorisation for a radiopharmaceutical.
CONTENTS
1. INTRODUCTION
2. PHYSICO-CHEMICAL, BIOLOGICAL OR MICROBIOLOGICAL TESTS OF

MEDICINAL PRODUCTS
3. TOXICOLOGICAL AND PHARMACOLOGICAL TESTS
4. CLINICAL DOCUMENTATION
5. RADIATION DOSIMETRY
6. LABELLING AND PACKAGING
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RADIOPHARMACEUTICALS
L INTRODUCTION
Applications for marketing authorisation in respect of radiopharmaceuticals should be
accompanied, as in the case of all medicinal products, by the particulars and documents
referred to in Directives 65/65/EEC and 75/319/EEC, as amended, and in the Annex of
Directive 75/318/EEC as amended. The provisions of Directive 89/343/EEC also apply. The
relevant provisions of the European Pharmacopoeia should be observed. Due account must be
taken of the other relevant CPMP guidelines.
Most radiopharmaceuticals are used for the purpose of medical diagnosis. They are usually
given only once, or sometimes on a few occasions, and contain only small amounts of the
active substances with a radionuclide attached to them to allow scintigraphic imaging or
measurement of biodistribution. Such radiopharmaceuticals do not often show any
measurable pharmacodynamic effect. Radiation is a general property of all
radiopharmaceuticals, which when administered give the patient an inevitable radiation
dose. In the case of therapeutic radiopharmaceuticals, the radiation effect is the wanted
property. Evaluation of the safety and efficacy of radiopharmaceuticals should include
radiopharmaceutical and radiation hygiene aspects and radiation dosimetry in addition to
general parameters.
Radiopharmaceuticals have changing composition with time, associated with the radioactive
decay. The physical half-life of the radionuclide is often so short that, in these cases, the
final preparation has to be done immediately before administration to the patient; this leads
to the use of semi-manufactured products such as radionuclide generators, precursors and
kits. Evaluation of the safety and efficacy of radiopharmaceuticals is also concerned with
the specifications of generators, kits and other semi-manufactured products. Specifications
may also require special attention in cases where samples from the patient are labelled with
a radioactive substance before readministration (precursor radiopharmaceuticals). When
radiopharmaceuticals go directly from the generator to the patient (e.g. ultra short-lived
radioactive gases), the consistency of the production process has a particularly great
importance.
This note for guidance covers the following products:
- ready-for-use radiopharmaceuticals;
- non-radioactive components (kits) for combination with a radioactive component
(usually the eluate from a radionuclide generator);
- radionuclide generators;
- precursors used for radiolabelling other substances prior to administration
(e.g. samples from patients).
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2. PHYSICO-CHEMICAL, BIOLOGICAL OR MICROBIOLOGICAL

TESTS OF MEDICINAL PRODUCTS
2.1 Qualitative and quantitative particulars of the constituents and
development pharmaceutics
For the radionuclides, details must be given of their source, i.e. whether fission or target
material is used.
Radioactivity should be expressed in Bequerels at a given date, and hour in appropriate cases
(other units may be added). If a calibration time is stated, the time zone used should be stated
(e.g. GMT/CET).
Where practicable, the proportion (specific activity, carrier free or carrier added) of inactive
isotopes in the carrier should be stated.
For radiopharmaceutical kits, any added compound (e.g. stannous salt for reduction of
pertechnetate ions in the eluate from a technetium 99mTc generator) and manipulation
essential for radiolabelling should be stated.
Where applicable, evidence to confirm the efficacy and specificity of the radiolabelling of the
labelling medium (e.g. 99mTc) should be supplied. A discussion of the necessary
specification (e.g. purity, pH) of radiolabelling medium should be stated for kits.
After radiolabelling, the compatibility of the product with the containers and closures should
be considered and validated where appropriate.
2.2 Description of method of preparation

Because of the complexity of the production of radiopharmaceuticals, it is important that
methods for obtaining and maintaining sterility during manufacture (preparation and
assembly) are adequately described. Information should be given on validation of those
processes.
a) Radiopharmaceutical kits: the instructions for final preparation should include:
- minimum and maximum for both volume and amount of radioactivity to be
added;
- any special quality requirement for the radiolabelling medium;
- the standing time and any other manipulation necessary during final
preparation (detailed and justified);
- details supporting recommendations on quality control procedures such as
checking radiochemical purity of the prepared radiopharmaceutical prior to
administration;
- as relevant, data on stability of particles (e.g. of colloidal size) after
reconstitution and justification for the quantity of added materials.
These aspects should be discussed and documented adequately in the Development
Pharmaceutics part of the dossier.
b) Generators
The recommendations for use of the generators should be discussed and documented.
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c) Precursors
The recommendations for use of the precursors should be discussed and documented.
2.3 Control of s t a r t i n g m a t e r i a l s
For the purposes of this section "starting materials" shall mean all the constituents of the
medicinal product, if necessary, all the constituents of its containers and closures and where
applicable, all constituents of the radionuclide source and any other materials used in the
final process prior to administration. A full description is required of the separation of
radionuclides and the control of radionuclide purity, as well as specific activity (with respect
to impurities and degradation products). Specifications of components of the container
(including the name of the approved producers) should be given. Specifications of' any
radiation shielding of the finished products should also be given.
For some radiopharmaceuticals, it is difficult to distinguish between control of starting
materials and control of the finished product. For such products, all the information should
preferably be placed in the section "Control tests on the finished product".
2.4 Control t e s t s o n t h e finished product

For products intended for intrathecal injection, regardless of volume, an appropriate
endotoxin test is required unless its omission can be fully and adequately justified.
For terminally-sterilised products, process parameter release* (sometimes called parametric
release) could well be justified. In the case of an aseptically-assembled product a sterility test
is required.
For some radiopharmaceuticals it may not be possible to obtain the results of certain tests,
e.g. sterility test, pyrogenicity tests, before the product is released. However, these tests should
be done as a monitor for the manufacturing process.
Potential and actual impurities should be considered not only for any direct effect on the
patient but also for their possible influence on the radiochemical purity or biodistribution of
the product.
2.5 Stability t e s t s
For all radiopharmaceuticals, the shelf life of the product as supplied by the manufacturer
should be specified and justified, as should a shelf life after reconstitution where applicable,
taking into account radiochemical and radionuclide degradation products.
For radiopharmaceutical kits, the shelf life of the prepared product should be defined; in this
case, data should be submitted which detail the minimum and maximum levels of
radioactivity (and maximum and minimum volumes) and other relevant factors that are
recommended for use in the preparation of the product to be administered to the patient.
For radiopharmaceuticals prepared in multiple-dose vials, the stability following removal of
successive doses should be discussed.
For the purpose of this guideline, process parameter release is denned as "the decision to release a batch of
product for sale or supply based on an assessment of measured and recorded information relating to the
validation, maintenance, operation and control of a process."
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3. Toxicological and Pharmacological Tests

It is appreciated that toxicity may be associated with a radiation dose. This toxicity is a
consequence of the use of radiopharmaceuticals in diagnosis and the wanted property of
radiopharmaceuticals used in therapy. The evaluation of safety and efficacy of
radiopharmaceuticals should, therefore, address both general parameters of the medicinal
product and radiation dosimetry aspects.
Toxicity studies should be designed to examine the chemical rather than the radiation
aspects of toxicity.
Knowledge of the toxicology of the ligand perse is of value. However, if the radiolabel is
likely to produce chemical changes in the ligand, it would be preferable to carry out the
toxicity studies on material in which radioactive decay has proceeded for long enough as to
expose the test animals to breakdown products as well as to the parent complex.
For other radiopharmaceuticals, consideration should be given to ascertaining the toxicity of
the parent molecule, either by reacting the ligand with a non-radioactive isotope of the
element in question, or, if appropriate, by allowing decay of the product to occur so that there
is no significant residual radioactivity.
Whatever the strategy and method chosen, it should be justified.
Distribution and elimination studies should be performed with the labelled compound.
For no-carrier-added radioactive elements and simple salts thereof, if the toxicity of the
element or simple salt is known and can be submitted in the application, no additional
toxicity studies would normally be required.
The contents in many final preparations (e.g. kit preparations) may be so small that is may
be justified to use a bulk preparation for toxicity testing but the stability of the bulk material
over the period of testing should be validated. The duration of animal toxicology testing will
be determined by the anticipated duration of clinical use. In cases where the
pharmacokinetic properties of the radiopharmaceutical (e.g. retention in certain organs)
may lead to long term exposure, the observation period of the toxicity study may have to be
extended.
Radiation dose should be evaluated with respect to target organs and physiological functions.
For radiopharmaceuticals, studies should be designed to assess:
a) the in vivo stability of the radionuclide complex;
b) the animal biodistribution of the radionuclide;
c) the potential chemical toxicity;
d) the radiation exposure of tissues resulting from administration of the
radiopharmaceutical.
3.1 Single dose/repeated dose toxicity

These tests should be carried out according to the principles applicable to other medicinal
products. The length of the studies should relate to the period of clinical use of the
radiopharmaceutical, e.g. for a single (day of) treatment to patients, the toxicity dosing
period would be two weeks, but observation for adverse effects may need to continue beyond
this time.
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3.2 E x a m i n a t i o n of r e p r o d u c t i v e f u n c t i o n a n d foetal t o x i c i t y
Although radiopharmaceuticals are not normally recommended for potentially pregnant
women, studies on reproduction may be required in certain cases, especially if the
radiopharmaceutical is intended for repeated use in women of child-bearing potential.
Otherwise the study on reproductive function may justifiably be limited to ascertaining the
effect on fertility.
3.3 M u t a g e n i c p o t e n t i a l
Mutagenicity testing may be limited to screening for gene and chromosome mutations and
should be performed to allow characterisation of the mutagenic potential of the non-
radioactive equivalent of the product.
3.4 C a r c i n o g e n i c p o t e n t i a l
An evaluation of any carcinogenic potential of the substances involved must be presented. If
no carcinogenicity tests are performed, this must be clearly indicated in the "Summary of
product characteristics".
3.5 Pharmacodynamics
Measurable pharmacodynamic effect is not normally expected to be seen for
radiopharmaceuticals. The likelihood of their absence may be deduced from toxicity testing,
but in reassurance information should be supplied that no pharmacological effect is seen i n
major organ systems.
3.6 Pharmacokinetics
Information should be provided as to the distribution and elimination of the radiolabelled
substances. If relevant, information should be provided on absorption and biotransformation.
Important pharmacokinetic parameters should be investigated in the animal species used in
the toxicological studies.
The animal pharmacokinetic studies should always provide data to allow estimation of
tissue and whole-body radiation doses, which can be extrapolated to man.
Diagnostic radiopharmaceuticals differ in many ways from therapeutic radiopharma-
ceuticals. Consequently clinical documentation on diagnostic radiopharmaceuticals will be
different from that relating to therapeutic radiopharmaceuticals.
Radiopharmaceuticals for diagnostic use are part of a diagnostic system where other factors
such as instrumentation, time schedule, etc. also play an important role which should be
discussed. The same criteria as for non-radioactive therapeutic medicinal products apply to
therapeutic radiopharmaceuticals.
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4.1 Clinical p h a r m a c o l o g y
Whenever possible, initial pharmacodynamic and pharmacokinetic studies with
radiopharmaceuticals should be performed in suitable patients, rather than in healthy
volunteers.
Pharmacodynamics:
It is expected that many radiopharmaceuticals will not have any pharmacological action.
During early studies, the subjects should be monitored for a sufficient period to ascertain
any change in major organ function. Any adverse events should be reported, giving nature
and frequency.
Pharmacokinetics:
Pharmacokinetic studies should always provide the data necessary for the calculation of
radiation doses.
The results should be presented in a form which allows evaluation of the proposed radiation
dose and discussion of the in vivo stability of any radionuclide/carrier complex.
4.2 Clinical trials

The main purpose of clinical trials with new radiopharmaceuticals is to prove their safety i n
use and their value as diagnostic or therapeutic agents. Comparison with existing agents or
with other relevant medicinal products and procedures should be the method of choice to prove
efficacy. In particular, radiopharmaceuticals for diagnostic use may need to be compared
with alternative techniques.
Diagnostic/therapeutic efficacy:
Each indication should be the subject of at least one separate trial.
Adverse reactions:
A summary should be given on the investigations performed to ascertain the nature, severity
and frequency of any adverse reactions.
Interactions:
Signs of interactions should be carefully observed during clinical trials and consideration
given to medicinal products likely to be used concurrently.
Dosage:
The clinical trials should provide a reliable basis for the dosing recommendations.
Information on pharmacokinetics should be sufficient for radiation dosimetry calculations.
Data from animal studies (extrapolated to estimated radiation doses in man) should be
confirmed as relevant or superseded by data obtained from patients. Radiation dose
estimates should consider the impact of age and clinical condition, particularly impairment
of hepatic or renal function.
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It is recommended that calculations of absorbed dose to organs should be carried out i n

accordance with the Medical Internal Radiation Dosimetry (MIRD) schedules. The model
used for calculations of the cumulated activity (time integral of the activity) in source
organs should be explained; the origin of data used, such as animal studies or
measurements in humans, should be stated. Physical parameters, such as absorbed dose to
target organs per unit of cumulated activity in source organs, should be taken from MIRD
tables.
The effective dose-equivalent should be calculated using the current weighting factors
established by the International Commission on Radiological Protection (ICRP). These
weighting factors are not applicable to children, pregnant women or elderly patients and
modifications should be given for radiopharmaceuticals intended for use in such patients.
If other methods of calculation of the absorbed dose in organs are used, details should be
given with reference to the original reports.
The absorbed dose in the organ receiving the highest exposure and in all organs included i n
the calculation of the effective dose-equivalent should be stated. The unit must be milligrays
per unit of activity administered: mGy/MBq.
The estimation of the radiation dose must be summarised in terms of the effective dose-
equivalent using the weighting factors given by ICRP. The unit must be millisieverts per
unit of activity: mSv/MBq.

6.1 Labelling
The label on the container should state:
- the name of the product and the name of the radionuclide;
- any product identification code;
the name of the manufacturer;
- an identification number (batch number);
- for liquid preparations, the total radioactivity in the container, or the radioactive
concentration per millilitre, at a stated date and, if necessary, hour, and the volume of
liquid in container;
- for solid preparations, such as freeze-dried preparations, the total radioactivity at a
stated date and, if necessary, hour;
- for capsules, the radioactivity of each capsule at a stated date and, if necessary, hour
and the number of capsules in the container;
- where relevant, the international symbol for radioactivity.
In addition, the label on the package should state:
- qualitative and quantitative composition;
- the route of administration;
- the expiry date;
- any special storage conditions.
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Information on batch coding should be provided to the authorities.
6.2 Packaging
The suitability of packaging material for the product and for the labelling procedure to be
carried out should be described. It may be necessary to describe special radiation shielding.
6.3 Package leaflets

Package leaflets play a particularly important role for semi-manufactured products such as
preparation kits and should include:
- the name of the product and a description of its use;
- a list of the contents of the kit;
- the name and the address of the manufacturer of the kit;
- identification and quality requirements concerning the radiolabelling materials that
can be used to prepare the radiopharmaceutical;
- directions for preparing the radiopharmaceutical including range of activity and
volume and a statement of the storage requirements for the prepared
radiopharmaceutical;
- a statement of the useful life of the prepared radiopharmaceutical;
- indications and contra-indications in respect of the prepared radiopharmaceutical;
- warnings and precautions in respect of the components and the prepared
radiopharmaceutical including radiation safety aspects;
- where applicable, the pharmacology and toxicology of the prepared radiopharmaceutical
including route of elimination and effective half-life;
- the radiation dose to the patient from the prepared radiopharmaceutical;
- precautions to be taken by the user and the patient during the preparation and
administration of the product and special precautions for the disposal of the container
and its unused contents;
- a statement of recommended use for the prepared radiopharmaceutical and the
recommended dosage;
- a statement of the route of administration of the prepared radiopharmaceutical;
- and, if it is appropriate for particular kits (i.e. those subject to variability beyond the
recommended limits) the leaflet should contain the methods and specifications needed
to check radiochemical purity.
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RADIOPHARMACEUTICALS BASED ON
MONOCLONAL ANTIBODIES
Guideline Title Radiopharmaceuticals based on Monoclonal Antibodies

Legislative basis Directives 65/65/EEC, 75/318/EEC as amended, Directive
89/343/EEC
Date of entry into January 1992
force
Status Last revised May 1991
references
radiopharmaceuticals based on monoclonal antibodies of
Directive 65/65/EEC and of parts 2, 3 and 4 of the Annex to
granting of a marketing authorisation.
It should be read in conjunction with the guideline
Production and Quality Control of Monoclonal Antibodies.
CONTENTS
1. INTRODUCTION
2. SOURCE MATERIALS
3. PRODUCTION FACILHTES
4. MANUFACTURING PROCEDURE FOR MONOCLONAL ANTIBODIES
5. MANUFACTURE OF MODIFIED AND DERrVATISED MONOCLONAL
ANTIBODIES
6. RADIOPHARMACEUTICAL ASPECTS
7. PRECLINICAL SAFETY TESTS
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RADIOPHARMACEUTICALS BASED ON
L INTRODUCTION
Monoclonal antibodies may form the basis of radiopharmaceuticals for in vivo diagnosis or
therapy. The antibody or antibody fragment is thus only one component of the medicinal
product and in the evaluation of quality and safety of this group of products, the
radiopharmaceutical and radiation protection aspects must be considered in addition to those
of the antibody component. The same principle would apply to monoclonal antibodies used in
conjunction with other agents e.g. toxins, though such products are not covered in this
document. The monoclonal antibodies used as the basis of such products may be of murine
origin, prepared in human cell lines or "humanised" using rDNA techniques. As regards
monoclonal antibodies and radiopharmaceuticals, different notes for guidance have already
been adopted by the CPMP: a note on "Production and quality control of monoclonal
antibodies", and a note on radiopharmaceuticals is published in this volume.
The notes for guidance are intended to be used by manufacturers submitting applications for
marketing authorisation. They are not intended for non-commercial producers. The notes
for guidance are advisory, not mandatory, and (as stated in the notes for guidance on
murine monoclonal antibodies) "a flexible approach" should be adopted.
A special consideration with this class of products is that chemical modification of the
antibody may be carried out. This may take the form of preparation of sub-fragments of
antibody (e.g. Fab or F(ab')2) and the antibody molecule (or a fragment of it) may also be
modified by addition of a conjugating agent for the radionuclide. These modified forms
require consideration with respect to quality, in addition to that for the monoclonal
antibodies from which they were derived.
Consideration of radiolabelling procedures encompasses the quality control of the
manufacturing steps and of the radiopharmaceutical aspects. In addition, the use of
radionuclides of short half-life will require specifications or instructions for the antibody
derivative/conjugate, the radionuclide (especially where specific purity requirements apply),
and the preparation and quality control of the final product intended for administration to
the patient, which typically, will be prepared by the user immediately prior to clinical use.
Frequently, the radionuclide and the monoclonal antibody components are marketed by
different manufacturers who are responsible independently for the marketing authorisation
and control of their product(s). The radionuclide (e.g. m i n ) may be authorised for use with a
number of monoclonal antibodies (or indeed with any antibody). In each specific case the
antibody manufacturer is responsible for providing the clinical and pharmaceutical data on
the radiolabelled antibody.
2. SOURCE MATERIALS
The development and establishment of cell lines for the production of monoclonal antibodies
in this field has often taken place in non-commercial institutions. The initial development
may therefore not be as well documented as is generally required in the pharmaceutical
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industry. In cases where the history of the myeloma cell line and parental cell line are
limited by available data, more emphasis must be put on the characterisation at the seed lot
stage and of the final product to ensure quality (e.g. freedom from adventitious agents).
However, every effort should be made to provide evidence of the origin and acceptability of
the cell line. Non-commercial organisations collaborating with industry in the production of
monoclonal antibodies that will form part of a marketed product should be strongly
encouraged to improve their record-keeping so that full information on production of
antigen, immunisation, establishment of cell lines, testing of antibodies, etc. can be
provided.
3. PRODUCTION FACILITIES
Even though production may be on a smaller scale than is usual in the pharmaceutical
industry, the appropriate good manufacturing practice should be followed for both the
radionuclide and the antibody components. A strategy for avoiding cross-contamination of
cell lines should be developed and specified if more than one antibody is produced within the
same facility.
4. MANUFACTURING PROCEDURE FOR MONOCLONAL

ANTIBODIES
Although the points to consider in the manufacture of the antibodies are in essence those
outlined in the notes for guidance on murine and human monoclonal antibodies, special
considerations apply in the case of antibodies intended for use with radiopharmaceuticals:
- for monoclonal antibodies prepared in only small amounts, it may not be essential to
have both a master cell bank and a manufacturer's working cell bank;
- where production of only a small number of batches is envisaged, evidence should be
provided of consistency of production of at least three production batches;
- where these batches are subdivided for further processing, evidence of consistency of
this must be provided.
Purification of the antibody remains crucial and, in particular, virus contamination should
be carefully attended to. Steps should be included that will inactivate or remove
contaminating viruses that may be present. The purified bulk antibody should be tested for
extraneous proteins and DNA. Reference should be made to the note for guidance on Virus
Validation Studies: The Design, Contribution and Interpretation of Studies Validating the
Inactivation and Removal of Viruses.
5. MANUFACTURE OF MODIFIED AND DERIVATISED

Radiopharmaceuticals based on monoclonal antibodies utilise either an unmodified
immunoglobulin or, more usually, a chemically modified form. Radiolabelling is either by
direct attachment of a radionuclide or attachment via a conjugating agent.
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Each relevant step in the production of chemically modified monoclonal antibodies requires
validation and quality control covering source materials, limits for impurities arising from
the production process, evidence for consistency of the process, etc.
Initial immunological studies may be carried out on the unmodified antibody but for the
purposes of market authorisation the determination of definitive immunological properties
should be performed at an appropriate stage. For example, characteristics such as class,
subclass, and interaction with Fc receptors can best be determined with the monoclonal
antibody in the unmodified form. In contrast, for regulatory purposes, any tests of toxicity,
biological half-life, immunoreactivity and tissue cross-reactivity should be carried out on a
form that is as close as possible to the product to be administered to the patient, e.g. for a
chemically modified antibody on the derivatised form rather than the parent antibody. For
the radiolabelled form, appropriate studies should be undertaken on the product intended for
clinical use using "Cold" non-radioactive labelled material wherever possible. It should be
noted that in the case of 99Tcm, there is no equivalent non-radioactive isotope and often a
small percentage of the modified antibodies actually carries the label.
In the instances in which the final product is a chemically modified or derivatised
monoclonal antibody, criteria and specification limits for purity and potency should be
applied to the derivatised form and include a test for immunoreactivity.
Material from an early batch that has been clinically evaluated should be retained as the
manufacturer's reference batch for purity and potency of subsequent batches. A secondary
working standard may be established providing that equivalence with the primary reference
is demonstrated.
6. RADIOPHARMACEUTICAL ASPECTS
6.1 Radionuclide
The radionuclide to be used for labelling the monoclonal antibody (whether derivatised or
not) needs to be an authorised medicinal product indicated for use for that purpose (see
Introduction). The radionuclide may be supplied as a component in the kit or separately.
Special purity measurements may apply and the radionuclide should have specifications for:
a) identity: radionuclide characteristics;
b) potency: radionuclide concentration.
c) purity: radionuclide purity, radiochemical purity, specific activity, chemical
composition, chemical impurities (e.g. metal ions, reducing substances);
d) chemical stability, in vitro.
6.2 R a d i o l a b e l l i n g method
Data on the radiolabelling method should be supplied by the antibody manufacturer.
a) Where this is carried out by the manufacturer: The process should be validated. This
includes quantitative relationships between the (derivatised) antibody and
radionuclide, purification of the labelled product and removal of excess reagents, tests
for radiochemical purity, quantity of radioactive material in the container, and
stability data.
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b) Where this is carried out by the user: This is likely to be in the form of a
radiopharmaceutical kit consisting of a (derivatised) monoclonal antibody, reagents
and materials necessary for the radiolabelling procedure including any necessary
purification of the product plus a package insert giving clear, precise instructions for
the use of the kit, quality control, and potential hazards. Radiolabelling methods and
quality requirements for the necessary reagents should form part of the product
marketing authorisation application.
The radiolabelling procedure for kit preparations should be validated under relevant
circumstances and the detailed specifications for the radiolabelling medium (e.g.
99Tcm or i n l n ) should be discussed. Quantitative relationships between the antibody (in
particular the immunoreactivity), the conjugating agent and the radionuclide should
be presented.
c) Specifications and quality control
Specifications to be fulfilled should be part of the application and could include the following:
- identity: product including protein, conjugating agent, radionuclide;
- potency: immunoreactivity, radionuclide concentration, protein concentration (specific
activity);
- purity: radiochemical purity, aggregation, chemical impurities (conjugating material,
reagents used in fragmentation of the antibody, labelling reagents, etc.), sterility,
pyrogens;
- stability: in vitro/in vivo.
If it is considered necessary for the user to carry out appropriate quality control tests on the
final radiolabelled product, the methods should be fully described and validated.
Samples of reference materials, antigens and special reagents should be made available
upon request.
7. P R E C L I N I C A L SAFETY TESTS
7.1 General
Due account should be taken of the note for guidance on Pre-clinical Biological Safety
Testing on Medicinal Products derived from Biotechnology.
Radiolabelled monoclonal antibodies may be used for diagnosis and therapy. While the
diagnostic use may cover many different types of diseases, the therapeutic use is currently
limited to treatment of cancer as a means of getting a high radiation dose to the target organ.
Testing requirements may therefore be different for the two uses. It is characteristic for the
diagnostic use that smaller amounts of antibodies are needed.
It is appreciated that toxicity may be associated with a radiation dose. This toxicity is a
consequence of the use of radiopharmaceuticals in diagnosis and the wanted property of
radiopharmaceuticals used in therapy. The evaluation of safety and efficacy of
radiopharmaceuticals should therefore address both general substance parameters and
radiation dosimetry aspects.
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The usefulness of established toxicological investigations may be questioned in particular

because of immunological incompatibility between the product and the animal species used.
The strategy and method chosen should be justified.
The content of material in many final preparations (e.g. kits) may be so small that it may
be justified to use a bulk preparation of the formulated product for toxicity testing but the
stability of the bulk material over the period of testing should be validated. The duration of
animal toxicology testing will be determined by the anticipated duration of clinical use.
7.2 Single dose/repeated dose toxicity

These tests should, if possible, be carried out according to the same principles as for other
radiopharmaceuticals. Some testing should be carried out, however the relevance of'these
tests may be discussed. Any conjugating material should be included in this discussion and
testing may be necessary.
7.3 E x a m i n a t i o n of r e p r o d u c t i v e f u n c t i o n ; f o e t a l t o x i c i t y ; m u t a g e n i c
potential; carcinogenic potential
Due account should be taken of the note for guidance on Radiopharmaceuticals.
7.4 Pharmacodynamics
Measurable pharmacodynamic effects are not normally expected to be seen from
radiopharmaceuticals for diagnostic or therapeutic purposes. The likelihood of their absence
may be deduced from toxicity testing and any observed effects should be reported.
Information should be provided as to the distribution and elimination of the radiolabelled
substance(s). Where appropriate, information should be provided on absorption and
biotransformation. The animal pharmacokinetic studies should always provide the
necessary data for estimating tissue and whole body radiation doses which can be
extrapolated to man. Studies in immunodeficient animals may be relevant.
8.1 General
There are two quite different types of radiopharmaceuticals; first radiopharmaceuticals
which are used to effect a medical diagnosis and which are part of a diagnostic system
where other factors such as instrumentation, time schedule etc., also play an important role
which should be discussed; second, radiopharmaceuticals which are used for the treatment of
diseases. Diagnostic radiopharmaceuticals differ in many ways from therapeutic
radiopharmaceuticals and consequently clinical documentation has to be different. The
same criteria as for non-radiolabelled therapeutic substances apply to therapeutic
radiopharmaceuticals.
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8.2 Clinical pharmacology

Whenever possible, initial pharmacodynamic and pharmacokinetic studies of the
radiolabelled material should be performed in suitable patients, rather than in healthy
volunteers.
Pharmacodynamics: it is expected that many radiopharmaceuticals will not have any
pharmacological action. During early studies, the subjects should be monitored for a
sufficient period to ascertain any change in major organ function. Any adverse events
should be reported, giving nature and frequency. Emphasis should be paid to the formation of
antibodies against the product (e.g. human anti-mouse antibodies: HAMA).
Pharmacokinetics: Pharmacokinetic studies should always provide the data necessary for
the calculation of radiation doses.
The results should be presented in a form which allows evaluation of the proposed radiation
dose and discussion of the in vivo stability of any radionuclide/carrier complex. The effect
of HAMA should be discussed.
8.3 Clinical trials

The main purpose of the clinical trials is to prove the safety of the new radiopharmaceutical
and its value as a diagnostic or therapeutic agent.
Comparison with existing agents or with other relevant medicinal products and procedures
should be the method of choice to prove efficacy. Particularly, radiopharmaceuticals for
diagnostic use may have to be compared to alternative techniques. Controlled and non
controlled studies should be separately summarised.
Diagnostic/therapeutic efficacy:
Where appropriate, each indication should be described separately and be the subject of at
least one separate trial including data on specificity and sensitivity.
Adverse reactions:
A summary should be given on the investigations performed to ascertain the nature, severity
and frequency of any adverse reactions.
Interactions:
Signs of interactions should be carefully observed during clinical trials and consideration
given to medicinal products likely to be used concurrently.
Dosage:
The clinical trials should provide a reliable basis for the dosing recommendations.
Information on pharmacokinetics should be sufficient for radiation dosimetry calculations.
Such data should preferably have been obtained in patients as appropriate animal models
may not exist. Radiation dose estimates should consider the impact of age and clinical
condition, particularly hepatic or renal function impairment.
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It is recommended that calculations of absorbed dose to organs should be carried out i n

accordance with the Medical Internal Radiation Dosimetry (MIRD) schedules. The model
used for calculations of the cumulated activity (time integral of the activity) in source
organs should be explained and the origin of data used, such as animal studies or
measurements in humans, should be stated. Physical parameters, such as absorbed dose to
target organs per unit of cumulated activity in source organs, should be taken from MIRD
tables.
The effective dose-equivalent should be calculated using the weighting factors established by
the International Commission for Radiological Protection (ICRP). These weighting factors
are not applicable to children, pregnant women or elderly patients and modifications should
be given for radiopharmaceuticals intended for use in such patients.
If other methods of calculation of the absorbed dose to target tissues/organs are used, details
should be given with reference to the original reports.
The absorbed dose to the organ receiving the highest exposure and to all organs included i n
the calculation of the effective dose-equivalent should be stated. The unit must be milliGrays
per unit of activity administered: mGy/MBq.
The estimation of the radiation dose should be summarised in terms of the effective dose-
equivalent using the weighting factors given by ICRP. The unit must be milliSieverts per
unit of activity: mSv/MBq.

10.1 Labelling
The label on the container should state:
- the name of the product and the name of any radionuclide;
- any product identification code;
- the name of the manufacturer;
- an identification number (batch number);
- for liquid preparations, the total radioactivity in the container, or the radioactive
concentration per millilitre, at a stated date and, if necessary, hour (and state the time
zone used), and the volume of liquid in the container;
- for solid preparations, such as freeze-dried preparations, the total radioactivity at a
stated date and, if necessary, hour (and state the time zone used);
- for capsules, the radioactivity of each capsule at a stated date and, if necessary, hour
(and state the time zone used), and the number of capsules in the container;
- where relevant, the international symbol for radioactivity.
In addition the label on the container should state:
- qualitative and quantitative composition;
- the route of administration;
- the expiry date;
- any special storage conditions.
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Information on batch coding should be provided to the authorities.
10.2 P a c k a g i n g m a t e r i a l
The suitability of packaging material for the product and for the radiolabelling procedure to
be carried out should be described. It may be necessary to describe special radiation
shielding.
10.3 P a c k a g e leaflets
Package leaflets play a particularly important role for semi-manufactured products such as
preparation kits for radiolabelled monoclonal antibodies. This is the responsibility of the
antibody manufacturer and should at least show:
- the name of the product and a description of its use;
- a list of the contents of the kit;
- the name and address of the manufacturer of the kit;
- identification and quality requirements concerning the radiolabelling materials that
can be used to prepare the radiopharmaceutical;
- directions for preparing the radiopharmaceutical including range of activity and
volume and a statement of the storage requirements for the prepared
radiopharmaceutical;
- a statement of the useful life of the prepared radiopharmaceutical;
- warnings and precautions in respect of the components and the prepared
radiopharmaceutical including radiation safety aspects;
- indications and contraindications in respect of the prepared radiopharmaceutical;
- precautions to be taken by the user and the patient during the preparation and
administration of the product and special precautions for the disposal of the container
and its unused contents;
- precautions to be taken if the patient has received monoclonal antibodies previously,
with regard to interference by antibodies and hypersensitivity;
- where applicable, the pharmacology and toxicology of the prepared radiopharmaceutical
including the route of elimination and effective half-life;
- the radiation dose to the patient from the prepared radiopharmaceutical;
- a statement of recommended use for the prepared radiopharmaceutical and the
recommended dose;
- a statement of the route of administration of the prepared radiopharmaceutical, and;
- if it is appropriate for particular kits (i.e. those subject to variability beyond the
recommended limits) the leaflet should contain the methods and specification needed
to check radiochemical purity.
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QUALITY OF HERBAL REMEDIES
Guideline Title Quality of Herbal Remedies

Date of first adoption November 1988
Date of entry into May 1989
force
references
Additional Notes This note for guidance concerns the application to herbal
based remedies of Part 2 of the Annex to Directive
75/318/EEC as amended, with a view to the granting of a
marketing authorisation for a medicinal product.
CONTENTS
A QUALITATIVE AND QUANTITATIVE PARTICULARS O F THE

CONSTITUENTS
DESCRIPTI
O N O F THE METHO D O F PREPARATIO N
C O
C NTR
O L O F STARTING MATERIALS
D O
C NTR
O L O F TESTS CARRHD O UT AT AN INTERMEDIATE STAGE O F THE
MANUFACTURING PRO CESS O F THE FINISHED PRO DUCT
E O
C NTR
O L TESTS O N FINISHED PRO DUCT
F STABILITY TESTS
ANNEX
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QUALITY OF HERBAL REMEDIES
Note for guidance concerning the application of Part 2 of the Annex to Directive 75/318/EEC
as amended. The special problems of herbal remedies and the differences between medicinal
products containing chemically defined active substances are described in this note for
guidance.
Consistent quality for products of vegetable origin can only be assured if the starting
materials are defined in a rigorous and detailed manner including especially the specific
botanical identification of the plant material used. It is also important to know, the
geographical source and the conditions under which the vegetable substance is obtained to
ensure material of consistent quality.
Reference substances used in the control of all stages of the manufacturing process should be
clearly defined.
A QUALITATIVE AND QUANTITATIVE PARTICULARS OF

THE CONSTITUENTS
L In the case of a vegetable substance
either a) the quantity of vegetable substance must be stated
or b) the quantity of a vegetable substance may be given as a range corresponding to
a defined quantity of constituents with known therapeutic activity.
EXAMPLE
a) Active substance
Name Quantity
Sennae folium 900 mg
or
b) Active substance
Name Quantity
Sennae folium 830-1000 mg, corresponding to 25 mg of
hydroxyanthracene glycosides, calculated as
Sennoside B
Other substance
Name Quantity
0-170 mg, corresponding to the quantity of Sennae
folium
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2. In the case of a vegetable substance preparation

either a) the equivalent quantity, or the ratio e.g. 8:1 of the vegetable substance to the
vegetable substance preparation must be stated (this does not apply to fatty or
essentials oils).
or b) the quantity of the vegetable substance preparation may be given as a range
corresponding to a defined quantity of constituents with know therapeutic
activity (see example).
The composition of any solvent or solvent mixture and the physical state of the extract must
be indicated.
If any other substance is added during the manufacture of the vegetable substance
preparation to adjust the vegetable substance preparation to a certain level of constituents
with known therapeutic activity, or for any other purpose, the added substance must be
mentioned as an "other substance" and the genuine extract as the "active substance".
EXAMPLE
a) Active substance
Name Quantity
Sennae folium 125 mg
dry 60% ethanolic extract (8:1)
or
Sennae folium 125 mg equivalent to 1000 mg Sennae folium
dry 60% ethanolic extract
or
b) Active substance
Name Quantity
Sennae folium 100-130 mg, corresponding to 25 mg of

hydroxyanthracene glycosides, calculated as
dry 60% ethanolic extract (8:1) Sennoside
Other substances
Name Quantity
Dextrin 20-50 mg
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B DESCRIPTION OF THE METHOD OF PREPARATION

The manufacturing process within the meaning of this section is the preparation of the
finished product from the starting materials. The description should include details of any
comminution or size reduction step, and details of any process such as fumigation etc. used
to reduce the levels of microbial contamination together with the controls exercised over the
process. If vegetable substance preparations are the starting material, the manufacture of the
vegetable substance preparations and their controls do not belong under this section but
under section C.
C CONTROL OF STARTING MATERIALS

L Control of the vegetable substance
A complete monograph for each vegetable substance must be submitted, even if the starting
material is a vegetable substance preparation. This also applies if the applicant is not the
manufacturer of the preparation. In the case of fatty or essential oils a complete monograph
for the vegetable substance is not required, only the scientific name of the parent plant and
its part(s) have to be stated.
If no monograph for the vegetable substance is given in a Pharmacopoeia referred to in
Directive 75/318/EEC as amended, Annex 1, a monograph on the vegetable substance must be
supplied and should be set out in the same way where practicable, as the monographs on
vegetable substances in the European Pharmacopoeia. This should include the botanical
name and authority and the common name if used for labelling purposes. Information on
the site of collection, the time of harvesting and stage of growth, treatment during growth
with pesticides etc., and drying and storage conditions should be included if possible. The
monograph should be established on the basis of recent scientific data. In the case of
vegetable substances with constituents of known therapeutic activity, assays of their content
(with test procedure) are required. The content must be included as a range, so as to ensure
reproducibility of the quality of the finished product.
As a general rule, vegetable substances must be tested for microbiological quality and for
residues of pesticides and fumigation agents, radioactivity, toxic metals, likely
contaminants and adulterants, etc., unless otherwise justified. Specifications and
descriptions of the analytical procedures must be submitted, together with the limits applied.
Reference samples of the vegetable substances must be available for use in comparative tests
e.g. macro and microscopic examination, chromatography etc.
2. Control of vegetable substance preparations

If the herbal remedy contains not the vegetable substance itself but a preparation, the
monograph on the substance must be followed by a description and validation of the
manufacturing process for the vegetable substance preparation.
For each vegetable substance preparation, a monograph must be submitted. This must be
established on the basis of recent scientific data and must give particulars of the
characteristics, identification tests and purity tests. This has to be done e.g. by different
appropriate chromatographic methods. If deemed necessary by the results of the analysis of
the starting material, tests on microbiological quality, residues of pesticides, fumigation
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agents, radioactivity, solvents and toxic metals have to be carried out. Quantitative
determination (assay) of characteristic constituents is required. The content must be
indicated with the lowest possible tolerance. The test methods must be described in detail.
If preparations from vegetable substances with constituents with known therapeutic activity
are standardised (i.e. adjusted to a certain level of constituents with known therapeutic
activity) it must be stated how such standardisation is achieved. If another substance is used
for this purpose, it is necessary to specify as a range the quantity that can be added.
D CONTROL OF TESTS CARRIED OUT AT AN

INTERMEDIATE STAGE OF THE MANUFACTURING
PROCESS OF THE FINISHED PRODUCT
Details of all control tests with details of test procedures and limits applied at any
intermediate stages of the manufacturing processes are required, especially if these tests
cannot be done in the finished product.
E CONTROL TESTS ON FINISHED PRODUCT

The control tests on the finished product must be such as to allow the qualitative and
quantitative determination of the composition of the active substances and a specification has
to be given which may be done by using markers if constituents with known therapeutic
activity are unknown. In the case of vegetable substances or vegetable substance preparations
with constituents of known therapeutic activity, these constituents must also be specified and
quantitatively determined.
If a herbal remedy contains several vegetable substances or preparations of several vegetable
substances and it is not possible to perform a quantitative determination of each active
substance, the determination may be carried out jointly for several active substances. The
need for this procedure must be justified.
F STABILITY TESTS
Since the vegetable substance or vegetable substance preparation in its entirety is regarded
as the active substance, a mere determination of the stability of the constituents with known
therapeutic activity will not suffice. It must also be shown, as far as possible e.g. by means of
appropriate fingerprint chromatograms, that other substances present in the vegetable
substance or in the vegetable substance preparation are likewise stable and that their
proportional content remains constant.
If a herbal remedy contains several vegetable substances or preparations of several vegetable
substances and if it is not possible to determine the stability of each active substance, the
stability of the medicinal product should be determined by appropriate fingerprint
chromatograms, appropriate overall methods of assay and physical and sensory tests or other
appropriate tests.
If the only evidence that can be submitted concerning the stability of the finished product
consists of results of trials in which each active substance was separately tested in a
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formulation corresponding to that of the finished product, the reasons why it is not possible to
carry out stability tests on the finished product must be stated in full. It must furthermore be
shown that interactions between the active substances and the excipients in the finished
product are unlikely to occur.
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ANNEX
Glossary-
Herbal remedies (herbal medicines) are medicinal products containing as active substances
exclusively plant material and/or vegetable substance preparations.

Vegetable substances are plant material used for a medicinal purpose. A vegetable substance
or a preparation thereof is regarded as one active substance in its entirety whether or not the
constituents with therapeutic activity are known.
Vegetable substance preparations are comminuted or powdered vegetable substances,
extracts, tinctures, fatty or essential oils, expressed juices etc. prepared from vegetable
substances, and preparations whose production involves a fractionation, purification or
concentration process. However, chemically defined isolated constituents or their mixtures
are not vegetable substance preparations. Other substances such as solvents, diluents,
preservatives may form part of vegetable substance preparations. These substances must be
indicated.
Constituents with known therapeutic activity are chemically defined substances or groups of
substances which are known to contribute to the therapeutic activity of a vegetable substance
or of a preparation.
Markers are chemically defined constituents of a vegetable substance which are of interest
for control purposes. Markers may serve to calculate the quantity of vegetable substance or
preparation in the finished product if that marker has been quantitatively determined in the
vegetable substance or preparation when the starting materials were tested.
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BIOTECHNOLOGY GUIDELINES
3AB1a
PRODUCTION AND QUALITY CONTROL OF

MEDICINAL PRODUCTS DERIVED BY
RECOMBINANT DNA TECHNOLOGY
Guideline Title Production and Quality Control of Medicinal Products

derived by recombinant DNA Technology
Date of first adoption First adopted June 1987
This version adopted December 1994
Date of entry into July 1995
force
Previous titles/other III/3477/92
references
Additional Notes This note for guidance is intended to facilitate the
collection and submission of data to support applications
for marketing authorisation within the EEC for
polypeptide based products derived by rDNA technology
and intended for medicinal use in man. It concerns the
application of Part 2, sections A- of the Annex to
granting of a marketing authorisation for a new m e d i c i n a l
product derived by rDNA technology.
CONTENTS
1. INTRODUCTION
2. POINTS TO CONSIDER IN PRODUCTION
3. DEVELOPMENT GENETICS
4. CONTROL OF CELL BANKS
5. FERMENTATION OR CELL CULTURE
6. PURD7ICATION OF THE PRODUCT
7. ACTrVE SUBSTANCE
8. CONSISTENCY AND ROUTINE BATCH CONTROL OF BULK FINAL ACTRTE
SUBSTANCE
9. SPECULATION AND REFERENCE MATERIALS
10. FINISHED PRODUCT AND DEVELOPMENT PHARMACEUTICS
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MEDICINAL PRODUCTS DERIVED BY
RECOMBINANT DNA TECHNOLOGY
1 INTRODUCTION
Developments in molecular genetics and nucleic acid chemistry enable the genes coding for
natural, biologically active proteins to be identified, analysed in fine detail, transferred
between organisms, and expressed under controlled conditions so as to obtain synthesis of
the polypeptide for which they code.
Sufficient quantities of medicinal products which were previously difficult to prepare from
natural sources can now be produced using such recombinant DNA (rDNA) technology. In
addition, the ability to synthesise and manipulate nucleic acids allows the construction of
genes coding for modified products possessing different properties from their natural
counterpart, or even entirely novel products.
A common strategy in the development of rDNA derived products is the insertion of
naturally occurring or intentionally modified natural sequences or novel nucleotide
sequences into a vector which is introduced into a suitable host organism so as to ensure the
efficient expression of the desired gene product. Both prokaryotic and eukaryotic vector/host
cell expression systems have been developed and are in use for production. The factors
affecting the expression of foreign genes introduced into a new host using a suitable vector
are complex and the efficient, controlled expression of stable, cloned DNA sequences is a n
important aspect of product development.
A flexible approach to the control of these products should be adopted so that recommendations
can be modified in the light of experience of production and use, and with the further
development of new technologies. Implementation of these recommendations for an
individual product should reflect its intended clinical use.
This note for guidance is intended to facilitate the collection and submission of data to
support applications for marketing authorisation within the European Union for polypeptide
based products derived by rDNA technology and intended for medicinal use in man. It
should be read in conjunction with the European Directives and other specialised guidelines
where appropriate.
2. POINTS TO CONSIDER IN PRODUCTION

Requirements relating to establishments in which biological products are produced (e.g.
GMP Directive 91/356/EEC and Directive 90/219/EEC on the contained use of genetically
modified micro-organisms) will apply to the production of products derived by rDNA
methodology as will several of the general recommendations for the quality control of
biological products.
Thus, appropriate attention needs to be given to the quality of all reagents used in production,
including components of fermentation media; specifications for these are to be included in
documentation and they must comply with any relevant European recommendations (e.g.
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note for guidance on Minimising the Risk of Transmitting Agents causing Spongiform
Encephalopathy via Medicinal Products).
Tests for potency, abnormal toxicity, pyrogenicity and sterility etc., which apply to products
made by conventional methods, will also apply to products made by rDNA technology. It is
undesirable to use in production agents which are known to provoke sensitivity in certain
individuals, such as, for example, penicillin or other -lactam antibiotics.
Although comprehensive characterisation of the final product is essential, considerable
emphasis must also be placed on "in-process" control, a concept which has been highly
effective in the quality control of bacterial and viral vaccines prepared by conventional
methods.
Certain factors may compromise the consistency, safety and efficacy of rDNA-derived
products; these should be given special attention and are outlined below:
a) All biological systems are inherently subject to genetic alteration through mutation
and selection and foreign genes inserted into new host cells may exhibit increased
genetic instability. The purpose of molecular genetic studies is to establish that the
correct sequence has been made and incorporated in the host cell and that both the
structure and the number of copies of the inserted sequence are maintained within the
cell during culture to the end of production. Such studies can provide valuable
information which should be considered in conjunction with tests performed at the
protein level for assuring the quality and consistency of the product.
b) Products expressed in foreign hosts may deviate structurally, biologically or
immunologically from their natural counterparts. Such alterations can arise at post-
translational level or during production or purification and may lead to undesirable
clinical effects. Therefore, their presence must be justified and shown to be
consistently controlled.
c) The choice of manufacturing procedure will influence the nature, range and amount
of potential impurities in the final product and which the purification processes must be
shown to be capable of removing. Examples of these are endotoxins in products
expressed in bacterial cells, and adventitious agents and DNA in products expressed
in mammalian cells.
d) Unintended variability in the culture during production may lead to changes which
favour the expression of other genes in the host/vector system or which cause alteration
in the product. Such variation might result in differing yield, in change to the product
itself (e.g. in the nature and degree of glycosylation) and/or in quantitative and
qualitative differences in the impurities present. Consequently, procedures to ensure
consistency of production conditions as well as the final product are imperative.
e) Extensive "scale-up" at the level of fermentation a n d ^ r purification occurs as
laboratory developments progress to full scale commercial production, and this may
have considerable consequences for the quality of the product including effects on its
conformational structure, yield and/or in quantitative and qualitative differences i n
impurities. Therefore, sufficient in-process controls and quality control tests during
each production run to show equivalency are required.
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Whilst the recommendations set out below should be considered to be generally applicable,
individual products may present particular quality control issues. Thus, the production and
control of each product must be given careful individual consideration taking fully into
account any special features.
3.1 Gene of interest, Vector and Host Cell
A detailed description of the cloned gene should be given. This should include details of its
origin, identification and isolation, as well as the details of the origin and structure of the
expression vector. A description of the host strain or cell line should be provided including
the history of the strain or cell line, its identification characteristics and potential viral
contaminants. Special attention should be given to the possibility of cross-contamination
with other cells or viruses.
3.2 Expression construct

Full details of the nucleotide sequence of the gene of interest and of the flanking control
regions of the expression vector should be provided to confirm that the construction is
identical to that desired. The steps in the assembly of the expression construct should be
described in detail. A detailed map and a complete annotated sequence of functionally
relevant regions of the vector should be given, indicating the regions which have been
sequenced during the construction and those deduced from the literature. All the junctions
created by ligation during construction directly impinging on the expression of the inserted
gene should be confirmed by sequencing. All known expressed sequences should be clearly
identified.
3.3 Status of the rDNA within the host cell

The method by which the vector is introduced into the host cell and the status of the rDNA
within the host (integrated or extrachromosomal, copy number, etc.) should be described. For
extrachromosomal expression systems, the percent of host cells retaining the expression
construct should be determined. The coding sequence for the recombinant product of the
expression construct should be verified at the cell bank stage. In systems where multiple
integrated copies of the gene exist, which may or may not be the result of amplification, a
detailed study using various restriction enzymes and Southern blot analysis should be used,
in addition to sequence analysis of mRNA or cDNA molecules in order to provide
convincing data on the integrity of the expressed gene(s).
3.4 Expression
The strategy by which the expression of the relevant gene is promoted and controlled during
production should be described in detail.
3.5 Stability of t h e e x p r e s s i o n s y s t e m
The stability of host/vector genetic and phenotypic characteristics should be investigated up
to and beyond the population doubling level or generation number used for routine
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production (End of Production Cells). The expression construct should be analysed in the
End of Production Cells, as described above, at least once for each MCB.
Stability studies should also provide detailed information on:
i) gene copy number in relation to productivity of the culture,
ii) deletions and/or insertions affecting any part of the expression vector
iii) the protein produced.
For this purpose, analysis should be performed in such a way that the results can confirm
that the number of variants is below an acceptable limit to be established on a case by case
basis depending on the nature and proposed use of the product. Analysis at the protein and/or
at the DNA level can be envisaged. Whichever method is used, it should be validated and the
detection limit given.
4. CONTROL OF CELL BANKS

It is essential that production is based on a well defined master and working cell bank
system. During the establishment of the banks no other cell lines should be handled
simultaneously in the same laboratory suite or by the same persons. The origin, form,
storage, use, and expected duration at the anticipated rate of use must be described in full for
all cell banks. New working cell banks should be fully characterised.
A critical part of quality control will involve the full characterisation of cells. Where
eukaryotic cells are used for production, distinguishing genetic, phenotypic and
immunological markers of the cell will be useful in establishing the identity of the cells.
Likewise, where microbial cultures are used, specific phenotypic features which form a basis
for identification should be described.
The cell banks should be examined for adventitious agents (viral, bacterial, fungal and
mycoplasmal). Special attention should be given to viruses which can commonly
contaminate the animal species from which the cell line has been derived. Certain cell lines
contain endogenous viruses, e.g. retroviruses, which may not readily be eliminated. The
possibility of mutations of endogenous viruses during prolonged culture should be
considered. Furthermore, the purification process should be shown to be capable of removing
and/or inactivating any such virus which may inevitably be present in the cells as an
endogenous agent.
Cell banks should be periodically tested for cell viability, genetic and phenotypic stability
and any other relevant parameters.
5. FERMENTATION OR CELL CULTURE

A clear definition of a "batch" of product for further processing should be provided.
Whatever the production process, details of the fermentation or culture with the in-process
controls should be provided. Criteria for rejection of harvests and premature termination of
the culture should be defined.
The presence, extent and nature of any microbial contamination in the culture vessels must
be thoroughly examined at a suitable stage at the end of each production run. Detailed
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information to confirm the adequate sensitivity of the methods used to detect contamination
should be provided and acceptable limits of contamination set.
Ideally not more than one cell line should be cultivated simultaneously in the same
production area. If other cell lines are cultivated in parallel, records must be kept of the cell
lines handled and validation data presented for the absence of cross-contamination between
them.
5.1 Single harvest production

The maximum permitted generation number or population doubling level for production
should be defined and should be based on information concerning the stability of the host
cell/vector system up to and beyond the level of production. Data on consistency of growth of
the culture and on the maintenance of yield within specified limits should be presented.
Appropriate monitoring of host cell/vector characteristics at the end of the production cycles
should also be undertaken. Evidence should be provided that the yield does not vary beyond
defined limits and that the nature and quality of the product does not change with respect to
specific parameters.
5.2 M u l t i p l e h a r v e s t p r o d u c t i o n
The period of continuous cultivation should be specified and this should be based on
information concerning the stability of the system and consistency of the product up to and
beyond this limit. Monitoring of the production system is necessary throughout the duration
of the culture. The required frequency and type of monitoring will depend upon several
factors including the nature of the expression system and product, as well as the total length
of the period of continuous cultivation undertaken. The acceptance of harvests for further
processing should be clearly linked to the schedule of monitoring applied. Evidence should
be provided that the yield does not vary beyond defined limits and that the nature and
quality of the product does not change with respect to specific parameters.
6. PURIFICATION OF THE PRODUCT

6.1 Methods
Methods used to purify the product and their in-process controls including their specification
limits should be described in detail, justified and validated. Procedures which make use of
affinity chromatography, for example employing monoclonal antibodies, should be
accompanied by appropriate measures to ensure that these substances, or any additional
potential contaminants arising from their use, do not compromise the quality and safety of
the final product. Attention is drawn to the notes for guidance "Production and Quality
Control of Monoclonal Antibodies" and "Virus validation studies: The design, contribution
and interpretation of studies validating the inactivation and removal of viruses".
The criteria for reprocessing of any intermediate or final bulk should be carefully defined,
validated and justified.
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6.2 Validation of t h e purification p r o c e d u r e

The capacity of the purification procedure to remove unwanted host cell derived proteins,
nucleic acids, carbohydrates, viruses and other impurities including product-related proteins
should be investigated thoroughly.
Studies using a carefully selected group of viruses which exhibit a range of physico-chemical
features relevant to their behaviour on purification (see note for guidance Virus Validation
Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and
Removal of Viruses) intentionally mixed with the crude preparation (spiking) should be
undertaken. The ability of the purification process to remove other specific contaminants
such as host-cell proteins, other potential impurities derived from the production process and
DNA should also be demonstrated using, where necessary, concentrations of those
contaminants in excess of that expected during normal production (spiking). A reduction
factor for such contaminants at each stage of purification, and overall, should be established.
Validation of the purification process should also include justification of the working
conditions such as column loading capacity , column regeneration and sanitisation and
length of use of the columns. Columns should also be validated regarding leaching of
ligands (e.g. dye, affinity ligand, etc.) and/or chromatographic material, throughout the
expected life span of the column.
7. ACTDTE S U B S T A N C E
7.1 C h a r a c t e r i s a t i o n of t h e active s u b s t a n c e
7.1.1 Physico-chemical characterisation, relative molecular mass, pi value
Rigorous characterisation of the active substance by chemical and biological methods will be
essential. Particular attention should be given to using a wide range of analytical
techniques exploiting different physico-chemical properties of the molecule; for instance,
size, charge, isoelectric point and hydrophobicity. A list of the analytical possibilities is
beyond the scope of this guideline. In the following there are only examples of the type of
analysis.
7.1.2 Structural evidence for the active substance (including comparison with
reference or natural product)
Sufficient sequence information to characterise the gene product adequately should be
obtained. The degree of sequence verification required will depend on the size and
complexity of the molecule, considering the extent of other characterisation tests. In most
instances, determination of the entire sequence can be obtained after HPLC separation and
sequencing of the peptides released by enzymatic digestion. Attention should be paid to the
possible presence of N-terminal methionine and N-formyl methionine, signal or leader
sequences, other possible N- and C-terminal modifications (proteolytical processing). It
should be considered integrating modern mass spectrometry techniques in characterising
the primary structure.
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7.1.3 Post-translational modifications

Apart from the proteolytical processing, potential types of post-translational modifications
are N- and O- glycosylation and for instance acetylation, hydroxylation and gamma-
carboxylation. In addition, there are post-translational modifications that occur as
degradation products such as deamidation and oxidation.
Some rDNA products are glycoproteins. There is a large range of oligosaccharide structures
and these substances are characterised by glycoform heterogeneity both in their natural
forms and those resulting from rDNA technology. The detail of this heterogeneity can be
affected by many factors and the glycosylation pattern may have an important role i n
determining activity, particularly in vivo. The extent of analysis undertaken should depend
on the role played by the carbohydrate moiety where this is known. A range of different
analytical techniques should be explored, for instance quantitative isoelectric focusing or
capillary electrophoresis, anion exchange chromatography for monosaccharide component
analysis and oligosaccharide determination, lectin affinity chromatography, mass
spectrometry.
7.1.4 Conformational data for macromolecules

It is desirable to include suitable tests to establish that the product has the desired
conformational structure and state of aggregation. Examples of techniques suitable for such
purposes are: Polyacrylamide gel electrophoresis; isoelectric focusing; size exclusion,
reversed phase ion exchange, hydrophobic interaction or affinity chromatography; peptide
mapping and subsequent amino acid sequencing; light scattering; UV spectroscopy; circular
dichroism and mass spectrometry. Additional characterisation of the product using for
example NMR spectra, X-ray crystallography or relevant immunochemical techniques may
provide valuable information.
7.1.5 Biological, immunological characterisation, expression of strength

Biological and immunological characterisation should include as wide a range of
techniques as necessary. The specific activity of highly purified material should be
determined (units of activity/weight of product).
When appropriate the biological activity of the product and its physical characteristics,
including the amino acid sequence, should be compared with that of a highly purified
preparation of the naturally occurring molecule.
7.2 Purity
Data should be provided on contaminants whose presence is anticipated in the final
processed product. The level of contamination considered as acceptable should be justified,
and criteria for acceptance or rejection of a production batch should be given. It is important
that the techniques used to demonstrate purity be assessed using as wide a range of methods
as possible, including physico-chemical and immunological techniques. Unwanted
materials of host origin, as well as materials which may have been added during the
production or purification processes, and where appropriate, viral and nucleic acid
contamination should be tested.
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8. CONSISTENCY AND ROUTINE BATCH CONTROL OF BULK

FINAL ACTIVE SUBSTANCE
A comprehensive analysis of the initial batches of a product should be undertaken to
establish consistency with regard to identity, purity and potency. Thereafter, a more limited
series of tests may be appropriate as outlined below. A clear difference should be made
between the analytical tests performed during the development, in order to fully characterise
the active substance and tests performed routinely on each production batch of purified bulk
product.
8.1. Consistency
An acceptable number, for example 5 (smaller numbers could be acceptable where justified),
of successive batches of the bulk processed product should be characterised as fully as
possible to determine consistency of composition. In the case of a production where multiple
harvests are applied, batches from different fermentation runs should normally be studied.
The studies should include biological, chemical and immunological methods to characterise
and assay the active substance (including methods showing the consistency of the
glycosylation pattern for glycoproteins) and methods to detect and identify impurities. Any
differences which occur between batches should be noted.
8.2. Routine batch control analysis

8.2.1 Identity
A selection of the tests used to characterise the purified active substance (see 7.1) should be
used to confirm the product identity for each batch. The methods employed should include
tests for the physico-chemical and immunological characteristics, together with test for the
anticipated biological activity. Depending on the extent of other identification tests, sequence
verification of a number of amino acids at the N- or C-terminus or other methods such as
peptide mapping should be performed.
82.2. Purity
The degree of purity desirable and attainable will depend on several factors; these include
the nature and intended use of the product, the method of its production and purification and
also the degree of consistency of the production process. In general, a very high degree of
purity can be achieved for most products by modern manufacturing procedures.
The purity of each batch should be established and be within specified limits. The analysis
should include sensitive and reliable assays for DNA of host cell origin and/or of the vector
applied to each batch of product prepared from cell lines of mammalian origin, in which
case upper limits should be set. It is recommended that DNA analyses are also performed on
each batch of bulk product obtained from other eukaryotic cell systems and limits set for
DNA content. DNA of prokaryotic expression systems should be tested for wherever
appropriate to consideration of the quality of the product. The residual cellular proteins
should also be determined by an assay with appropriate sensitivity (e.g. ppm) and strict
upper limits set. In some instances, potential impurities such as DNA can only be
determined on intermediates of purification, at an earlier step.
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8.2.3. Test for potency

The potency of each batch of the product should be established (e.g. units of biological activity
per ml) using, wherever possible, an appropriate national or international reference
preparation calibrated in units of biological activity (see section 9).
In addition, information on specific activity (units of biological activity per unit weight of
product) will be of considerable value and should be reported. A highly purified reference
preparation is required to standardise measurements of specific activity (see section 9).
It is recommended that correlation between potency measurements, involving biological
tests, and the results of physico-chemical methods of assay are made and the information
reported. If possible, batches should be calibrated using accurate physico-chemical tests, and
the biological assays used to confirm -within stated limits- that the product is biologically
potent.
9. SPECIFICATION AND REFERENCE MATERIALS

The studies described in section 7 will contribute to a definitive specification for the product
when justified by the information obtained from the examination of successive batches and
results of batch analysis, as indicated in section 8.
A suitable batch of the product, preferably one which has been clinically evaluated, should be
fully characterised in terms of its chemical composition, purity, potency and biological
activity, including where possible full amino acid sequencing, and retained for use as a
chemical and biological reference material.
Criteria for expiration and possible re-testing and re-qualification of reference standards
should be established.
10. FINISHED PRODUCT AND DEVELOPMENT

PHARMACEUTICS
The development of the formulation should be described in detail and justified, particularly
with regard to the presence and amount of stabiliser such as albumin and/or detergents. The
product in final containers should be shown to comply with the requirements of the European
directives and pharmacopoeias. In circumstances where this is not possible the omission of
tests should be justified by the manufacturer.
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GLOSSARY
1. Cell Banks
a) Master cell bank (MCB)
A homogeneous suspension of the original cells already transformed by the expression
vector containing the desired gene, aliquoted into individual containers for storage (e.g. in
a liquid nitrogen refrigerator). In some cases it may be necessary to establish separate
master cell banks for the expression vector and the host cells.
b) Working cell bank (WCB)
A homogeneous suspension of cells derived from the master cell bank(s) by a finite passage
level, aliquoted into individual containers for storage (e.g. in a liquid nitrogen
refrigerator).
In both cell banks, all containers are treated identically during storage, and once removed
from storage, the containers are not returned to the cell bank stock.
2. Production method
a) Production at finite passage (single harvest)
This cultivation method is defined by a limited number of passages or population doublings
which must not be exceeded during production.
b) Continuous culture production (multiple harvest)
The number of population doublings (or duration of culture for certain production systems)
are specified based on information concerning the stability of the system and the
consistency of the product. Criteria for the termination has to be defined by the
manufacturer.
3. Bulk harvest
This is a homogeneous pool of individual harvests or lysates which is processed in a single
purification run.
4. Bulk final active substance

This is the final product, after completion of the production process, obtained from a bulk
harvest. It is maintained in a single container or multiple identical containers where
necessary and used in the preparation of the final dosage form. The generation of this final
batch has to be clearly defined and unambiguously recorded by the producer.
5. Finished product
The active substance is formulated and filled into final, sealed containers which hold the
product in its final dosage form, i.e. the finished product. The containers of a filling lot are
processed together and uniform in their contents and biological potency.
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QUALITY OF BIOTECHNOLOGICAL PRODUCTS:

ANALYSIS OF THE EXPRESSION CONSTRUCT IN
CELLS USED FOR PRODUCTION OF RDNA DERIVED
PROTEIN PRODUCTS *)
Guideline Title Quality of Biotechnological Products: Analysis of the

Expression Construct in Cells used for the Production o f
rDNA derived Protein Products *)
Date of entry i n t o June 1996
force
Previous titles/other ICH Q5 : Genetic stability I CPMP/ICH/139/95
references
Additional Notes This document presents guidance regarding the
characterisation of the expression construct for the
production of rDNA protein products in eukaryotic and
prokaryotic cells. This document is intended to describe
the types of information that are considered valuable i n
assessing the structure of the expression construct used to
produce rDNA derived proteins.
CONTENTS
I. INTRODUCTION
II. RATIONALE FOR ANALYSIS OF THE EXPRESSION CONSTRUCT
III. CHARACTERISATION OF THE EXPRESSION SYSTEM
IV. CONCLUSION
V. GLOSSARY
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ANALYSIS OF THE EXPRESSION CONSTRUCT IN
CELLS USED FOR PRODUCTION OF R-DNA
DERIVED PROTEIN PRODUCTS*)
I. INTRODUCTION
This document presents guidance regarding the characterisation of the expression construct
for the production of recombinant DNA (rDNA) protein products in eukaryotic and
prokaryotic cells. This document is intended to describe the types of information that are
considered valuable in assessing the structure of the expression construct used to produce
rDNA derived proteins. This document is not intended to cover the whole quality aspect of
rDNA derived medicinal products.
The expression construct is defined as the expression vector containing the coding sequence
of the recombinant protein. Segments of the expression construct should be analysed using
nucleic acid techniques in conjunction with other tests performed on the purified
recombinant protein for assuring the quality and consistency of the final product. Analysis
of the expression construct at the nucleic acid level should be considered as part of the
overall evaluation of quality, taking into account that this testing only evaluates the coding
sequence of a recombinant gene and not the translational fidelity nor other characteristics
of the recombinant protein, such as secondary structure, tertiary structure, and post-
translational modifications.
II. RATIONALE FOR ANALYSIS OF THE EXPRESSION

CONSTRUCT
The purpose of analysing the expression construct is to establish that the correct coding
sequence of the product has been incorporated into the host cell and is maintained during
culture to the end of production. The genetic sequence of recombinant proteins produced i n
living cells can undergo mutations that could alter the properties of the protein with potential
adverse consequences to patients. No single experimental approach can be expected to detect
all possible modifications to a protein. Protein analytical techniques can be used to assess
the amino acid sequence of the protein and structural features of the expressed protein due to
post-translational modifications such as proteolytic processing, glycosylation,
phosphorylation, and acetylation. Data from nucleic acid analysis may be useful since
protein analytical methods may not detect all changes in protein structure resulting from
mutations in the sequence coding for the recombinant protein. The relative importance of
nucleic acid analysis and protein analysis will vary from product to product.
Nucleic acid analysis can be used to verify the coding sequence and the physical state of the
expression construct. The nucleic acid analysis is performed to ensure that the expressed
protein will have the correct amino acid sequence but is not intended to detect low levels of
variant sequences. Where the production cells have multiple integrated copies of the
expression construct, not all of which may be transcriptionally active, examination of the
transcription product itself by analysis of mRNA or cDNA may be more appropriate than
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analysis of genomic DNA. Analytical approaches that examine a bulk population of nucleic
acids, such as those performed on pooled clones or material amplified by the polymerase
chain reaction, may be considered as an alternative to approaches that depend on selection of
individual DNA clones. Other techniques could be considered that allow for rapid and
sensitive confirmation of the sequence coding for the recombinant protein in the expression
construct.
The following sections describe information that should be supplied regarding the
characterisation of the expression construct during the development and validation of the
production system. Analytical methodologies should be validated for the intended purpose of
confirmation of sequence. The validation documentation should at a minimum include
estimates of the limits of detection for variant sequences. This should be performed for either
nucleic acid or protein sequencing methods. The philosophy and recommendations for
analysis expressed in this document should be periodically reviewed to take advantage of
new advances in technology and scientific information.
IH. C H A R A C T E R I S A T I O N O F T H E E X P R E S S I O N SYSTEM
A. Expression Construct a n d Cell Clone Used to Develop t h e Master
Cell B a n k (MCB)
The manufacturer should describe the origin of the nucleotide sequence coding for the
protein. This should include identification and source of the cell from which the nucleotide
sequence was originally obtained. Methods used to prepare the DNA coding for the protein
should be described.
The steps in the assembly of the expression construct should be described in detail. This
description should include the source and function of the component parts of the expression
construct, e.g. origins of replication, antibiotic resistance genes, promoters, enhancers,
whether or not the protein is being synthesised as a fusion protein. A detailed component
map and a complete annotated sequence of the plasmid should be given, indicating those
regions that have been sequenced during the construction and those taken from the
literature. Other expressed proteins encoded by the plasmid should be indicated. The
nucleotide sequence of the coding region of the gene of interest and associated flanking
regions that are inserted into the vector, up to and including the junctions of insertion,
should be determined by DNA sequencing of the construct.
A description of the method of transfer of the expression construct into the host cell should be
provided. In addition, methods used to amplify the expression construct and criteria used to
select the cell clone for production should be described in detail.
B. Cell B a n k System
Production of the recombinant protein should be based on well-defined Master and Working
Cell Banks. A cell bank is a collection of ampoules of uniform composition stored under
defined conditions each containing an aliquot of a single pool of cells. The Master Cell
Bank (MCB) is generally derived from the selected cell clone containing the expression
construct. The Working Cell Bank (WCB) is derived by expansion of one or more ampoules
of the MCB. The cell line history and production of the cell banks should be described i n
detail, including methods and reagents used during culture, in vitro cell age, and storage
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conditions. All cell banks should be characterised for relevant phenotypic and genotypic
markers which could include the expression of the recombinant protein or presence of the
expression construct.
The expression construct in the MCB should be analysed as described below. If the testing
cannot be carried out on the MCB, it should be carried out on each WCB.
Restriction endonuclease mapping or other suitable techniques should be used to analyse the
expression construct for copy number, for insertions or deletions, and for the number of
integration sites. For extrachromosomal expression systems, the percent of host cells
retaining the expression construct should be determined.
The protein coding sequence for the recombinant protein product of the expression construct
should be verified. For extrachromosomal expression systems, the expression construct
should be isolated and the nucleotide sequence encoding the product should be verified
without further cloning. For cells with chromosomal copies of the expression construct, the
nucleotide sequence encoding the product could be verified by recloning and sequencing of
chromosomal copies. Alternatively, the nucleic acid sequence encoding the product could be
verified by techniques such as sequencing of pooled cDNA clones or material amplified by
the polymerase chain reaction. The nucleic acid sequence should be identical, within the
limits of detection of the methodology, to that determined for the expression construct as
described in Section III.A. and should correspond to that expected for the protein sequence.
C. Limit for In vitro Cell Age for Production

The limit for in vitro cell age for production should be based on data derived from
production cells expanded under pilot or full scale conditions to the proposed in vitro cell age
or beyond. Generally, the production cells are obtained by expansion of the Working Cell
Bank; the Master Cell Bank could be used to prepare the production cells with appropriate
justification.
The expression construct of the production cells should be analysed once for the MCB as
described in Section III.B, except that the protein coding sequence of the expression construct
in the production cells could be verified by either nucleic acid testing or analysis of the final
protein product. Increases in the defined limit for in vitro cell age for production should be
supported by data from cells which have been expanded to an in vitro cell age which is equal
to or greater than the new limit for in vitro cell age.
IV. CONCLUSION
The characterisation of the expression construct and the final purified protein are both
important to ensure the consistent production of a rDNA derived product. As described above,
it is considered that analytical data derived from both nucleic acid analysis and evaluation
of the final purified protein should be evaluated to ensure the quality of a recombinant
protein product.
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GLOSSARY
Expression Construct
The expression vector which contains the coding sequence of the recombinant protein and the
elements necessary for its expression.
Flanking Control Regions

Non-coding nucleotide sequences that are adjacent to the 5' and 3' end of the coding
sequence of the product which contain important elements that affect the transcription,
translation, or stability of the coding sequence. These regions include, e.g. promoter,
enhancer, and splicing sequences and do not include origins of replication and antibiotic
resistance genes.
Integration Site
The site where one or more copies of the expression construct is integrated into the host cell
genome.
In vitro Cell Age

Measure of time between thaw of the MCB vial(s) to harvest of the production vessel
measured by elapsed chronological time in culture, by population doubling level of the cells,
or by passage level of the cells when subcultivated by a defined procedure for dilution of the
culture.
Master Cell Bank (MCB)

An aliquot of a single pool of cells which generally has been prepared from the selected cell
clone under defined conditions, dispensed into multiple containers and stored under defined
conditions. The MCB is used to derive all working cell banks. The testing performed on a
new MCB (from a previous initial cell clone, MCB or WCB) should be the same as for the
MCB unless justified.
Pilot Plant Scale

The production of a recombinant protein by a procedure fully representative of and
simulating that to be applied on a full commercial manufacturing scale. The methods of cell
expansion, harvest, and product purification should be identical except for the scale of
production.
Relevant Genotypic and Phenotypic Markers

Those markers permitting the identification of the strain of the cell line which should
include the expression of the recombinant protein or presence of the expression construct.
Working Cell Bank (WCB)

The Working Cell Bank is prepared from aliquots of a homogeneous suspension of cells
obtained from culturing the MCB under defined culture conditions.
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CYTOKINE PRODUCTS DERIVED BY
BIOTECHNOLOGICAL PROCESSES
Guideline Title Production and Quality Control of Cytokine Products

derived by Biotechnological Processes
force
references
for marketing authorisation for cytokine products derived
from biotechnological processes, particularly with
respect to Part 2, sections A- of the Annex to Directive
75/318/EEC as amended.
CONTENTS
1. INTRODUCTION
2. POINTS TO CONSIDER IN MANUFACTURE
3. MANUFACTURING REQUIREMENTS
4. FINISHED PRODUCT
5. GLOSSARY
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CYTOKINE PRODUCTS DERIVED BY
BIOTECHNOLOGICAL PROCESSES
1 INTRODUCTION
Cytokines are a heterogeneous group of biologically active proteins or glycoproteins which
regulate cell growth, differentiation and other functions. They are soluble (non-antibody)
mediators which are active in very small quantities. Much research has centred on those
cytokines which appear to modulate the growth and function of cells contained within the
immune system.
The roles of cytokines in health and disease are likely to be complex. In general,
measurable amounts of cytokines are rarely found in body fluids of healthy individuals.
Much increased, detectable concentrations may, however, be found in body fluids during
episodes of acute microbial infections and, in some cases, during the course of chronic
invasive disease. It has been postulated therefore that cytokines are primarily involved i n
the induction and maintenance of host defence mechanisms against microbial infection
and invasive disease. On the other hand, their continued presence in some chronic diseases
may contribute to pathology.
Cytokines are highly pharmacologically active and thus may be effective agents for
modifying biological responses (cytokines are often referred to as biological response
modifiers). Since only human cytokines are likely to be used clinically, they should be of
very low immunogenicity. So far, cytokines have mostly been used for the therapy of
neoplastic disease (cancer). Clinical investigations, mainly carried out with interferon
alpha or beta, have confirmed that cytokine treatment can be beneficial for a limited number
of cancers. However, the full clinical potential of many cytokines, used individually or i n
combination with other agents, has yet to be explored.

support applications for marketing authorisation within the European Union for cytokine
products derived from biotechnological processes. Cytokines may be produced in quantity by
the large scale cultivation either of (i) transformed cell lines producing particular cytokines
or (ii) prokaryotic or eukaryotic, including mammalian, cells in which the relevant
cytokine genes have been inserted by rDNA techniques. The large scale production of
cytokines by these procedures may affect the quality of particular cytokine products, and thus
have implications for control testing. Although comprehensive characterisation of the final,
purified cytokine product will be essential, particular emphasis must also be placed on "in-
process" control and the consistency of the manufacturing process, a concept which has been
very effective in the control of other biological products.
Requirements relating to establishments in which biological products are manufactured

(e.g. Revised Requirements for Biological Substances No 1; WHO TRS 323) will apply to the
manufacturers of cytokine products as will several of the general requirements for the
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quality control of biological products. Thus, appropriate attention needs to be given to the
quality of all reagents used in production, including components of fermentation or
cultivation media; specifications for these are to be included in documentation and they
must comply with any relevant European requirements. Tests for potency, abnormal
toxicity, pyrogenicity and sterility, etc., which apply to products made by conventional
methods, will also apply to products of biotechnological processes. It is undesirable to use in
production agents which are known to provoke sensitivity reactions in certain individuals,
such as, for example, penicillin or other beta-lactam antibiotics.
Certain factors may compromise the safety and efficacy of cytokine products; these should be
given special attention and are outlined below:
a) The use of a transformed cell line, particularly one of neoplastic origin, as the
substrate for the manufacture of particular cytokines raises questions of safety. These
questions, pertaining mainly to the potential oncogenicity of contaminating
heterogeneous DNA derived from the substrate, have been debated for a number of
years at many national and international scientific meetings. There is a growing
international consensus that transformed cell lines can be used for the manufacture of
biologicals, although their use as yet has only been approved in certain cases (e.g. the
manufacture of interferon derived from the Namalwa human lymphoblastoid cell
line) where the product may be subjected to rigorous purification.
b) Cytokines encoded by naturally occurring genes expressed in foreign hosts may
deviate structurally, biologically or immunologically from their natural counterparts.
Such alterations can arise either at the genetic or post-translational level or during
production or purification. Some cytokine products may be entirely novel in structure,
since they may be produced by manipulation of naturally occurring genes or by
chemical synthesis of new ones. These products may have enhanced biological
properties and/or diminished undesirable characteristics compared with their
naturally-occurring counterparts. It should be recognised, however, that they could
have unexpected and undesirable biological properties.
c) The choice of manufacturing procedure may influence the nature and range of
potential contaminants. Examples of these are endotoxins in products expressed in
bacterial cells and DNA or oncogenic potential in products expressed in transformed
mammalian cells. Thus cytokine products may contain potentially hazardous
contaminants which the purification processes must be shown to be capable of
removing.
d) Unintended variability in the culture during production may lead to changes which
favour the expression of other genes in the host/vector system or which cause
alterations in the polypeptide product. Such variation might result in decreased yield of
the products and/or quantitative and qualitative differences in the impurities present
in the product. Consequently, procedures to ensure the consistency of production
conditions as well as the consistency of the final product are imperative.
Whilst the requirements set out below should be applied wherever they are appropriate for
safety and efficacy, individual cytokine products may present particular quality control
problems. Thus, the production and control of each cytokine product must be given careful
individual consideration taking fully into account any special features.
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3. MANUFACTURING REQUIREMENTS
3.1 Definitions
3.1.1 International name
A cytokine shall be named in accordance with international consensus. Proper names shall
be the equivalent of the international name in the language of the country of origin. The use
of the international name should be limited to suitably purified preparations.
3.12, Descriptive definition

A particular cytokine product shall be a preparation containing the intended cytokine
prepared by a designated production process; the cytokine will be harvested in supernatants
or extraction fluids derived from the cell substrate, purified and prepared in a form suitable
for clinical application and should satisfy all the criteria specified in this note for guidance.
3.1.3 International standards and international unit

A number of international standards for particular cytokines have been or are being
developed. These have assigned potencies in terms of biological activity which are quoted i n
International Units (IU).
3.2 Strategy for p r o d u c t i o n

32.1 Cytokine production by transformed cell lines
A full description of the biological characteristics of the transformed cell line and any
additions, e.g. the inducer and any enhancer or other substances (e.g. antibiotics) used in
production, shall be given. The information includes:
a) documentation concerning the origin of the cell line, and the nature of any known
relevant condition of the donor, e.g. Burkitt's lymphoma, active infectious
mononucleosis, infection with viruses or mycoplasma in general;
b) data which can be used to establish the identity of the cell line;
c) the growth characteristics of the cell line;
d) data which document the stability of the cell line under the cultivation conditions used,
especially if cells are to be sub cultured for an extended or indefinite period;
e) if serum is included in the medium of production cell cultures, evidence that it is free
from bacteria, fungi, mycoplasma and viruses.
3.2.2 Cytokine production by genetically engineered organisms - strategy for

cloning and expression
a) Expression vector and host cell: A description of the host cell and expression vector
used in production should be given. This should include details of the origin and
identification of the gene which is being cloned and the construction, genetics and
structure of the expression vector. The method by which the vector is introduced into the
host cell and the state of the vector within the host should be described. The association
of the vector and host cell may be permanent, allowing continuous expression of the
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product, or self-limiting, for example where the vector is an acceptable cytopathogenic

virus.
b) Sequence of the cloned gene: Full details of the nucleotide sequence of the gene insert
and of flanking control regions of the expression vector should be provided. All
relevant expressed sequences should be clearly identified. The DNA sequence of the
cloned gene should normally be confirmed at the seed lot stage and at least once after
a full scale fermentation. In certain systems, for example where multiple copies of the
gene are inserted into the genome of a continuous cell line, it may be inappropriate to
sequence the cloned gene at the production level. Under these circumstances, Southern
blot analysis of total cellular DNA or sequence analysis of the mRNA may be helpful
and particular attention should be paid to the characterisation of the final product.
c) Expression: The strategy by which the expression of the relevant gene is promoted and
controlled during production should be described in detail.
3.3 Validation and control of manufacturing process

3.3.1 Control of cell bank
It is essential that production is based on a well defined cell bank system involving a
master cell bank and manufacturer's working cell bank(s) (MWCB). During the
establishment of the cell bank, no other cell lines should be handled simultaneously in the
same laboratory suite or by the same persons. The origin, storage, use and details of life
expectancy at the anticipated rate of use must be described in full for all cell bank materials.
Attention should be paid to the stability of the host-vector expression system in the cell bank
under conditions of storage and recovery. Any known instability should be reported. New
cell banks should be fully characterised.
A critical part of quality control is the full characterisation of cell bank material. Where
higher eukaryotic cells are used for production, distinguishing markers of the cell, such as
specific isoenzyme and immunological features or karyology will be useful in establishing
the identity of the cell bank. Likewise, where microbial cultures are used, specific phenotypic
features which form a basis for identification should be described.
Evidence that the cell bank is free from infective adventitious agents (viral, bacterial,
fungal or mycoplasma) shall be provided. Special attention should be given to viruses which
can commonly contaminate the species from which the cell line has been derived. For
instance, cell lines of murine origin should be checked for contamination according to
Annex I of the note for guidance Production and Quality control of Monoclonal Antibodies.
Certain cell lines contain endogenous viruses, e.g. retroviruses, which may not readily be
eliminated. The expression of these organisms, under a variety of conditions known to
cause their induction, should be tested for and reported. Furthermore, the purification process
should be shown to be capable of removing and/or inactivating any such virus which may be
present in the cell bank as an endogenous agent or as part of the expression vector.
3.3.2 Production
production area or the cultivation should be carried out in closed vessels with effective
barriers which would prevent contamination with other adventitious agents or other cell
lines. If other cell lines are cultivated in parallel, records must be kept of the cell lines
handled and evidence presented for the absence of cross-contamination between them.
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a) Production at finite passage: Details of the fermentation or culture used to manufacture

the product should be provided. For each production run, the presence, extent and nature
of any microbial contamination in the culture vessels immediately prior to all
harvesting shall be thoroughly examined. Detailed information to confirm the
adequate sensitivity of the methods used to detect contamination should be provided
and acceptable limits of contamination set.
Maximum permitted passage levels for production should be defined and should be
based on information concerning the stability of the host cell/vector system on serial
sub-cultivation up to and beyond the level of production. Data on consistency of yield of
the product should be presented. Criteria for the rejection of culture lots should be
established.
Monitoring of the host cell/vector characteristics at the end of a number of production
cycles should also be undertaken. For example, detailed information on plasmid copy
number and degree of retention of the expression vector within the host cell, as well as
restriction mapping of the vector containing the gene insert, may be of value.
b) Continuous culture production: This approach should only be undertaken when special
consideration has been given to the control of production based on continuous culture.
Where it is undertaken, monitoring of the production system is necessary throughout
the life of the culture. The required frequency and type of monitoring will depend upon
several factors including the nature of the expression system and product. Information
should be obtained on the molecular integrity of the gene being expressed and of the
phenotypic and genotypic characteristics of the host cell after long term cultivation.
Evidence should be provided that the yield does not vary beyond defined limits and that
the nature and quality of the product does not change with respect to specific
parameters. The acceptance of harvests for further processing should be clearly linked
to the schedule of monitoring applied. The period of continuous cultivation should be
specified and this should be based on information concerning the stability of the
system and consistency of the product up to and beyond this limit. In cases on long
term continuous cultivation, the cell line and product should be completely re-evaluated
at intervals based on information concerning the stability of the system and the
characteristics of the product.
A clear definition of a "batch" of product for further processing should be provided.
Regular tests for microbial contamination should be performed in relation to the
strategy for harvesting. Criteria for rejection of harvests and premature termination of
the culture should be defined.
3.4 P u r i f i c a t i o n of t h e p r o d u c t
3.4.1 Methods
Methods used to purify the product should be described in detail. Procedures which make use
of affinity chromatography, for example employing monoclonal antibodies, should be
accompanied by appropriate measures to ensure that these substances, or any additional
potential contaminants arising from their use, do not compromise the quality and safety of
the final product. Attention is drawn to the note for guidance Production and Quality Control
of Monoclonal Antibodies.
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3.4.2 Validation of the purification procedure

nucleic acids, viruses and other impurities should be investigated thoroughly, as should the
reproducibility of the purification process as regards its ability to remove specific
contaminants and the consistent composition of the purified product with respect to any
impurities which may be present. Laboratory scale pilot studies which mimic the production
process using, for example, a carefully selected group of viruses which exhibit a range of
physico-chemical features relevant to their behaviour during the process of purification, or
radioactively labelled DNA intentionally mixed with the crude preparation (spiking) should
be undertaken. A reduction factor for such contaminants at each stage of purification should
be established by using, if necessary, concentrations of DNA and viruses in excess of that
expected during normal production.
3.5 Final processed product

3.5.1 Characterisation of the purified cytokine
Rigorous characterisation of the cytokine by chemical and biological methods will be
essential. Routine detailed characterisation of the final product may be required if the
nature of the expression system makes it impossible to characterise the gene at the
production level.
Particular attention should be given to using a wide range of analytical techniques
exploiting different physico-chemical properties of the molecule; for instance, size, charge,
isoelectric point, amino acid composition and hydrophobicity. It may be desirable to include
suitable tests to establish that the product has the desired conformational structure and state
of aggregation. Examples of techniques suitable for such purposes are: Polyacrylamide gel
electrophoresis, isoelectric interaction or affinity chromatography; peptide mapping; amino
acid analysis; light scattering; UV spectroscopy; circular dichroism and other spectroscopic
techniques. Additional characterisation of the product using, for example, immunochemical
techniques may provide valuable information. Biological and immunological
characterisation should include as wide a range of techniques as possible appropriate to the
anticipated biological activity, use, system of administration of the product and duration of
treatment. The determination of the specific activity of highly purified material is of
particular value (units of activity/weight of product).
Sufficient sequence information to characterise the gene product adequately should be

obtained. The degree of sequence verification required will depend on the extent of other
characterisation tests. For some purposes, partial sequence determination and peptide
mapping may suffice, for others full sequence determination may be necessary. Attention
should be paid to the possible presence of N-terminal methionine, signal or leader sequences
and other possible N- and C- terminal modifications (for instance acetylation, amidation or
partial degradation by exopeptidases). Other post-translational modifications, such as
glycosylation should be indicated. Special consideration should be given to the possibility
that such modifications are likely to differ from those found in a natural counterpart and
may influence the biological and pharmacological properties of the product.
3.5.2 Purity
Data should be provided on contaminants which might be present in the final processed
product. The level of contamination considered as acceptable and criteria for acceptance or
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rejection of a production batch should be given. It is important that the techniques used to
demonstrate purity be assessed using as wide a range of methods as possible, including
physico-chemical and immunological techniques. Particular emphasis should be placed on
tests for viral and nucleic acid contamination and for other unwanted materials of host
origin, as well as on materials which may have been added during the production or
purification processes.
3.6 Routine hatch control of hulk product

series of tests may be appropriate as outlined below.
3.6.1 Consistency
An acceptable number of successive batches of the purified cytokine bulk solution should be
characterised as fully as possible to determine consistency of composition. The studies
should include biological, chemical and immunological methods to characterise and assay
the cytokine and methods to detect and identify impurities. Any differences which occur
between batches should be noted. The data obtained from these consistency studies should be
used as the basis for product specification.
3.6.2 Identity
A selection of the tests used to characterise the purified cytokine (see section 3.4) should be
used to confirm the product identity for each batch. The methods employed should include
tests for the anticipated biological activity as well as physico-chemical and immunological
methods. Depending on the extent of other identification tests, sequence verification of a
number of amino acids at the N- and C- terminus or other methods such as peptide mapping
should be performed.
3.6.3 Purity
The purity of each batch should be established and be within specified limits. The analysis
should include sensitive and reliable assays for DNA of host cell origin applied to each
batch of product prepared from continuous lines of transformed mammalian cells. Strict
upper limits should be set for DNA in the product. It is recommended that DNA analyses are
also performed on each batch of product obtained from other eukaryotic cells, and limits set
for DNA content, until further information on safety is obtained. DNA of prokaryotic
expression systems (e.g. of vector or plasmid origin) should be tested for when considering
the quality and safety of the product. For products to be administered chronically, or in high
doses, the residual cellular proteins should also be determined by an assay with appropriate
sensitivity (e.g. ppm) and limits be established for these.
3.6.4 Potency
The potency of each batch of the cytokine product should be established (e.g. units of
biological activity per ml) using, wherever possible, an appropriate national or international
reference preparation calibrated in units of biological activity (see section 3.7).
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In addition, information on specific activity (units of biological activity per unit weight of
product) is of considerable value and should be reported. A highly purified reference
preparation is required to standardise measurements of specific activity (see section 3.7).
It is recommended that correlations between potency measurements, involving biological
tests, and the results of physico-chemical methods of assay are made and the information
reported. If possible, batches should be calibrated using accurate physico-chemical tests, and
the biological assays used to confirm - within stated limits - that the product is biologically
potent.
3.7 Specification and reference materials

The studies described in section 3.5 will contribute to a definitive specification for the
product when considered together with the information obtained from the examination of
successive batches, as indicated under section 3.6.
A suitable batch of the cytokine product, preferably one which has been clinically evaluated,
should be fully characterised in terms of its chemical composition, purity, potency and
biological activity, including where possible full amino acid sequencing, and retained for
use as a chemical and biological reference material.
When appropriate, the biological activity of the cytokine product and its physical
characteristics, including the amino acid sequence, should be compared with that of a highly
purified preparation of the naturally occurring molecule.
4. FINISHED PRODUCT
Where appropriate, the product in final containers should be shown to comply with the
requirements of the relevant European directives. In circumstances where this is not
possible, the omission of tests should be justified by the manufacturer.
The manufacturer shall take samples from each final lot for the following tests.
4.1 Sterility test

Sterility shall be tested according to Ph. Eur. requirements.
4.2 Identity test

Cytokine products shall be identified as the intended cytokine by appropriate methods as
approved by the competent authority. Methods such as immunoassays or neutralisation
assays are useful in this context.
4.3 Potency
The test for potency should utilise a bioassay unless otherwise justified. An appropriate
reference preparation should be tested in parallel. These tests shall be performed on samples
representative of the final filling lots. Statistical analysis of the data should show that the
mean potency value obtained has the confidence limits accepted by the competent authority.
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4.4 Safety tests

Each final lot shall be tested for safety in mice and guinea pigs as required by the European
Pharmacopoeia.
4.5 Test for pyrogenic substances

Each final lot shall be tested for pyrogenic substances. It should be noted that some cytokines
may themselves induce a pyrogenic response in vitro and, where applicable, this should be
taken into account when evaluating the product.
4.6 Test for preservatives/additives

Each final lot shall be tested to determine the levels of preservatives/additives. The tests
used and the permitted concentration shall be approved by the competent authority.
4.7 Moisture content

For lyophilised products, the acceptable level of moisture content per container shall be
approved by the competent authority.
4.8 Colour, clarity and pH

The colour, clarity, and pH of the cytokine solution in the final containers or in the re-
constituted final containers should be approved by the competent authority.
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GLOSSARY
1. Master Cell Bank

A quantity of cells able to produce the intended cytokine stored at -70C or below in aliquots of
uniform composition, one or more of which is used for the production of a Manufacturer's
Working Cell Bank.
2. Manufacturer's Working Cell Bank (MWCB)

A single uniform suspension of cells which have been dispensed in a single working
session into a number of containers which are stored at -70C or below. Cells revived from
one or more of these containers are used as a source of Production Cell Cultures.
3. Production Cell Cultures

The cell cultures derived from one or more containers of MWCB which in the production
process are able to form or are induced to form the intended cytokine.
4. Inducer
A substance added to a cell culture which leads to or stimulates the production of the intended
cytokine.
5. Enhancers
Any substances added to an induced cell culture which improve the final yield of the
intended cytokine.
6. Single Harvest
The biological material prepared from a single production run.
7. Purified Cytokine Solution

The resultant cytokine solution from a single harvest, which has been taken through a
designated purification process.
8. Purified Cytokine Bulk Solution

Either the purified cytokine solution of the resultant of blending two or more batches of
purified cytokine solution.
9. Formulated Cytokine Bulk Solution

The purified cytokine bulk solution to which excipients, e.g. stabilisers, have been added.
10. Final Bulk

The formulated cytokine bulk solution (or the finished biological material) present in the
container from which the final containers are filled.
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11. Final Lot

A collection of sealed final containers that are homogeneous with respect to the risk of
contamination during filling or preparation of the finished product. A final lot must
therefore consist of finished material obtained during one working session from a single
final bulk.
12. Manufacturers Reference Material

Numerous samples from one or more final lots of material which has been shown to be
active in clinical use, or are directly related to such material e.g. purified cytokine bulk
solution, and which has been fully characterised in ways to be specified by the competent
authority, appropriately stored by the manufacturer to serve as a reference material for
several years. For certain critical tests, such reference material shall be included i n
parallel with each lot of production material, which must match the specification of the
reference batch within limits to be agreed by the competent authority.
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Guideline Title Production And Quality Control Of Monoclonal

Antibodies
Date of first adoption See previous titles/other references
This version adopted December 1994
force
Previous titles/other Originally published as two guidelines: Production and
references Quality Control of Human Monoclonal Antibodies (July
1990) and Production and Quality Control of Monoclonal
Antibodies of Murine Origin (June 1987). The previous
reference of the combined version was III/5271/94
Additional Notes This guideline outlines the requirements for murine,
human and engineered monoclonal antibodies for
therapeutic (including ex vivo application) and in v i v o
diagnostic use in humans. It concerns the application o f
Part 2, sections A, B, C, D and E of the Annex to D i r e c t i v e
75/318/EEC as amended with a view to the granting of a
CONTENTS
1. INTRODUCTION
3. SOURCE CELLS
4. CELL LINE PRODUCING THE MONOCLONAL ANTIBODY
5. CELL LINE PRODUCING THE RECOMBINANT MONOCLONAL ANTD30DY
6. CELL BANK SYSTEM
7. CHARACTERISATION OF THE MONOCLONAL ANTIBODY
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8. PROD
U CTION
9. U
P RIFICATION OF THE ANTD30DY
10. THE BU LK FINAL PROCESSED PRODU CT
11. CONSISTENCY AND ROU TINE BATCH CONTROL OF BU LK PROCESSED

PRODUCT
12. SPECIFICATIONS AND REFERENCE MATERIALS
13. MODIFIED MONOCLONAL ANTIBODIES
14. FINISHED PRODU CT AND DEVELOPMENT PHARMACEU TICS
15. PRODU CT EQU IVALENCE
ANNEX I
ANNEX II
ANNEX III (GLOSSARY)
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L INTRODUCTION
In this document the requirements for murine, human and engineered monoclonal
antibodies for therapeutic (including ex vivo application) and in vivo diagnostic use i n
humans are outlined. Monoclonal antibodies intended for use in the purification of other
products should be shown to be pure and free from adventitious agents by the methods
described. Monoclonal antibodies to be used for diagnostic purposes in vitro are not the
concern of this note for guidance.
Monoclonal antibodies are antibodies with a defined specificity derived from cloned cells or
organisms. They can be obtained from immortalised lymphocytes that are cloned and
expanded as continuous cell lines (murine and human monoclonal antibodies) or from
rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies).
Important considerations for the clinical use of monoclonal antibodies include the possible
unintentional immunological cross-reactivity of the antibody with human tissue antigens
other than those desired, and the possible presence of viruses in the products.
11 M o n o c l o n a l a n t i b o d i e s of m u r i n e o r i g i n
Murine monoclonal antibodies are obtained from murine hybridomas produced by fusion of
B-lymphocytes from immunised mice or rats with murine myeloma cells.
A general problem with the therapeutic use of murine monoclonal antibodies in man is the
possible induction of antibodies in the recipient against murine immunoglobulin (human
anti murine antibody or HAMA response). This may result in adverse reactions and limit
the duration of effective antibody therapy. In addition the in vivo half life of murine
monoclonal antibodies is relatively short. It may be prudent to minimise the murine protein
load administered to the patient by the use of a parental myeloma cell lines which does not
itself synthesise immunoglobulin chains.
1.2 Human monoclonal antibodies

The advantages of human monoclonal antibodies over murine monoclonal antibodies are
that human recipients are less likely to develop antibodies against them (although anti-
idiotypic and possibly anti-allotypic antibodies may still be produced) and that human
antibodies are likely to have the full range of biological functions, such as those of the Fc
region which may be species specific. There may be other advantages such as selection of a
subclass of antibody with particular properties.
Murine monoclonal antibodies are almost always prepared using cell lines (hybridomas)
made by fusion of lymphocytes from an immunised donor with myeloma cells. This is not
the case for human monoclonal antibodies as, despite encouraging early reports, there is
still no really satisfactory human myeloma fusion partner. As a result, a major difficulty
with the production of human monoclonal antibodies has been the generation of hybridoma
lines of acceptable stability. It is also difficult in many cases to obtain antigen-primed
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lymphocytes suitable for fusion. In view of this, a number of alternative strategies have been
devised for production of human monoclonal antibodies. These are:
a) Fusion of human lymphocytes (usually peripheral blood or lymph-node derived) with a
murine myeloma or hybrid human-murine myeloma line. This procedure is
essentially similar to the hybridoma technique used to produce murine monoclonal
antibodies, but presents some technical problems in that a lower fusion efficiency is
usually found and human chromosomes are lost preferentially. This procedure may be
regarded as a compromise due to the absence of a suitable human myeloma fusion
partner.
b) Transformation of human lymphocytes with Epstein-Barr virus (EBV). This procedure
has been used for many years to produce continuous, rapidly growing human cells.
c) Fusion of human B-lymphocytes with a human lympho-blastoid B-cell line.
d) Fusion of an EBV-transformed human B-lymphocyte line with a mouse myeloma cell
line.
Other methods for generating stable lines secreting human antibodies may be developed or
exploited in future.
1.3. Engineered monoclonal antibodies

An alternative approach to circumvent the HAMA response, the limited duration of effective
murine antibody therapy and several manufacturing problems in the production of human
monoclonal antibodies is the production of so called chimeric and humanised monoclonal
antibodies using recombinant DNA (rDNA) technology and eukaryotic gene expression
methods. Both types of rDNA-engineered monoclonal antibodies contain human sequences.
In chimeric antibodies the variable heavy and light chain domains of a human antibody are
replaced by those of a rodent (usually murine) antibody, which possesses the desired antigen
specificity. In humanised antibodies only the three short hypervariable sequences
(complementarity determining regions or CDR's) of the rodent variable domains for each
chain are engineered into the variable domain framework of a human antibody producing
mosaic variable regions. Humanised antibodies contain a minimum of rodent sequence.
Suitable cells for expression of the rDNA monoclonal antibody genes are mammalian cell
lines such as immunoglobulin non-producing myeloma cell lines, that are capable of high-
level expression of exogenous heavy and light chain genes and the glycosylation,
assemblage and secretion of functional antibodies.
Engineered monoclonal antibodies may have the advantages of decreased immunogenicity,
enhanced in vivo circulating half life in combination with optimised specificity and effector
functions.
Certain aspects of the control requirements likely to apply to rDNA derived chimeric and
humanised monoclonal antibody usage will be similar to those already described for
products derived by rDNA technology (Note for guidance Production and Quality Control of
Medicinal Products derived by rDNA Technology) with which the applicants should be
familiar. These control requirements concern e.g. status of the rDNA within the host cell,
expression regulation and stability of the expression system and the purification procedure.
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Several of the requirements relating to establishments in which biological products are
manufactured (e.g. WHO technical Report series 822, 1992: Annex 1 Good Manufacturing
Practices for Biological Products) apply to the manufacture of monoclonal antibodies.
Additional information can be found in WHO technical Report Series 822, 1992: Annex 3
Guidelines for Assuring the Quality of Monoclonal Antibodies for use in Humans.
Manufacturers should also refer to the EU Guide for Good Manufacturing Practice for
Medicinal Products. Attention is drawn to the following points.
2.1. Production process

Many of the general requirements for the quality control of biological products, such as
potency, abnormal toxicity testing, freedom from contaminants, stability and freedom from
detectable levels of antibiotics will apply to monoclonal antibodies. It is undesirable to use
agents which are known to provoke sensitivity reactions in certain individuals such as, for
example, penicillin or other beta-lactam antibiotics.
2.2. Biological materials used in the production

Any reagents of biological origin (e.g. sheep erythrocytes, foetal calf serum, bovine serum
albumin, human transferrin, insulin, trypsin) used in the generation of the monoclonal
antibody producing cell line and/or during routine production, should be free of microbial
contamination such as mycoplasma, fungi and bacteria. Special consideration should be
given to possible viral contamination and tests for relevant viruses should be performed, e.g.
trypsin should be tested for porcine parvovirus.
Bovine sera should be checked and found negative for potentially dangerous viruses (at least
bovine diarrhoea virus, infectious bovine rhinotracheitis and parainfluenza 3). In addition,
bovine sera and other bovine derived biologicais should comply with the requirements in the
note for guidance Minimising the Risk of Transmitting Agents causing Spongiform
Encephalopathy via Medicinal Products.
The following points, set out below, should de considered.
3. SOURCE CELLS
Whenever possible, murine tissue and animals used as source materials should be shown to
be free of viruses as indicated in Annex I (a), table 2.
Monoclonal antibodies obtained from human cells present particular concerns regarding
safety. Human monoclonal antibodies for use in humans are currently unique in that they
are often derived from cells which are likely immortalised by the deliberate introduction of
EBV, a potential human pathogen. They are likely to be obtained from a transformed human
cell line which is potentially oncogenic. Evidence of contamination with viruses originating
from the donor is cause for concern, as they will by definition be viruses capable of infecting
humans. Cells from human origin should be shown free of viruses indicated in Annex I (a)
table 3.
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3.1 C h a r a c t e r i s a t i o n of n o n - s p e c i f i c c e l l s
3.1.1 Feeder cells
Whenever appropriate, the origin of feeder cells used should be defined. Feeder cells should
be derived from SPF (specific pathogen free) animals or cell seed stocks shown to be free of
microbial contamination such as mycoplasma, bacteria and fungi and special consideration
should be given to possible exogenous viral contamination.
3.1.2 Fusion partner(s)

The fusion partner used (e.g. myeloma, human lymphoblastoid B-cell line) should be fully
described and documented. The source, name and characteristics of the parental cell line
should be given. It should be shown that the cell line is a pure culture and is not
contaminated with cells of other types. If possible the cell line used as fusion partner should
be selected as one which does not synthesise any immunoglobulin chains. Cryopreserved
samples of the cell line used as fusion partner should be retained in case retrospective
investigations become necessary.
3.1.3 Host cell for the expression of the recombinant monoclonal antibody
A description of the starting host strain or cell line should be provided including the history
of the strain or cell line, its identification characteristics and potential viral contaminants.
Special attention should be given to the possibility of unintended cross-contamination with
other cell lines or viruses not endogenous to a particular cell line.
The cell line used should not synthesise any endogenous immunoglobulin chains before and
after transfection.
Cryopreserved samples of the host cell line should be retained in case retrospective
investigations become necessary.
3.2 G e n e r a t i o n a n d c h a r a c t e r i s a t i o n of t h e specific p a r e n t a l cell

(murine and human monoclonal antibodies)
The source of the immune parental cells should be documented. If an immunogen has been
deliberately used, information on its source and preparation and on the immunisation
procedure should be provided.
If the immune parental cells are derived from a human donor, information concerning the
health of the donor should be provided. Any relevant clinical data on the donor must be
reported, especially data on possible virological infections. Preferably, the description of the
state of health of the donor should cover a period of some months before and after derivation
to establish that blood borne viruses such as HIV, hepatitis and hepatitis C were not in the
process of incubation. If these conditions can not be completely fulfilled, this should be
justified and it should be demonstrated that the cell bank system is devoid of any relevant
viruses (e.g. HIV 1/2, HBV, HCV).
For the production of monoclonal antibodies of major therapeutic value it may be necessary
to use cells potentially contaminated by a virus. In such a case, it will be necessary to look at
the possible detection of the virus in the cell bank and to add one or more steps dedicated to
inactivate this virus in the processing of monoclonal antibody.
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4. CELL LINE PRODUCING THE MONOCLONAL ANTD30DY

4.1. Generation of the cell line
A complete description of the production of the cell line secreting the monoclonal antibody
should be provided including details of cell fusion, EBV transformation and cloning
procedures where appropriate. Sufficient data should be given to allow an assessment of the
efficiency of the cloning procedure.
Agents used in the fusion and selection procedure should be described (e.g. PEG).
4.2. Characterisation of the cell line

The characteristics of the monoclonal antibody producing cell line should be detailed. These
should include the specificity, class and, where appropriate, subclass of the immunoglobulin
secreted, together with any distinguishing features, such as isoenzyme/immunochemical
markers. The production of immunoglobulin chains originating from the fusion partner
should be determined. The antibody secretion should be stable in respect to both the type of
antibody (class switch) and level of expression up to and beyond the population doublings
used for routine production. Appropriate precautions should be taken to avoid cross-
contamination with other cells.
5. CELL LINE PRODUCING THE RECOMBINANT

MONOCLONAL ANTIBODY
5.1 Cloning and characterisation of the DNA coding for the non-
specific part of the recombinant mAb
For both chimeric and humanised monoclonal antibody a description of the origin, isolation
and cloning strategy of the heavy and the light chain genes should be provided. In addition
the following information is required:
i) the introduced framework residue substitutions in humanised monoclonal antibodies
to improve the CDR conformation (where applicable).
ii) a justification of the choice of the immunoglobulin isotype.
iii) a characterisation of the human constant domain genes (e.g. by restriction
endonuclease maps).
5.2 Selection, cloning and characterisation of the DNA coding for

the specific part of the recombinant mAb
The origin of the hybridoma cell line and the characteristics of the rodent monoclonal
antibody used should be described.
A description of the cloning of the rodent heavy and light chain variable region genes from
the hybridoma cell line and the characterisation of the coding regions of the cloned genes
should be provided. For humanised monoclonal antibodies a description of the identification,
the method of isolation, either by cloning or synthesis, and the characterisation of the rodent
CDR genes for both heavy and light chain should be provided.
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5.3 Construction of the gene coding for the recombinant mAb

A description of the strategy followed either to join the rodent variable fragment to the
human constant region, or, in the case of humanised antibody, to insert the rodent CDR
genes into the human framework region sequences, is required. This documentation should
include:
i) cell lines and vectors used in the generation of the monoclonal antibody, and a
description of the expression vectors used for the transfection of rDNA antibody genes
into the mammalian host cell line, including the origin, structure and selection
markers.
ii) for both the heavy and light chain expression vectors the nucleotide sequences of the
genes of interest and the flanking control regions. A detailed map indicating the
regions which have been sequenced during construction and those deduced from the
literature should be given. All the junction regions created by ligation during
construction should be confirmed by sequencing.
iii) a clear identification of all known expressed sequences.
iv) an indication of any additional modifications.
5.4 Generation of the cell line expressing the recombinant

monoclonal antibody
In addition to the documentation concerning the starting host strain or cell line, the
following information is required:
i) the methods used for introducing the vector into the host cell, the selection and cloning
of the transformants.
ii) the status of the vectors within the host.
iii) a detailed study using various restriction enzymes and Southern blot analysis
providing convincing data on the integrity in the host cell. Useful information is
provided on the expression system by Northern blot analysis.
iv) a detailed description of the strategy by which expression of the relevant gene is
promoted and controlled (during production).
5.5 Genetic stability

The stability of the host/vector genetic and phenotypic characteristics should be investigated
up to and beyond the population doubling level or generation number expected during full
scale production. Such stability studies should provide detailed information on:
i) gene copy number in relation to productivity of the culture.
ii) characterisation of the monoclonal antibody. Analysis at the protein level and/or DNA
level can be envisaged. Whichever method is used, it should be validated and the
detection limit should be given.
iii) the level and consistency of expression of both the heavy and light chain.
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6. CELL BANK SYSTEM

6.1 E s t a b l i s h m e n t of t h e cell b a n k s y s t e m (MCB a n d MWCB)
It is essential that production is based on a well defined cell bank system. This will
normally involve a Master Cell Bank (MCB) and a Manufacturer's Working Cell Bank
(MWCB). During the establishment of the cell bank no other cell lines should be handled
simultaneously in the same laboratory suite or by the same persons. The origin, form,
storage, use, and details of life expectancy at the anticipated rate of use must be described i n
full for all cell banks. New working cell banks should be fully characterised.
Samples of the working cell bank should be retained until at least after the expiry date of the
resulting final lot.
6.2 C o n t r o l of v i r o l o g i c a l a n d m i c r o b i a l c o n t a m i n a t i o n
The various cell levels, including MCB, MWCB and PPCB (Post Production Cell Bank; see
6.5) should be examined for adventitious agents (viral, bacterial, fungal or mycoplasmal).
Special attention should be given to viruses which can commonly contaminate the species
from which the cell line has been derived. Appropriate tests to demonstrate the absence of
virus contamination as described in Annex I should be performed.
Certain cell lines contain endogenous viruses, e.g. retroviruses, which may not readily be
eliminated. Furthermore, potential viral contamination may take the form of complete viral
genomes or subgenomic fragments resulting in the expression of infectious viral particles.
Therefore the possibility of mutations of endogenous viruses during prolonged culture should
be considered. The presence of sequences from viral genomes may not disqualify use of the
cells, but any exogenous viral nucleic acid found should be identified. If the
heterohybridoma approach is used for construction of the antibody secreting cell line the cell
bank should be examined for the presence of murine and human viruses.
A cell line which produces any infectious virus capable of infecting human cells would be
acceptable only in exceptional circumstances. All products derived from such lines would
have to be considered on a case by case basis. If the cell line secretes infectious virus,
appropriate precautions should be taken to protect personnel involved in production from
infection.
There is special concern about the use of cell lines transformed by the deliberate introduc
tion of EBV for the production of human monoclonal antibodies. Despite the fact that EBV
transformed human cells in general do not secrete viral particles these cells contain
complex copies of the viral genome and EBV sequences should be sought by PCR or by co-
cultivation with suitable indicator cell lines.
6.3 Characterisation
A critical part of quality control will involve the full characterisation of cells. The identity
of the cells should be established by distinguishing markers of the cell, such as specific
isoenzyme and immunological features, and phenotypic characterisation.
If the EBV transformation procedure is used alone for the generation of a cell line for the
production of human monoclonal antibodies difficulties can arise in the cloning procedure.
It is therefore essential that manufacturers show convincing evidence that the cell line i s
monoclonal.
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6.4 Secretion of cytokines

Manufacturers should be aware that lymphocytes and/or feeder cells can secrete a number of
biological mediators which have diverse functions and may cause adverse effects when
administered to humans. Consideration should be given to the ability of the production
process to remove immune mediators such as interferons and other cytokines.
6.5 Establishment of the post production cell bank

For validation purposes a post production cell bank should be established. For single harvest
production 10 or more population doublings beyond the maximum population doubling level
used for routine production should be used. For multiple harvest production at a time which
exceeds the total length of the cultivation period by one third is suggested.
7. CHARACTERISATION OF THE MONOCLONAL ANTIBODY

The monoclonal antibody should be characterised thoroughly. This characterisation must
include both biochemical/physico-chemical and biological/immunological properties of the
antibody. In addition the specificity and crossreactivity of the monoclonal antibody should be
assessed.
7.1 Biochemical/physico-chemical characterisation

The biochemical/physico-chemical properties of the antibody should be described in detail. At
least the following parameters should be determined: class, subclass (when appropriate) and
light-chain composition, molecular weight and either N- and C-terminal amino acid
sequence, secondary and tertiary structure.
7.2 Biological/immunological characterisation

The immunological properties of the antibody should be described in detail. Therefore the
biological/immunological characterisation should include: antigenic specificity including
the characterisation of the epitope the antibody recognises, binding capacity, ability for
complement binding and activation, cytotoxic properties, antibody dependent cytotoxicity,
ability to modify relevant antigens, capacity to stimulate immunocompetent cells and the
ability to induce secretion of cytokines or other mediators.
7.3 Specificity and crossreactivity

The analysis should include the determination of unintentional reactivity with or
cytotoxicity for human tissues distinct from the intended target, and cross-reactivity with a
range of human tissues (listed in Annex II) by immunohistochemical procedures.
8. PRODUCTION
In vitro production is the preferred method of production. During the last few years the
technology for in vitro production of monoclonal antibodies has been considerably improved.
In vitro production of monoclonal antibodies offers a high degree of control and
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standardisation and has important advantages over in vivo production with respect to viral
safety, consistency of production and the absence of contaminant immunoglobulins in the
crude harvest. In vitro production in serum free medium is also now feasible. Another
advantage of this method of production is the considerable reduction of animal usage.
Manufacturers should be aware of Directive 86/609/EEC concerning the protection of
animals used for experimental and other scientific purposes.
If in vivo production is chosen it must be justified by the manufacturer.
A clear definition of a "batch" of product for further processing should be provided. A
production batch should normally originate from a fresh ampoule of the MWCB. Details of
the culture with the in-process controls should be provided. Criteria for rejection of the
harvests and premature termination of the culture should be defined.
8.1 In vitro production

For each production run, the presence, extent and nature of any microbial contamination i n
the culture vessels immediately prior to all harvesting must be thoroughly examined.
Detailed information to confirm the adequate sensitivity of the methods used to detect
contamination should be provided and acceptable limits of contamination set. The bulk
culture fluid should be shown to be free from mycoplasmal, mycotic and bacterial
contamination and should be tested for the presence of viruses using a general test
involving inoculation into suitable cell substrates (see Annex I, b).
The composition and source of the cell culture medium used for production should be
recorded. If animal serum-derived additives are used, they should be shown to be free from
adventitious agents (See 2.2.).
production area. If other cell lines are cultivated in parallel, records must be kept of the cell
lines handled and evidence presented for the absence of cross contamination between them.
8.1.1 Single harvest production

The maximum permitted generation number for production should be defined and should be
based on information concerning the stability of the cell line or the up to and beyond the
level of production. Data on consistency of growth of the culture and on the maintenance of
yield within specified limits should be presented. Appropriate monitoring of the cell line
characteristics at the end of the production cycles should also be undertaken. Evidence
should be provided that the yield does not vary beyond defined limits and that the nature and
quality of the product does not change with respect to specific parameters.
8.12 Multiple harvest production

The period of continuous cultivation should be specified and this should be based on
information concerning the stability of the system and consistency of the product up to and
beyond this limit. Monitoring of the production system is necessary throughout the life of the
culture. The required frequency and type of monitoring will depend upon several factors
including the nature of the expression system and monoclonal antibody, as well as the total
length of the period of continuous cultivation undertaken. The acceptance of harvests for
further processing should be clearly linked to the schedule of monitoring applied. Evidence
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should be provided that the yield does not vary beyond defined limits and that the nature and
quality of the monoclonal antibody does not change with respect to specific parameters.
8.2 In vivo production

In vivo production should comply to the additional requirements set below.
82.1 Characterisation of the animals used

The strain and origin of the animals used for production should be specified, together with
their genotype and age. They should be from a closed, specific pathogen-free (SPF) colony
which is routinely monitored for those viruses listed in Annex I Table 2. The long term
records of the breeding colony in respect of freedom from viral contamination should be
considered in relation to the reliability of maintenance of the colony. Evidence should also
be presented that animals are maintained under SPF conditions at all times during transfer
and use.
8.2.2 Harvest and handling of ascitic fluid

Each production batch should originate from a fresh ampoule of the MWCB. The maximum
permissible number of serial passages in vivo during normal production should be defined
and restricted: justification of this limit should include information concerning the yield of
monoclonal antibody and the stability of the hybridoma characteristics on in vivo passage up
to beyond that used in production. Indefinite passage in animals is not acceptable. A scheme
of priming, inoculation and harvesting should be provided.
The number of animals and the procedure used to prepare the bulk ascitic harvest should be
given in detail. Full details should be provided on any substances used to pre-treat mice or
rats to facilitate growth of hybridomas. Description, volume and concentration of cell
inoculum should be given. Data concerning the titre of the antibody in and storage
conditions of the bulk ascitic fluid should be provided (e.g. temperature, duration, details of
any proteolytic enzyme inhibitors added). Particular attention should be paid to the degree
and nature of any microbial contamination (bacterial, mycotic and mycoplasmal) in the
bulk ascitic fluid. Testing procedures capable of detecting all of the murine viruses listed i n
Annex I Table 2 should be performed, as indicated in Annex 1(a) and (b), on at least the first
five bulk harvests of the product. However, it may be expected that general testing methods
for viruses may be sufficient as experience of production is gained. Consequently, after the
first five production runs, general testing for viruses, limited to those described in Annex I
(b), may be considered adequate. Any infectious agent should be identified and tests for
viruses in Group I Table 2 should be negative. If the source of mice is changed to a different
colony or supplier, tests described in Annex 1(a) should be performed on at least the first five
bulk harvests to re-establish consistency of freedom from contaminant agents.
8.3 Virological aspects: in-process controls

The bulk harvest should be tested for the presence of viruses using a general test involving
inoculation into suitable cell substrates as described in procedures given in Annex I (b).
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9. PURIFICATION OF THE ANTIBODY

9.1 Methods
Methods used to purify the product and their in-process controls including their specification
limits should be described in detail, justified and validated. It is important to ensure that
purification procedures do not impair relevant immunobiological features of the
immunoglobulin. Procedures which make use of affinity chromatography, for example
employing monoclonal antibodies, should be accompanied by appropriate measures to ensure
that these substances, or any additional potential contaminants arising from their use, do
not compromise the quality and safety of the final product. Cross reference is made to the
note for guidance Virus Validation Studies: The Design, Contribution and Interpretation of
Studies Validating the Inactivation and Removal of Viruses.
The criteria for reprocessing of any intermediate or final bulk should be carefully defined,
validated and justified.
Consideration should be given to incorporating procedures which inactivate/eliminate
potential viral contaminants where such methods will not compromise the biological activity
of the product.
9.2 Validation of the purification

nucleic acids, carbohydrates, viruses and other impurities including product-related proteins
should be investigated thoroughly. Any inactivation process used should be shown to be
effective and not compromise the biological activity of the product. The reproducibility of the
purification process with respect to its ability to remove specific contaminants, should also be
demonstrated. Studies using, for example, a carefully selected group of viruses which exhibit
a range of physico-chemical features relevant to their behaviour on purification (see note for
guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies
Validating the Inactivation and Removal of Viruses), host-cell proteins, other potential
impurities derived from the production process (e.g. heavy or light chain immunoglobulin
fragments) and DNA intentionally mixed with the crude preparation (spiking) should be
undertaken. The choice of the nucleotide probe to detect DNA contamination should be
relevant to the system used. A reduction factor for such contaminants at each stage of
purification, and overall, should be established by using, if necessary, concentrations of
viruses, host cell proteins, other potential impurities and DNA in excess of that expected
during normal production.
Where a cell line contains viral subgenomic fragments (see section 6.2) consideration
should be given to using appropriate viral nucleic acid in DNA spiking studies. Where a
hybridoma line has been established by transformation with Epstein-Barr virus, specific
EBV sequences should be sought by sensitive techniques such as the polymerase chain
reaction.
Validation of the purification process should also include justification of the working
conditions such as column loading capacity, column regeneration and sanitisation and
length of use of the columns.
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10. T h e b u l k final p r o c e s s e d p r o d u c t
10.1 T h e monoclonal a n t i b o d y
Rigorous characterisation of the purified monoclonal antibody by chemical and biological
methods will be essential. At least the following parameters should be determined: class,
subclass and light-chain composition, glycosylation pattern, integrity of the molecule by
analysis of the ratio heavy/light chain, microheterogeneity, molecular weight, N- and C-
terminal amino acid sequence, and secondary and tertiary structure of the antibody. With
increasing experience, th tests for subclass, light chain composition, N- and C- terminal
amino acid sequence and secondary and tertiary structure could be omitted. The total protein
content, the degree of aggregation and molecular fragmentation of the immunoglobulin
should be determined. Appropriate specifications for these parameters, with acceptance
limits, should be set. Especially for engineered and humanised antibodies sufficient
sequence information to characterise the gene product adequately should be obtained by
peptide mapping or amino acid sequencing.
Particular attention should be given to use a wide range of analytical techniques exploiting
different physico-chemical properties of the molecule. Examples of suitable techniques are:
SDS-polyacrylamide gel electrophoresis under reducing and non reducing conditions,
isoelectric focusing, column chromatography (including HPLC), peptide mapping, amino
acid analysis, circular dichroism and carbohydrate mapping. The manufacturer should
provide clear photographs of the gels, etc..
The immuno-reactivity of the antibody should be assessed. The specific activity of the
purified monoclonal antibody should be determined (units of activity/weight of product).
A clear difference should be made between the analytical tests performed during
development, in order to fully characterise the monoclonal antibody and tests performed
routinely on each batch of purified bulk product. Quality control tests should be carried out
routinely on each batch of purified bulk product according to the Guide to GMP.
10.2 P u r i t y
Data should be provided on contaminants whose presence is anticipated in the final
processed product. The level of contamination considered as acceptable should be justified,
and criteria of acceptance or rejection of a production batch should be given. It is important
that the techniques used to demonstrate purity be assessed using as wide a range of methods
as possible, including physico-chemical and immunological techniques. These should
include tests for viral and cellular nucleic acid and protein contamination of parental,
hybridoma, or host cell origin, as well as on materials derived from tissue culture medium
or materials which have been added during the production or purification processes.
Measurements of total protein and cellular DNA concentrations, specific activity,
microbiological and chemical purity should be reported for the final product. Assays of
endotoxin level should also be carried out.
10.3 A d v e n t i t i o u s a g e n t s
The final bulk product should be shown to be free from bacterial, fungal and mycoplasmal
contamination. Evidence should be presented to show that any viral contaminant known to
be possibly present in the bulk harvest has been eliminated or inactivated (see Annex I).
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IL CONSISTENCY AND ROUTINE BATCH CONTROL OF BULK

PROCESSED PRODUCT
series of tests may be appropriate as outlined below.
111 Consistency
Evidence should be provided on the consistency of production, for example on at least five
consecutive full scale production batches. This should include information on the bulk
harvest and final dosage form as well as on in-process controls. In the case of a production
where multiple harvests are applied, batches from different fermentation runs are needed.
The studies should include biological, chemical and immunological methods to characterise
and assay the monoclonal antibodies and methods to detect and identify impurities. Any
differences which occur between batches should be noted.
112 Routine batch control analysis

112.1 Identity
A selection of the tests used to characterise the purified monoclonal antibody should be used
to confirm the product identity for each batch. The methods employed should include tests for
the biological activity as well as physico-chemical and immunological methods. Engineered
antibody should be subjected to sequence verification of the peptide backbone by adequate
methods such as peptide mapping.
11.2.2 Purity
the nature and intended use of the product, method of its production and purification and also
the degree of consistency of the production process. The purity of each batch should be
established and be within specified limits. For engineered monoclonal antibodies the
analysis should include sensitive and reliable assays for DNA of host cell origin and the
vectors applied to each batch of product. Strict upper limits should be set for DNA in the
product.
The product should be shown to be free from microbial contamination. Evidence should be
presented to show that any viral contaminant known to be present in the bulk harvest has
been eliminated or inactivated (see Annex I). Pyrogenicity should be tested for.
Particular attention should be given to assessment of the degree of aggregation or molecular
fragmentation of the immunoglobulin. All possible steps should be taken to prevent
aggregation. Limits for the presence of oligomeric immunoglobulin molecules should be
justified.
11.2.3 Test for potency

When appropriate, the biological activity of the monoclonal antibody should be established by
a biological assay. In addition information on specific activity will be of considerable value
and should be reported. A fully characterised reference preparation is required to
standardise measurements of specific activity (see section 13).
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12. SPECIFICATIONS AND REFERENCE MATERIALS

The studies described in section 10 will contribute to a definitive specification for the product
when justified by the information obtained from the examination of successive batches and
results of batch analysis, as indicated in section 11.
The reference preparation should be produced from a suitable batch of the product, which has
been clinically evaluated and fully characterised in terms of chemical composition, purity,
potency and biological activity. Criteria for establishing the reference preparation and
criteria for re-testing and prolongation of the shelf life should be stated.
13. MODIFIED MONOCLONAL ANTIBODIES

The preparation of sub-fragments of the antibody (Fab or F(ab')2 fragments) may have
advantages for some applications. Where such fragments are preferred for clinical use,
their molecular and antigenic properties should be defined. Appropriate analytical tests
should be performed. Specified limits for impurities such as fragments other than those
desired or intact immunoglobulin, should be defined. Specifications, with limits, should be
given for each contaminant (e.g. residual levels of enzymes used, such as pepsin or papain),
specific activity, immunoreactivity, and antigen cross-reactivity. A reference batch should
be prepared and all assays should be validated.
The therapeutic and diagnostic uses of monoclonal antibodies and antibody subfragments
can sometimes be enhanced by chemical modifications (e.g. radiolabelling, conjugation
with a toxin, attachment to specific substances for "targeting" or chemically linking of two
antibody molecules or their derivatives to generate a bispecific antibody). For these a
detailed description of their preparation and purification should be supplied. Each relevant
step in the production process requires validation and quality control covering source
materials, limits for impurities arising from the production process, evidence for
consistency etc. Modifications can change the properties of the monoclonal antibody and
general requirements for such products must include information concerning the biological
half-life of the antibody, of the medicinal product or toxin, and also of the conjugate after
injection into a recipient. Information about the specificity, the toxicity and stability of the
conjugate should also be supplied.
Criteria and specification limits for purity and potency of the final product should be applied
and immunoreactivity and antigen cross-reactivity should be determined. Additional
specific control procedures may be required, but these are dealt with best on a case by case
basis.
The preparation of a reference batch is required and all assays should be validated.
Detailed information for the production of radiolabelled monoclonal antibodies can be found
in the note for guidance Radiopharmaceuticals based on Monoclonal Antibodies.
14 FINISHED PRODUCT AND DEVELOPMENT

PHARMACEUTICS
The development of the formulation should be described in detail and justified, particularly
with regard to the presence and amount of stabilisers such as albumin and/ or detergents.
The product in final containers should be shown to comply with the requirements of the
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European directives and pharmacopoeias. In circumstances where this is not possible the
omission of tests should be justified by the manufacturer.
15. PRODUCT EQUIVALENCE

Some changes or adaptations in the production of a monoclonal antibody during clinical
development or subsequent to product approval can lead to an altered form of the antibody
with identical specificity. Examples of such changes are: transition of in vivo production to
in vitro production, changes in culture procedures or culture conditions, changes i n
purification procedure, or additional modifications of the monoclonal antibody molecule. In
these cases, studies to prove product equivalence should be performed to show that both,forms
of the antibody are essentially identical.
In all cases these studies should include a complete physico-chemical and biological
characterisation of both antibodies.
15.1 I n v i t r o s t u d i e s o n p r o d u c t e q u i v a l e n c e
The physico-chemical characteristics of the monoclonal antibody, like isotype, subclass,
microheterogeneity, molecular weight, primary structure, secondary structure, glycosylation
pattern, structural integrity, should be determined. The biological characterisation should
include immunoreactivity and crossreactivity, the determination of relevant functional
characteristics and binding studies to determine affinity.
When there are changes in the cell culture procedure/conditions without changes in the
MCB, relevant parameters such as morphology, cell growth, viability, isoenzymes, and
stability of production should be analysed.
152 In vivo studies on product equivalence

The decision on the selection of in vivo tests depends on the results of the analytical
characterisation. In the case of identical analytical results of both forms of antibody, at least
the pharmacokinetic, biodistribution and half life should be determined.
15.3 Clinical studies

When both monoclonal antibodies are demonstrated to have identical physico-chemical,
biological and pharmacological characteristics, clinical studies performed with the former
monoclonal antibody can be accepted. However, an essential prerequisite is that the
production is based on the same MCB. Otherwise, clinical trials have to be carried out with
the second form of antibody.
15.4 Manufacturing procedure

Consistency of the manufacturing procedure of the monoclonal antibody, including
validation of the production process and quality control in accordance to the requirements
should be demonstrated.
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ANNEX I
Testing for viruses should be performed in laboratories with experience in routine virus
testing and should be performed in accordance with good laboratory practice.
Table 1 lists the tests for viruses to be performed at the different stages of production.
Table 2 lists viruses which should be considered as potential contaminants in the
manufacture of monoclonal antibodies produced by cell lines of murine origin.
Table 3 lists viruses which should be considered as potential contaminants in the
manufacture of monoclonal antibodies produced by cell lines of human origin.
Testing for viral Contamination

a) Tests for detection of specific viruses
(i) Monoclonal antibodies produced by cell lines of murine origin
Tests for detection of specific viruses listed in table 2, for example Mouse
Antibody Production (MAP) or Rat Antibody Production (RAP) tests or other tests
of at least equivalent sensitivity and reliability. Additional specific tests may
need to be carried out for lymphocytic choriomeningitis virus (LCMV), mouse
cytomegalo virus, mouse rotavirus (EDIM), thymic virus and lactic
dehydrogenase virus. Tests capable of detecting murine retrovirus should be
included, for example the XC plaque assay or the S+ L- focus assay for the
detection of ecotropic or xenotropic retrovirus respectively.
(ii) Monoclonal antibodies produced by cell lines of human origin
For human monoclonal antibodies the viruses which may be found in the cell
line depend to some extent on the nature and health of the donor. They may be
specifically able to infect lymphocytes. As a minimum, the viruses which are
known to persist in lymphocytes and are listed in table 3 should be tested for.
Viruses should be sought by culture methods employing cell lines including
virus free lymphoblastoid cells as well by examination of the lymphocyte line
itself by use of immunochemical procedures, electron microscopy, Southern
blotting, polymerase chain reaction or other sensitive techniques.
(iii) Engineered monoclonal antibodies produced by mammalian cell lines
For engineered monoclonal antibodies the viruses which may be found in the
cell line depend on the origin of the cell line. Relevant viruses should be tested
for.
b) Inoculation of cell cultures capable of detecting a wide range of murine, human, and,
if relevant, bovine viruses. Examples of useful cell types (substrates) are: murine
fibroblast cultures, e.g. mouse embryo cultures; human fibroblast cultures, e.g. human
diploid cells such as MRC5; continuous cell lines of human, murine and bovine
origin. The indicator cell lines should additionally be tested for haemadsorbing
viruses (with erythrocytes from human blood group O, guinea pig, chicken) at the end
of the observation time. Tests for retroviruses should be included.
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c) Tests in animals for adventitious agents should include the inoculation by the
intramuscular route of each of the following groups of animals with the test material
or with disrupted cells from the seed lot propagated beyond the maximum level (or
population doubling, as appropriate) used for production:
- 2 litters of suckling mice, comprising at least 10 animals less than 24 hours old
- 10 adult mice
- 5 guinea-pigs
Test material should be injected intracerebrally into each of 10 adult mice.
The animals should be observed for at least 4 weeks. Any animals that are sick or show any
abnormality should be investigated to establish the cause of illness. Test material can be
considered to be suitable for production if at least 80 % of the animals inoculated remain
healthy and survive the observation period and none of the animals shows evidence of the
presence in the tested material of any adventitious agent.
Fertilised eggs may also act as useful substrates. Test material should be injected into eggs
by appropriate routes, the chorioallantoic membrane, amniotic cavity and yolk sack of each
of 10 embryonated chicken eggs, 9-11 days old. The embryonated eggs should be examined
after not less than 5 days incubation. The allantoic fluids should be tested with guinea-pig
and chick or other avian red cells for the presence of haemaglutinins.
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TABLE 1
TESTING SCHEME FOR VIRAL CONTAMINANTS
Annex I sections which are applicable
MCB or MWCB (a) (b) (c)

Mouse breeding Colony (a)
Ascitic fluid harvest (a)* (b)
In vitro bulk harvest (b)
Bulk final processed product Specified tests of (b) if virus contamination was detected i n
the bulk harvest
* It is proposed that these tests should be carried out on at least the first five production
runs.
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TABLE 2
MURINE VIRUSES
Group Virus Species Affected

I Hantavirus (Haemorrhagic fever with renal syndrome)* M, R
Lymphocytic choriomeningitis virus (LCMV) * M
Rat rotavirus * R
Reovirus type 3 (reo 3)* M, R
Sendai virus* M, R
II Ectromelia virus* M
K virus M
Kilham rat virus (KRV) R
Lactic dehydrogenase virus (LDH) M
Minute virus of mice (MVM) M, R
Mouse adenovirus (MAV) M
Mouse cytomegalovirus (MCMV) M
Mouse encephalomyelitis virus (MEV, Theiler's or GDVII) M
Mouse hepatitis virus (mhv) M
Mouse rotavirus (EDIM) M
Pneumonia virus of mice (PVM) M, R
Polyoma virus M
Rat cornavirus (RCV) R
Retrovirus* M, R
Sialodacryoadenitis virus (SDA) R
Thymic virus M
Toolan virus (HI) R
M - mouse
R-rat
Viruses for which evidence exists of a capacity to infect man or primates are to be found i n
Group I. Those viruses for which there is no evidence of infection in man but which could
nevertheless pose a potential danger, for example in immunocompromised individuals, are
listed in Group II. Viruses which are known to be capable of replicating in vitro in cells of
human and monkey origin are indicated by * in Table 2.
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TABLE 3
HUMAN VIRUSES
Virus
Human Immunodeficiency Virus (Type 1, Type 2)

Human cell Leukaemia Virus (Type I, Type II)
Cytomegalo virus
HHV6
Epstein - Barr virus
Hepatitis virus
Hepatitis C virus
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ANNEX II
Suggested list of human tissues to be used for immunohistochemical or cytochemical

investigations of cross reactivity of monoclonal antibodies. This list should reflect the
specificity of the antibody and its particular use.
Tonsil, thymus, lymph node

Bone marrow, blood cells
Lung, liver, kidney, bladder, spleen, stomach, intestine
Pancreas, paratid, thyroid, para-thyroid, adrenal, pituitary
Brain, peripheral nerve
Heart, striated muscle
Ovary, testis
Skin
Blood vessels
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ANNEX III
Glossary
1. Murine
"Murine" means derived from an animal belonging to the M uridae family which includes
mice and rats.
2. Cell Banks
a) M aster cell bank (M CB)
A homogeneous suspension of the original cells on which production is based, aliquoted into
individual containers for storage (e.g. in a liquid nitrogen refrigerator). The original cell
line may not necessarily have been produced by the manufacturer.
For engineered products the cells in the master cell bank are already transformed by the
expression vector containing the desired gene. In some cases it may be necessary to esta
blish separate master cell banks for the expression vector and the host cells.
b) M anufacturers working cell bank (M WCB)
A homogeneous suspension of cells derived from the master cell bank(s) by a finite passage
level, aliquoted into individual containers for storage (e.g. in a liquid nitrogen
refrigerator).
In both cell banks, all containers are treated identically during storage, and once removed
from storage, the containers are not returned to the cell bank stock.
c) Post production cells (PPC)
Post production cells are the cells cultured up to 10 or more population doublings beyond the
maximum population doubling level used for routine production (single harvest production)
or cells cultured for a period of time which exceeds the total length of the cultivation period
by one third (multiple harvest production).
3. Production Method
a) Production at finite passage (single harvest)
This cultivation method is defined by a limited number of passages or population doublings
which must not be exceeded during production.
b) Continuous culture production (multiple harvest)
The number of population doublings are specified based on information concerning the
stability of the system and the consistency of the product criteria for the termination has to be
defined by the manufacturer.
4. Bulk Harvest
This is a homogeneous pool of individual harvests, lysates or ascitic fluids which is
processed in a single manufacturing run.
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5. Bulk Final Active Substance

This is the final product, after completion of the production process, obtained from a bulk
harvest. It is maintained in one or more containers and used in the preparation of the final
dosage form. The generation of this final batch has to be clearly defined and unambiguously
recorded by the producer.
6. Finished Product
The active substance is formulated and filled into final, sealed containers which hold the
product in its final dosage form, i.e. the finished product. The containers of a filling lot are
processed together and uniform in their contents and biological potency.
7. Engineered Monoclonal Antibody

A human monoclonal antibody in which critical amino acid residues are replaced by
molecular technology.
8. Fusion Partner
A cell line fused with the antibody producing cell with the intention to immortalise this cell.
9. Feeder Cells
Cells or cell line co-cultivated with the antibody producing cell line to provide optimal
growth conditions.
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STABILITY TESTING OF BIOTECHNOLOGICAL/
BIOLOGICAL PRODUCTS *)
Guideline Title Quality of Biotechnological Products:

Stability Testing of Biotechnological/Biological
Products *)
force
Previous titles/other ICH Q5C, CPMP/ICH/138/95
references
sections C and F of the Annex to Directive 75/318/EEC as
authorisation for a new medicinal product of biological
or biotechnological origin.
It is an annex to the Stability Testing of New Active
Substances and Medicinal Products guideline w h i c h
CONTENTS
1. PREAMBLE
2. SCOPE OF THE ANNEX
3. TERMINOLOGY
4. SELECTION OF BATCHES
5. STABILITY INDICATING PROFILE
6. STORAGE CONDITIONS
7. TESTING FREQUENCY
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8. SPECIFICATIONS
9. LABELLING
GLOSSARY
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STABILITY TESTING OF BIOTECHNOLOGICAL/
BIOLOGICAL PRODUCTS *)
L PREAMBLE
The guidance stated in the ICH Harmonised Tripartite Guideline Stability Testing of New
Drug Substances and Products, hereafter called Tripartite Guideline on Stability, (published
in this volume under the title Stability Testing of New Active Substances and Medicinal
Products) applies in general to biotechnological/biological products. However,
biotechnological/biological products do have distinguishing characteristics to which
consideration should be given in any well-defined testing program designed to confirm
their stability during the intended storage period. For such products, in which the active
components are typically proteins and/or polypeptides, maintenance of molecular
conformation and, hence of biological activity, is dependent on noncovalent as well as
covalent forces. The products are particularly sensitive to environmental factors such as
temperature changes, oxidation, light, ionic content, and shear. In order to ensure
maintenance of biological activity and to avoid degradation, stringent conditions for their
storage are usually necessary.
The evaluation of stability may necessitate complex analytical methodologies. Assays for
biological activity, where applicable, should be part of the pivotal stability studies.
Appropriate physico-chemical, biochemical and immunochemical methods for the analysis
of the molecular entity and the quantitative detection of degradation products should also be
part of the stability program whenever purity and molecular characteristics of the product
permit use of these methodologies.
With the above concerns in mind, the applicant should develop the proper supporting stability
data for a biotechnological/biological product and consider many external conditions which
can affect the product's potency, purity and quality. Primary data to support a requested
storage period for either an active substance or medicinal product should always be based on
long-term, real-time, real-condition stability studies. Thus, the development of a proper long-
term stability program becomes critical to the successful development of a commercial
product. The purpose of this document is to give guidance to applicants regarding the type of
stability studies that should be provided in support of marketing applications. It is understood
that during the review and evaluation process, continuing updates of initial stability data
may occur.
2. SCOPE OF THE ANNEX

The guidance stated in this annex applies to well-characterised proteins and polypeptides,
their derivatives and products of which they are components, and which are isolated from
tissues, body fluids, cell cultures, or produced using rDNA technology. Thus, the document
covers the generation and submission of stability data for products such as cytokines
(interferons, interleukins, colony-stimulating factors, tumour necrosis factors),
erythropoietins, plasminogen activators, blood plasma factors, growth hormones and growth
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factors, insulins, monoclonal antibodies, and vaccines consisting of well-characterised

proteins or polypeptides. In addition, the guidance outlined in the following sections may
apply to other types of products, such as conventional vaccines, after consultation with the
appropriate regulatory authorities. The document does not cover antibiotics, allergenic
extracts, heparins, vitamins or whole blood.
3. TERMINOLOGY
For the basic terms used in this annex the reader is referred to the "Glossary" in the
Tripartite Guideline on Stability. However, since manufacturers of
biotechnological/biological products sometimes use traditional terminology, traditional
terms are specified in parentheses to assist the reader. A supplemental glossary is also
included that explains certain terms used in the production of biotechnological/biological
products.
4. SELECTION OF BATCHES
4.1 Active substance (Bulk Material)
Where bulk material is to be stored after manufacture but prior to formulation and final
manufacturing, stability data should be provided on at least three batches for which
manufacture and storage are representative of the manufacturing scale of production. A
minimum of six months stability data at the time of submission should be submitted in cases
where storage periods greater than six months are requested. For active substances with
storage periods of less than six months, the minimum amount of stability data in the initial
submission should be determined on a case by case basis. Data from pilot-plant-scale batches
of active substance produced at a reduced scale of fermentation and purification may be
provided at the time the dossier is submitted to the regulatory agencies with a commitment to
place the first three manufacturing scale batches into the long-term stability program after
approval.
The quality of the batches of active substance placed into the stability program should be
representative of the quality of the material used in pre-clinical and clinical studies and of
the quality of the material to be made at manufacturing scale. In addition, the active
substance (bulk material) made at pilot-plant scale should be produced by a process and
stored under conditions representative of that used for the manufacturing scale. The active
substance entered into the stability program should be stored in containers which properly
represent the actual holding containers used during manufacture. Containers of reduced
size may be acceptable for active substance stability testing provided that they are constructed
of the same material and use the same type of container/closure system that is intended to be
used during manufacture.
4.2 Intermediates
During manufacture of biotechnological/biological products, the quality and control of
certain intermediates may be critical to the production of the final product. In general, the
manufacturer should identify intermediates and generate in-house data and process limits
that assure their stability within the bounds of the developed process. While the use of pilot-
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plant-scale data is permissible, the manufacturer should establish the suitability of such data
using the manufacturing-scale process.
4.3 Medicinal product (Final Container Product)

Stability information should be provided on at least three batches of final container product
representative of that which will be used at manufacturing scale. Where possible, batches of
final container product included in stability testing should be derived from different batches
of bulk material. A minimum of six months data at the time of submission should be
submitted in cases where storage periods greater than six months are requested. For
medicinal products with storage periods of less than six months, the minimum amount of
stability data in the initial submission should be determined on a case by case basis. Product
expiration dating will be based upon the actual data submitted in support of the application.
Since dating is based upon the real-time/real-temperature data submitted for review,
continuing updates of initial stability data should occur during the review and evaluation
process. The quality of the final container product placed on stability studies should be
representative of the quality of the material used in the preclinical and clinical studies.
Data from pilot-plant scale batches of medicinal product may be provided at the time the
dossier is submitted to the regulatory agencies with a commitment to place the first three
manufacturing scale batches into the long-term stability program after approval. Where
pilot-plant scale batches were submitted to establish the dating for a product and, in the event
that product produced at manufacturing scale does not meet those long-term stability
specifications throughout the dating period or is not representative of the material used i n
pre-clinical and clinical studies, the applicant should notify the appropriate regulatory
authorities to determine a suitable course of action.
4.4 Sample selection criteria

Where one product is distributed in batches differing in fill volume (e.g. 1 millilitre (ml),
2 ml, or 10 ml), unitage (e.g. 10 units, 20 units, or 50 units), or mass (e.g. 1 milligram (mg),
2 mg, or 5 mg) samples to be entered into the stability program may be selected on the basis
of a matrix system and/or by bracketing.
Matrixing, i.e. the statistical design of a stability study in which different fractions of
samples are tested at different sampling . points, should only be applied when appropriate
documentation is provided that confirms that the stability of the samples tested represents the
stability of all samples. The differences in the samples for the same medicinal product
should be identified as, for example, covering different batches, different strengths, different
sizes of the same closure and possibly, in some cases, different container/closure systems.
Matrixing should not be applied to samples with differences that may affect stability, such as
different strengths and different containers/closures, where it cannot be confirmed that the
products respond similarly under storage conditions.
Where the same strength and exact container/closure system is used for three or more fill
contents, the manufacturer may elect to place only the smallest and largest container size
into the stability program, i.e., bracketing. The design of a protocol that incorporates
bracketing assumes that the stability of the intermediate condition samples are represented
by those at the extremes. In certain cases, data may be needed to demonstrate that all
samples are properly represented by data collected for the extremes.
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5. STABILITY-INDICATING PROFILE
On the whole, there is no single stability-indicating assay or parameter that profiles the
stability characteristics of a biotechnological/biological product. Consequently, the
manufacturer should propose a stability-indicating profile that provides assurance that
changes in the identity, purity and potency of the product will be detected.
At the time of submission, applicants should have validated the methods that comprise the
stability-indicating profile and the data should be available for review. The determination of
which tests should be included will be product-specific. The items emphasised in the
following subsections are not intended to be all-inclusive, but represent product
characteristics that should typically be documented to adequately demonstrate product
stability.
5.1 Protocol
The dossier accompanying the application for marketing authorisation should include a
detailed protocol for the assessment of the stability of both active substance and medicinal
product in support of the proposed storage conditions and expiration dating periods. The
protocol should include all necessary information which demonstrates the stability of the
biotechnological/biological product throughout the proposed expiration dating period
including, for example, well-defined specifications and test intervals. The statistical
methods that should be used are described in the Tripartite Guideline on Stability.
5.2 Potency
When the intended use of a product is linked to a definable and measurable biological
activity, testing for potency should be part of the stability studies. For the purpose of stability
testing of the products described in this guideline, potency is the specific ability or capacity of
a product to achieve its intended effect. It is based on the measurement of some attribute of
the product and is determined by a suitable quantitative method. In general, potencies of
biotechnological/biological products tested by different laboratories can be compared in a
meaningful way only if expressed in relation to that of an appropriate reference material.
For that purpose, a reference material calibrated directly or indirectly against the
corresponding national or international reference material should be included in the assay.
Potency studies should be performed at appropriate intervals as defined in the stability
protocol and the results should be reported in units of biological activity calibrated, whenever
possible, against nationally or internationally recognised standard. Where no national or
international standards exists, the assay results may be reported in in-house derived units
using a characterised reference material.
In some biotechnological/biological products, potency is dependent upon the conjugation of
the active substance(s) to a second moiety or binding to an adjuvant. Dissociation of the
active substance(s) from the carrier used in conjugates or adjuvants should be examined i n
real-time/real-temperature studies (including conditions encountered during shipment). The
assessment of the stability of such products may be difficult since, in some cases, in vitro
tests for biological activity and physico-chemical characterisation are impractical or provide
inaccurate results. Appropriate strategies (e.g. testing the product prior to
conjugation/binding, assessing the release of the active compound from the second moiety,
in vivo assays) or the use of an appropriate surrogate test should be considered to overcome
the inadequacies of in vitro testing.
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5.3 Purity and Molecular Characterisation

For the purpose of stability testing of the products described in this guideline, purity is a
relative term. Due to the effect of glycosylation, deamidation, or other heterogeneities, the
absolute purity of a biotechnological/biological product is extremely difficult to determine.
Thus, the purity of a biotechnological/biological product should be typically assessed by more
than one method and the purity value derived is method-dependent. For the purpose of
stability testing, tests for purity should focus on methods for determination of degradation
products.
The degree of purity, as well as individual and total amounts of degradation products of the
biotechnological/biological product entered into the stability studies, should be reported and
documented whenever possible. Limits of acceptable degradation should be derived from the
analytical profiles of batches of the active substance and medicinal product used in the pre-
clinical and clinical studies.
The use of relevant physico-chemical, biochemical and immunochemical analytical
methodologies should permit a comprehensive characterisation of the active substance
and/or medicinal product (e.g. molecular size, charge, hydrophobicity) and the accurate
detection of degradation changes that may result from deamidation, oxidation,
sulphoxidation, aggregation or fragmentation during storage. As examples, methods that
may contribute to this include electrophoresis (SDS-PAGE, Immunoelectrophoresis, Western
blot, isoelectrofocusing), high-resolution chromatography (e.g. reversed-phase
chromatography, gel filtration, ion exchange, affinity chromatography), and peptide
mapping.
Wherever significant qualitative or quantitative changes indicative of degradation product
formation are detected during long-term, accelerated and/or stress stability studies,
consideration should be given to potential hazards and to the need for characterisation and
quantification of degradation products within the long-term stability program. Acceptable
limits should be proposed and justified, taking into account the levels observed in material
used in pre-clinical and clinical studies.
For substances that can not be properly characterised or products for which an exact analysis
of the purity cannot be meaningfully determined through routine analytical methods, the
applicant should propose and justify alternative testing procedures.
5.4 Other Product Characteristics

The following product characteristics, though not specifically relating to
biotechnological/biological products, should be monitored and reported for the medicinal
product in its final container:
Visual appearance of the product (colour and opacity for solutions/suspensions; colour,
texture and dissolution time for powders), visible particulates in solutions or after the
reconstitution of powders or lyophilised cakes, pH, and moisture level of powders and
lyophilised products.
Sterility testing or alternatives (e.g. container/closure integrity testing) should be
performed at a minimum initially and at the end of the proposed shelf life.
Additives (e.g. stabilisers, preservatives) or excipients may degrade during the dating
period of the medicinal product. If there is any indication during preliminary stability
studies that reaction or degradation of such materials adversely affect the quality of
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the medicinal product, these items may need to be monitored during the stability
program.
The container/closure has the potential to adversely affect the product and should be
carefully evaluated (see below).
6. STORAGE CONDITIONS
6.1 Temperature
Since most finished biotechnological/biological products need precisely defined storage
temperatures, the storage conditions for the real-time/real-temperature stability studies may
be confined to the proposed storage temperature.
6.2 Humidity
Biotechnological/biological products are generally distributed in containers protecting them
against humidity. Therefore, where it can be demonstrated that the proposed containers (and
conditions of storage) afford sufficient protection against high and low humidity, stability
tests at different relative humidities can usually be omitted. Where humidity-protecting
containers are not used, appropriate stability data should be provided.
6.3 Accelerated and stress conditions

As previously noted, the expiration dating should be based on real-time/real-temperature
data. However, it is strongly suggested that studies be conducted on the active substance and
medicinal product under accelerated and stress conditions. Studies under accelerated
conditions may provide useful support data for establishing the expiration date, provide
product stability information for future product development (e.g. preliminary assessment of
proposed manufacturing changes such as change in formulation, scale-up), assist in
validation of analytical methods for the stability program, or generate information which
may help elucidate the degradation profile of the active substance or medicinal product.
Studies under stress conditions may be useful in determining whether accidental exposures
to conditions other than those proposed (e.g. during transportation) are deleterious to the
product and also for evaluating which specific test parameters may be the best indicators of
product stability. Studies of the exposure of the active substance or medicinal product to
extreme conditions may help to reveal patterns of degradation; if so, such changes should be
monitored under proposed storage conditions. While the Tripartite Guideline on Stability
describes the conditions of the accelerated and stress study, the applicant should note that
those conditions may not be appropriate for biotechnological/biological products. Conditions
should be carefully selected on a case by case basis.
6.4 Light
Applicants should consult the appropriate regulatory authorities on a case by case basis to
determine guidance for testing.
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6.5 Container/Closure
Changes in the quality of the product may occur due to the interactions between the
formulated biotechnological/biological product and container/closure. Where the lack of
interactions cannot be excluded in liquid products (other than sealed ampoules), stability
studies should include samples maintained in the inverted or horizontal position (i.e., in
contact with the closure), as well as in the upright position, to determine the effects of the
closure on product quality. Data should be supplied for all different container/closure
combinations that will be marketed.
In addition to the standard data necessary for a conventional single-use vial, the applicant
should demonstrate that the closure used with a multiple-dose vial is capable of withstanding
the conditions of repeated insertions and withdrawals so that the product retains its full
potency, purity, and quality for the maximum period specified in the instructions-for-use on
containers, packages, and/or package inserts. Such labelling should be in accordance with
relevant national/regional requirements.
6.6 Stability after R e c o n s t i t u t i o n of F r e e z e - D r i e d P r o d u c t

The stability of freeze-dried products after their reconstitution should be demonstrated for the
conditions and the maximum storage period specified on containers, packages, and/or
package inserts. Such labelling should be in accordance with relevant national/regional
requirements.
7. TESTING FREQUENCY
The shelf-lives of biotechnological/biological products may vary from days to several years.
Thus, it is difficult to draft uniform guidelines regarding the stability study duration and
testing frequency that would be applicable to all types of biotechnological/biological products.
With only a few exceptions, however, the shelf-lives for existing products and potential
future products will be within the range of 0.5 to five years. Therefore, the guidance is based
upon expected shelf-lives in that range. This takes into account the fact that degradation of
biotechnological/biological products may not be governed by the same factors during
different intervals of a long storage period.
When shelf-lives of one year or less are proposed, the real-time stability studies should be
conducted monthly for the first three months and at three-month intervals thereafter.
For products with proposed shelf-lives of greater than one year, the studies should be
conducted every three months during the first year of storage, every six months during the
second year, and annually thereafter.
While the testing intervals listed above may be appropriate in the pre-approval or pre-license
stage, reduced testing may be appropriate after approval or licensure where data are
available that demonstrate adequate stability. Where data exist that indicate the stability of a
product is not compromised, the applicant is encouraged to submit a protocol which supports
elimination of specific test intervals (e.g. nine-month testing) for post-approval/post-
licensure, long-term studies.
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8. SPECIFICATIONS
Although biotechnological/biological products may be subject to significant losses of activity,
physico-chemical changes, or degradation during storage, international and national
regulations have provided little guidance with respect to distinct release and end of shelf life
specifications. Recommendations for maximum acceptable losses of activity, limits for
physico-chemical changes, or degradation during the proposed shelf life have not been
developed for individual types or groups of biotechnological/biological products but are
considered on a case by case basis. Each product should retain its specifications within
established limits for safety, purity, and potency throughout its proposed shelf life. These
specifications and limits should be derived from all available information using the
appropriate statistical methods. The use of different specifications for release and expiration
should be supported by sufficient data to demonstrate that clinical performance is not
affected as discussed in the Tripartite Guideline on Stability.
9. LABELLING
For most biotechnological/biological substances and products, precisely defined storage
temperatures are recommended. Specific recommendations should be stated, particularly for
active substances and medicinal products that cannot tolerate freezing. These conditions,
and where appropriate, recommendations for protection against light and/or humidity,
should appear on containers, packages, and/or package inserts. Such labelling should be i n
accordance with relevant national regional requirements.
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GLOSSARY
Conjugated Product
A conjugated product is made up of an active substance (for example, peptide, carbohydrate)
bound covalently or noncovalently to a carrier (for example, protein, peptide, inorganic
mineral) with the objective of improving the efficacy or stability of the product.
Degradation Product
A molecule resulting from a change in the active substance (bulk material) brought about
over time. For the purpose of stability testing of the products described in this guideline, such
changes could occur as a result of processing or storage (e.g. by deamidation, oxidation,
aggregation, proteolysis). For biotechnological/biological products some degradation
products may be active.
Impurity
Any component of the active substance (bulk material) or medicinal product (final container
product) which is not the chemical entity defined as the active substance, an excipient, or
other additives to the medicinal product.
Intermediate
For biotechnological/biological products, a material produced during a manufacturing
process which is not the active substance or the medicinal product but whose manufacture is
critical to the successful production of the active substance or the medicinal product.
Generally, an intermediate will be quantifiable and specifications will be established to
determine the successful completion of the manufacturing step prior to continuation of the
manufacturing process. This includes material which may undergo further molecular
modification or be held for an extended period of time prior to further processing.
Manufacturing-Scale Production
Manufacture at the scale typically encountered in a facility intended for product production
for marketing.
Pilot-Plant Scale
The production of the active substance or medicinal product by a procedure fully
representative of and simulating that to be applied at manufacturing scale. The methods of
cell expansion, harvest, and product purification should be identical except for the scale of
production.
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GENE THERAPY PRODUCT QUALITY ASPECTS

IN THE PRODUCTION OF VECTORS AND
GENETICALLY MODIFIED SOMATIC CELLS
Guideline Title Gene Therapy Product Quality Aspects in the Production

of Vectors and Genetically Modified Somatic Cells
force
Previous titles/other Gene Therapy Products - Quality, Safety and Efficacy
references Aspects in the Production of Vectors and Genetically
Modified Somatic Cells/ III/5863/93
for marketing authorisation within the EC for gene
therapy products derived by biotechnology/high
technology and intended for medicinal use in man.
CONTENTS
1. INTRODUCTION
2. POINTS TO CONSDDER IN MANUFACTURE
4. PRODUCTION
5. PURIFICATION
6. PRODUCT CHARACTERISATION
7. CONSISTENCY AND ROUTINE BATCH CONTROL OF FINAL PROCESSED

PRODUCT
8. SAFETY REGULATIONS
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GENE THERAPY PRODUCT QUALITY ASPECTS

IN THE PRODUCTION OF VECTORS AND
GENETICALLY MODIFIED SOMATIC CELLS
L INTRODUCTION
Somatic gene therapy encompasses medical interventions which involve the deliberate
modification of the genetic material of somatic cells. Scientific progress over the past decade
has led to the development of novel methods for the transfer of new genetic material into
patients' cells. The aims of these methods include the efficient transfer and functional
expression or manifestation of the transferred genetic material in a target somatic cell
population for therapeutic, prophylactic or diagnostic purposes.
Although in the majority of cases the intention is the addition and expression of a gene to
yield a protein product, the transfer of nucleic acids with the aim of modifying the function
or expression of an endogenous gene, e.g. by homologous recombination, is also included i n
the definition of gene therapy. This will also include transfer of genetic material that
specify nucleic acid products, e.g. ribozymes, anti-sense nucleotides, designed to modify
endogenous gene expression at either transcriptional or translational levels. The transfer of
genetic material for the purposes of (i) marking or following the migration of particular
somatic cell populations, and (ii) protective vaccination against foreign antigens should be
included, since the products used to achieve these ends will have the same or similar
characteristics to those used in gene therapy.
There are several approaches to the introduction of genetic material into a somatic cell.
These include the transfer of naked nucleic acid, nucleic acid complexed with a carrier and
the use of replication deficient viruses. Defective viruses and nucleic acid complexes used
for nucleic acid transfer into cells are called gene transfer vectors and the nucleic acid
transferred is called the expression construct. Modification of somatic cells can be achieved
by in vivo administration of nucleic acids, with or without a carrier or transfer vector, or
performed ex vivo, after which the genetically modified autologous, allogenic or xenogenic
somatic cells are administered (Table 1).
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TABLE 1
POTENTIAL PRODUCTS FOR GENE THERAPY
(a) Naked nucleic acid natural or enzymatically synthesised nucleic acid, ligated
into appropriate plasmids or cassettes.
(b) Complexed nucleic acid (i) as above, but complexed with salts, proteins (e.g.
transferrin) or other polymers (e.g. DEAE-Dextran,
polylysine).
(ii) as above, but encapsulated in liposomes.
(iii) as above, but coated on gold particles
(c) Replication-deficient usually retroviruses or adenoviruses but probably other
viruses including adeno-associated virus, herpes simplex
virus and vaccinia virus will also form the basis of
vectors.
(d) Genetically-modified fibroblasts, myoblasts or other cell which could somatic
cells be introduced/engrafted into appropriate patient's
tissues/organs.
This document covers quality aspects in the production of the gene transfer vectors and
genetically modified somatic cells included in Table 1. However, it is not intended to apply
to chemically synthesised, short polynucleotides, e.g. anti-sense nucleotides, where quality
control in manufacture will be different.
support applications for marketing authorisation within the EC for gene therapy products
derived by biotechnology/high technology and intended for medicinal use in man. It should
be read in conjunction with the European Directives and other specialised guidelines where
appropriate. Any commercially manufactured gene therapy products will require marketing
authorisation by the European Medicines Evaluation Agency through the centralised
procedure. A flexible approach to the control of these products has been adopted so that
recommendations can be modified in the light of experience of production and use, and of
further developments. Implementation of these recommendations for an individual product
should reflect its intended clinical use.

2.1 General considerations
Since gene therapy products contain genetic and other biological materials, many of the
quality, efficacy and safety considerations which apply to recombinant DNA (rDNA)
products and other biologicals manufactured by modern biotechnological methods will apply
to some stages in their manufacture. Requirements relating to establishments in which
biological products are manufactured (e.g. Directive 91/536/EEC on GMP and Directive
90/219/EEC on the contained use of genetically modified micro-organisms; see Section 8)
will apply to the manufacture of gene therapy products as will several of the general
recommendations for the quality control of biological products.
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Appropriate attention needs to be given to the quality of all reagents used in production:
specifications for these are to be included in documentation and they should comply with any
relevant EU recommendations (e.g. note for guidance on Minimising the Risk of Transmitting
Agents causing Spongiform Encephalopathy via Medicinal Products.
It is undesirable to use in production agents which are known to provoke sensitivity i n
certain individuals, such as, for example, penicillin or other -lactam antibiotics.
Although comprehensive characterisation of the final product is essential, considerable
emphasis must also be placed on 'in-process' control, a concept which has been highly
effective in the quality control of bacterial and viral vaccines prepared by conventional
methods and, more recently, of rDNA-derived products.
Certain factors may compromise the quality, and thus the safety and efficacy, of gene
therapy products and should be given special attention:
a) The genetic material involved, a defined nucleic acid will require amplification
within a replicating organism or by an in vitro technique, e.g. polymerase chain
reaction (PCR). Uncertainties over the fidelity of the replication systems raise
concerns about the homogeneity of the amplified product. For example, a gene
containing errors in base sequences may specify an abnormal protein which may
have undesirable biological and immunological activities. Transference procedures
are intended to introduce copies of the genetic material involved into large numbers of
target cells. Therefore, it is essential to purify and characterise the genetic material
involved as thoroughly as possible before use. Where possible, evidence should be
obtained that the correct nucleotide sequence, or that at least the correct coding capacity,
has been made and that this has been stably maintained during the amplification steps
before transfer and that the sequence/coding capacity remains unmodified following
transfer.
b) In most instances, the genetic material (nucleic acid) involved will be ligated into
appropriate plasmids or cassettes having promoters which regulate its expression. The
resulting expression constructs may be complexed with salts, proteins (e.g.
transferrin) or polymers (e.g. polylysine), or linked to carriers (e.g. liposomes or
gold), or adsorbed to replication-deficient viruses (e.g. adenovirus), to increase the
specificity or efficiency of transfer of genetic material (Table 1). This may mean that
some products are manufactured as components of the final vector, which is constituted
just prior to use (cf. monoclonal antibodies which are radiolabelled just before
application). In these cases, all components of the final transfer vector should be
thoroughly characterised.
c) Virus vectors raise particular issues regarding manufacture and safety. For example,
viruses proposed as vectors are themselves likely to produce pathological effects under
certain circumstances. It is however expected that viral vectors will have been
'engineered' to lack viral genes (encoding structural and enzymatic proteins) that are
required for replication and viral particle formation. Viral nucleic acid sequences
known to be associated with pathological effects should also be deleted. Replication-
deficient viruses are propagated in special "packaging" cell lines genetically
modified to express the viral proteins necessary for the recombinant genomes to be
replicated and packaged. The aim should be the construction of packaging cell lines
which make the production of replication-competent (infectious) virus(es) by
recombination with the viral genome of the gene transfer vector used impossible. One
way to do this (e.g. for retroviruses) is to separate the genes encoding the viral
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structural and enzymatic proteins and to express them from separate constructs which
are inserted into separate chromosomal integration sites. To further minimise the risk
of recombination within the packaging cell line, packaging cell lines containing any
endogenous viral sequences that could complement the recombinant viral genome
should be avoided. Precautions must also be taken to prevent infection of the packaging
cell line by wild-type viruses that might also lead to the formation of replication-
competent recombinant viruses. In addition, the recombinant genome may be subject to
mutation during replication in the packaging cell line. Complete characterisation and
safety-testing of such vectors may be difficult, especially because purification to
homogeneity, e.g. for retroviral vectors, may not be readily attainable.
d) In some cases, genetically-modified somatic cells might themselves be perceived to be
products. For example, a gene may be transferred to and expressed in fibroblasts,
myoblasts, epithelial cells or other cell types and these expanded in vitro to sufficient
numbers for inoculation into one or more patients having the same condition.
Alternatively, the genetically modified cells may be grown in collagen-lattices or
other appropriate matrices to produce 'neo-organs' that secrete a particular 'therapeutic'
protein. The transplantation of genetically-modified somatic cells and the
implantation of neo-organs is governed by the same considerations of
histocompatibility and immunology which apply to conventional tissue-transplants. To
reduce the immunogenicity of neo-organs, they could be encapsulated.
e) Potential impurities in the final product will be influenced by the choice of
manufacturing procedure and the purification processes, where applicable, must be
shown to be capable of removing them. An example is the presence of endotoxin in
products expressed in bacterial cells; another is of adventitious agents and DNA in
products expressed in mammalian (including human) cells.
f) Unintended variability in culture during production may lead to changes which cause
alteration of the product, reduce the yield of product and/or result in quantitative and
qualitative differences in the impurities present. Procedures to ensure consistency of
production conditions as well as of the final product are imperative.
g) Scale-up of culture and/or purification occurs as laboratory developments progress to
full scale commercial production, and this may have significant consequences for the
quality of the product including effects on its biochemical and biological properties,
and thus implications for control testing.
Whilst the recommendations set out below should be considered to be generally applicable,
individual products may present particular quality control problems. The production and
control of each product will be considered on a case by case basis.
2.2 U n i n t e n d e d a n d u n e x p e c t e d consequences of gene transfer

2.2.1 Insertional mutagenesis
Most existing vectors can only transfer genetic material into target cells leading to either
random integration with chromosomal DNA or to localisation in extra-chromosomal sites,
suggesting a number of undesirable possibilities. Random integration of vector nucleic acid
could result in:
(i) integration in the middle of a tumour suppressor gene, so abolishing its expression.
(ii) integration at sites which induce cis- or trans-activation of proto-oncogenes or other
growth promoting genes.
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(iii) integration at sites which affect cellular responsiveness to exogenous agents, such as
growth factors, cytokines or hormones.
In all three examples cited, the affected cells may acquire tumourigenic potential. However,
oncogenesis (transformation of a cell to the tumorigenic phenotype) is generally regarded as
a multi-step process involving the disruption of many genes, and thus occurrence of single-
site insertional mutagenesis may only carry a very low risk of the development of tumour
cells. Nevertheless, a high vector nucleic acid copy number per cell, randomly integrated
into chromosomal DNA, may be cause for concern. Vector nucleic acid copy number could
increase in cases where repeated vector application is necessary.
2.2.2 Induced cellular changes

Following transfer of vector nucleic acid to target cells, certain unintended and unexpected
observable changes to target cell appearance, functions and behaviour, e.g. migratory
characteristics, may occur compared with the original unmodified target cell population.
These should be well-documented. In addition, certain non-observable changes may occur.
For example, several members of the herpesvirus family which are latent in human cells
are also reactivatable under certain conditions leading to the production of infectious virus.
Therefore, where possible, transduced target cells should be screened for the presence of
likely re-activatable viruses such as herpes zoster, Epstein-Barr virus and cytomegalovirus.
There may also be the possibility that transfer of vector nucleic acid increases the
immunogenicity of the target cells. For example, this could be the case where the vector
nucleic acid encodes viral or other non-human proteins, or proteins that were previously not
expressed within the patient treated due to the specific genetic defect.
2.2.3 Vector DNA mobilisation

Vector nucleic acid mobilisation is not likely to occur for those vectors which have no
replication potential. Replication-deficient viral vectors however while not normally
expected to replicate may infrequently be rescued either by co-infection with wild-type,
replication-competent viruses or by recombination with endogenous viral nucleic acid
sequences. There may also be a low risk that the recombinant viral genome itself
recombines with the genomes of co-infecting viruses to produce novel recombinant viruses.
Vector nucleic acid mobilisation may lead to non-target cells receiving this (e.g. germline
cells being transduced with vector nucleic acid) and a risk of its horizontal spread to
clinical staff and members of the public.
3.1 Genetic material involved
A detailed description of the functionally relevant genetic material involved should be
given. This should include details of its origin, identification and isolation as well as,
where appropriate, its coding capacity, and where possible, its nucleotide sequence. Any
truncations or other intended modifications, e.g. site-specific mutation, deletions,
rearrangements, to functional genes compared with their natural counterparts included in
the genetic material should also be detailed.
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3.2 Amplification of genetic material

Full details of how the genetic material is amplified for incorporation into the final product,
or into a vector for secondary amplification, should be given. Where possible, this should
include details of all nucleotide sequences within the genetic material which are required
for its replication in prokaryotic or eukaryotic cells. Cells used in amplification of the
genetic material should be fully characterised; this includes the history of the cell line, its
identification characteristics and potential viral contaminants. Special attention should be
given to the possibility of cross-contamination with other cells or viruses.
3.3 Vector construction

3.3.1 Complexed nucleic acid as vector
A complete description of the manufacturing procedures used in vector production, including
in-process controls, should be provided together with a complete description and
characterisation of all of the materials used to form these vectors. Where appropriate,
materials should be of pharmaceutical quality. A full description and characterisation of all
the genetic material's nucleic acid sequences included in the vector and which are
transferred into the somatic cells should be provided. Where appropriate, a plasmid and/or
cell bank should be established and characterised.
3.3.2 Replication-deficient viruses as vector

Full documentation on the origin, history and other characteristics of the parental virus,
current virus stocks and methods of propagation should be provided. A full description and
characterisation of the genetic material, the part(s) of the viral genome to which the genetic
material is inserted or ligated, modifications of remaining viral nucleic acid sequences
and any other nucleic acid sequences (e.g. promoters) to be included in the recombinant
viral genome should be provided.
Details of the construction of the packaging cell line should be given, including the nature
and, where possible, the location of the helper viral nucleic acid and its encoded
proteins/functions. The origin, identity and biological characteristics of the cell line
together with details of the presence or absence of endogenous viral particles and sequences
should be described. A well-defined master and working cell bank should be established.
Evidence of freedom from contamination with adventitious microorganisms, including
viruses, bacteria, mycoplasma, yeasts, moulds (fungi), is essential.
A complete description of the procedures used to transfect/transfer the recombinant viral
genome containing the genetic material into the packaging cells should be provided. Where
the packaging cells do not contain integrated helper viral nucleic acid sequences, but
packaging of the recombinant viral genome is reliant on transfecting an additional nucleic
acid construct, full details of this construct should be given.
The processes resulting in recombinant viral genome replication and its packaging into
virus particles should be described. Where possible, the stability of the recombinant viral
genome in the packaging cells should be assessed.
Where selection techniques are required to isolate cells producing replication-deficient viral
vector, details of the methods used should be provided.
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3.4 G e n e t i c a l l y - m o d i f i e d s o m a t i c c e l l s a s p r o d u c t s
Full documentation of the origin, history, construction and characteristics of the genetically-
modified somatic cells should be provided. The homogeneity and genetic stability of the cells
should be demonstrated or thoroughly characterised. Any observable changes in cell
morphology, functions and behaviour, e.g. migration characteristics, of the genetically-
modified somatic cells compared with the original unmodified cells should be well
documented. A well-defined master and working cell bank should be established, where
appropriate. Evidence of freedom from contamination with adventitious microorganisms,
including viruses, bacteria, mycoplasma, yeasts, moulds (fungi), is essential.
4. PRODUCTION
Details of the production process including volumes, times, harvest and storage should be
given. A clear definition of a "batch" of product, which may be subjected to further
processing, should be provided. Acceptable limits for the purity, consistency and yield of
product should be specified and justified.
5. PURIFICATION
A complete description of methods used in purification should be provided where applicable
together with full details of in-process controls. The capacity of the purification procedure to
remove potential contaminants should be thoroughly investigated. The consistency of the
purification process should be demonstrated together with its capacity to remove specific
contaminants.
6. PRODUCT CHARACTERISATION
Rigorous characterisation of the product and of its stability by a range of molecular and
biological methods is essential. It is desirable to include suitable tests to establish that
complexed nucleic acid has the desired biochemical and biological characteristics required
for its intended use. Immunological and immunochemical tests may provide valuable
information. In the case of replication-deficient viral vectors tests should, where possible, be
included to show integrity and homogeneity of the recombinant viral genome. Tests to
establish the cellular tropism and, if expected, tissue-specific transcription of gene transfer
vectors and, where appropriate, the inducibility of the desired gene, should also be
undertaken.
When appropriate, the purity of the final processed product should be determined, and the
level of contamination considered as acceptable should be justified. The criteria for
acceptance or rejection of a production batch must be given. In the case of replication-
deficient viral vectors, tests to show they are free from replication-competent viruses are
essential. For example, replication-competent retroviruses, even of xenogenic origin, are
able to promote oncogenesis, probably because they can randomly integrate many copies of
their genome through multiple-infection cycles in target cells. It is essential therefore that
all measures/steps be taken to exclude the possibility that replication-deficient retroviral
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vectors become contaminated with replication-competent retroviruses during manufacturing

processes.
7. CONSISTENCY AND ROUTINE BATCH CONTROL OF

FINAL PROCESSED PRODUCT
Analysis of the initial batches of a product should be undertaken to establish consistency
with regard to identity, purity and potency. Thereafter, a more limited series of tests may be
appropriate as outlined below. A clear difference should be made between the analytical tests
performed during product development, in order to fully characterise the product, and tests
performed routinely on each production batch of (purified) bulk product.
7.1 Consistency
An acceptable number, e.g. five, of successive batches of the bulk product should be
characterised as fully as possible to determine consistency of composition. The studies
should include molecular, biological, and immunological methods to characterise and assay
the product as well as methods to detect and identify impurities. Any differences which occur
among batches should be noted.
7.2 Routine batch control analysis

7.2.1 Identity
A selection of tests used to characterise the purified product (see section 6) should be used to
confirm product identity for each batch. The methods employed should include tests for the
genetic composition and physico-chemical and immunological characteristics, together with
tests for the expected biological activity (see section 7.2.3).
7.2.2 Purity
the nature and intended use of the product, the method of its production and purification and
also the degree of consistency of the production process. The purity of each batch should be
established and be within specified limits. Tests should be applied to determine levels of
contaminants of cellular origin, e.g. from the packaging cell line, as well as materials
which may have been added during the production processes. A strict upper limit for each
identifiable contaminant should be set.
72.3 Efficacy potency tests

For estimating the efficacy/potency of vectors, biological tests should be applied that permit
the efficiency of transfer and the level and stability of expression of genetic material, or its
effects, to be determined. Wherever possible, a reference batch of vector of assigned potency
should be established and used to calibrate tests.
The efficiency with which vectors transfer the genetic material to target/test cells together
with information on the resulting level of gene expression will provide the basis for
assessing their potency. When tests are conducted in vitro, the target cell population should
be carefully characterised. The variability of the biological system as a whole should be
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monitored, particularly where target cells may be derived from different sources/donors and
long term expression or manifestation of the transfected genetic material is being followed.
Where appropriate and for vectors intended for direct in vivo application, biological potency
tests in animal tissues maintained ex vivo or in whole animals should be carried out.
Transgenic animals or animals with transplanted human tissues or systems may be
suitable for this purpose.
Where possible, suitable ways for expressing potency of vectors should be established and
results reported in a reference unitage. It is recommended that the reference unitage be
correlated if possible with a physico-chemical parameter of the vector, e.g. weight of DNA, to
provide information on the 'specific activity" of the vector. Stated limits for the potency and
specific activity of batches of vector should be provided.
Where possible, the particle :infectivity ratio of replication-deficient viruses should be
determined and when this is excessively high rejection of the batch should be considered.
72.4 Safety tests

In products containing replication-deficient viruses, tests to detect replication competent
viruses in supernatant fluids and virus pellets at appropriate stages of production are
essential. Tests must be carried out on each production run and batch of product and where
replication-competent viruses are detected the whole batch should be rejected.
8. SAFETY REGULATIONS
Currently, gene therapy products with viruses as vectors fall under the scope of Directive
90/219/EEC on contained use of genetically modified microorganisms and Directive
90/220/EEC on the deliberate release of genetically modified organisms. The group of
competent authorities for the implementation of these Directives have adopted the following
approach:
a) actions under Directive 90/219/EEC:
(i) genetic modification of somatic cells, as well as the culture, storage and use of
the genetically modified somatic cells carried out in laboratory or hospital
facilities.
(ii) preparation of genetically modified viruses carried out in contained facilities.
(iii) treatment of patients with genetically modified viruses in contained facilities,
provided the virus is no longer capable of producing infectious particles.
b) actions under Directive 90/220/EEC:
where products such as recombinant viruses in the form of aerosol spray are used for
the treatment of genetic diseases, Directive 90/220/EEC applies in addition to any other
relevant legislation.
Since 1 January 1995, the deliberate release of medicinal products containing or consisting
of GMOs for the purpose of placing them on the market falls within the scope of Council
Regulation (EEC) 2309/93, which provides for a specific environmental risk assessment
similar to that laid down in Directive 90/220/EEC. Thus, in its opinion on applications for
marketing authorisation of such medicinal products, the CPMP shall ensure that all
appropriate measures are taken to avoid adverse effects on human health and the
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environment which might arise from the deliberate release or placing on the market of
genetically modified organisms.
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USE OF TRANSGENIC ANIMALS IN THE

MANUFACTURE OF BIOLOGICAL MEDICINAL
PRODUCTS FOR HUMAN USE
Guideline Title Use Of Transgenic Animals In The Manufacture Of

Biological Medicinal Products For Human Use
force
Previous titles/other none/III/3612/93
references
Additional Notes This document is concerned with the use of transgenic
animals to produce biological pharmaceutical materials
for use in human recipients.
CONTENTS
1. INTRODUCTION
2. DEFINITIONS
3. SCOPE OF THE NOTE FOR GUIDANCE
4. THE TRANSGENIC ANTMAL
5. PRODUCTION
6. CONCLUSION
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USE OF TRANSGENIC ANIMALS IN THE

MANUFACTURE OF BIOLOGICAL MEDICINAL
PRODUCTS FOR HUMAN USE
L INTRODUCTION
Transgenic organisms contain a foreign gene which has been experimentally inserted into
the normal genetic component, and currently include many plants and a number of a n i m a l
species. They have been used experimentally to investigate gene function, development and
disease. Transgenic animals have also been proposed as a means of testing agents for
oncogenicity or virulence.
This document is concerned with the use of transgenic animals to produce biological
pharmaceutical materials for use in human recipients. Transgenic animals may produce
higher quantities of material in more concentrated form than existing culture methods, and
therefore have considerable advantages in both the cost of producing the starting material
and in its downstream processing. In some instances where very large amounts of material
are required for therapy the use of transgenic animals may be one of the few viable
production strategies. However in some respects the products resemble classical biologicais
in that they derive from a whole animal rather than from definable culture systems. The
considerations which apply are therefore a blend of those relevant to recombinant DNA
(rDNA) derived materials and materials from less defined sources.
2. DEFINITIONS
Forebears: the animals from which the egg and sperm used to create the genetic founder
were derived
Host: the recipient mother in whom the embryonic genetic founder was implanted
Genetic founder: the transgenic animal resulting from the introduction of the foreign DNA
into the embryo or fertilised egg
Production founder: a transgenic animal used as a source for the generation of production
animal herds
Production animals: the immediate offspring of the production founder

Many different species have been considered or developed for the production of biological
medicinal products and by use of appropriate targeting sequences the transgene has been
expressed in body fluids such as blood or in milk as well as in other source tissues. A wide
range of host animals and source materials are therefore possible each raising specific
concerns. All products must be considered on a case by case basis. However the strategy
adopted should be such as to minimise potential microbiological contamination during the
creation of the transgenic line including potential contamination from the host and founder
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animals. Maintenance of the production animals should be such as to minimise

contamination of the starting materials such as milk or blood from which the final product
will be purified. The purity and microbiological safety of the final product is of major
concern.
The production facilities used will probably employ agricultural animals and techniques. It
is important to bear in mind that the requirements for manufacture of pharmaceutical
products will be more stringent than those for agricultural production, and the production
process should be designed accordingly. This document emphasises products derived from
fluids of transgenic animals, particularly milk, as there is at present considerable interest
in such sources, but many of the considerations will also apply to other source tissues. Other
relevant notes for guidance should be taken into account including those concerned with the
validation of virus removal and inactivation procedures, minimising the risk of
transmission of agents causing spongiform encephalopathy via medicinal products, the
production and quality control of medicinal products derived by rDNA technology and the
Biotech headings for Notice to Applicants (Part II of application file).
The veterinary and environmental issues relevant to animal welfare and the consequences
of release have been considered elsewhere, (see for example Directives 90/219/EEC on
contained use and 90/220/EEC on deliberate release of genetically modified organisms) and
the animals used in production must comply with existing regulations concerning the
development of transgenic animals.
4. THE TRANSGENIC ANIMAL

4.1 Origin
Animals which have been proposed as hosts for production include among others sheep, cows,
pigs, rabbits and mice, and much interest currently centres on the use of transgenes
expressed in milk or colostrum. The choice of animal will be determined by a variety of
factors. For example pigs breed rapidly and produce large litters, so that establishing a
suitable transgenic line of animals may be technically simpler than if the same process is
attempted in cows. On the other hand pigs are difficult to milk, while milk production in
cows is well understood.
Each species will raise its own microbiological and virological concerns which should be
addressed. Many of the potential host animals are not conventional laboratory animals, but
infectious agents of agricultural significance are likely to be well known. The
microbiological status of the production animal, its forebears and host animals involved i n
derivation of the transgenic line should be documented as far as is possible. Consideration
should be given to the use of breeds of animals resistant to specific agents such as scrapie
resistant breeds of sheep. The founder animals and their offspring should be shown to
comply with the existing guidelines Minimising the Risk of Transmitting Agents causing
Spongiform Encephalopathy via Medicinal Products.
4.2 The expression system

The isolation and characterisation of the gene and associated control elements should be
described as should the process by which the final construct was made. The strategy used to
develop the particular expression system should be described and justified. In particular the
rationale for the use of regulatory sequences to ensure correct expression of the gene in the
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appropriate tissue should be clearly described. The complete sequence of the final construct
should be determined.
4.3 C r e a t i o n of t h e t r a n s g e n i c a n i m a l
A number of methods are currently in use for the creation of transgenic animals. One
favoured method involves the inoculation of the DNA into the pronucleus of a fertilised
ovum, followed by implantation into pseudo pregnant females. This results in a proportion of
animals carrying the transgene in the germ line which may be high in some species (e.g.
mouse 5-30%) or low in others (e.g. cows and sheep, 1-5%). Depending on the time when the
transgene is incorporated into the cellular DNA, mosaic animals may develop in which
certain cell lineages carry the transgene while others do not. Other methods of creating
transgenic animals involving retroviral infection of the embryo at an early cleavage stage
in the blastocyst result in only a proportion of the cells carrying the transgene, and therefore
a high proportion of mosaic animals some of which may not have the transgene incorporated
in the germ line at all. Methods for predictable site specific integration of sequences into the
host genome would have advantages for both controlled expression and safety.
The method used to create the transgenic animal should be described in detail, including the
isolation of ova, in vitro fertilisation, insertion of the transgene, reimplantation and
delivery. The use of retroviral vectors raises additional quality considerations related to
preparation of the vector, its virological purity and its persistence. Consideration of
guidelines related to regulatory aspects of gene therapy is advisable.
The genealogy of the production animals must be documented. A transgenic line will derive
from a single genetic founder animal, and materials from different transgenic lines
should not be mixed. The founder animal and the production animals should be defined as
diploid or haploid with respect to the inserted sequence.
The level of expression of the incorporated gene should be assessed and the tissue
distribution of expression should wherever possible be shown to be consistent with the chosen
strategy of expression. Estimates of the copy number should be made and evidence as t the
accuracy of the incorporated gene squence should be presented. It is believed that while
multiple copies of the transgene are usually incorporated, there is usually only a single site
of integration. Thus, even where multiple copies are introduced it will be possible to identify
the expressed sequence or sequences with confidence at the level of the genomic DNA.
It is of doubtful value to determine multiple sequences of the insert but evidence that the
correct sequence is present should be obtained. Some sequence data for example of cDNA
clones will be valuable as will restriction endonuclease maps, which will serve to
demonstrate that the site of integration has not changed in offspring of the founder a n i m a l
where these are used. It should be clearly stated whether the animals used for production are
haploid or diploid for the transgene. The animals used in production should be characterised
to ensure an acceptable level of consistency.
The virological status of the donors and host animals should be shown to be acceptable; for
example calves born to mothers infected with BVDV are likely to be persistently infected,
and vertical transmission of BSE has not been eliminated as a possibility. Similarly bovine
immunodeficiency virus (BIV) may be transmissible through semen. These are examples
only.
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4.4 S t a b i l i t y of t h e g e n e
Transgenic animals produced by microinjection of DNA have the highest probability of
incorporating the transgene into the germ line and therefore expressing it in the appropriate
intended tissue. However this method often results in the insertion of multiple head to tail
copies of the transgene, and rearrangements and eliminations may occur on breeding. The
stability of the gene on breeding will be an issue where numbers of animals derived from a
founder animal are to be used. Greater consistency of production will be achievable if a
uniform production herd can be bred in a reproducible manner. The strategy used to
generate a herd of animals of similar productivity should be clearly delineated. Evidence
should be presented that the animals are similar, in the yield of product and genetically i n
terms of numbers of copies of the gene incorporated and the site of integration in the genome.
Restriction length polymorphisms may be of value in providing evidence for a constant
integration site.
5. PRODUCTION
5.1 Housing and animal care
There are major veterinary and ethical difficulties in raising and maintaining
agricultural animals under specific pathogen free conditions although this is desirable if it
can be achieved. Otherwise good husbandry and agricultural practice may contribute to
virological and microbiological safety. However the general conditions suitable for
satisfactory agricultural production are likely to be less stringent than those applicable to the
manufacture of pharmaceutical materials, so that good husbandry and agricultural practice
are unlikely to be sufficient alone to ensure adequate safety of a pharmaceutical product.
The conditions under which the animals are bred and maintained should be described and
precautions taken to ensure that the site is free of disease likely to affect the production
animal species prior to use. Potential sources of infection may include foodstuff, a n i m a l
handlers and veterinary surgeons, and the environment especially if the animals are kept
outside. The health and virological status of the animals should be documented and animals
subjected to regular veterinary examination. If the source material is milk the health of the
udder should be subject to special examination. Administration of antibiotics and hormones
for prophylactic or therapeutic reasons at any time when they may contaminate the product is
not permitted. Cows should be shown to be free of bovine tuberculosis.
Many cow herds are known to be infected with bovine viral diarrhoea virus, and other
infections include bovine polyoma and infectious rhinotracheitis virus which may or may
not be apparent. Sheep are susceptible to many agents including orf virus and Louping 111
virus, and pigs to swine vesicular disease and porcine parvovirus. These examples do not
constitute an exhaustive list. Many infectious agents of agricultural animals may establish
persistent infections, and some are also able to infect humans. In general animals which
are known to be infected with an agent should not be used for production.
5.2 T h e s o u r c e m a t e r i a l
Different litter mates have been reported to express the transgene to different levels for
unknown reasons unrelated to copy number or accuracy of the incorporated sequence.
During the period of lactation the expression of the gene may vary, and it may also vary
between different lactations. The source material may therefore be variable, m a k i n g
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purification procedures potentially less consistent. The nature of the source material (for
example milk or colostrum) should be clearly stated and justified. There is wide variation
in the composition of milk and the purification process must be shown to be satisfactory i n
dealing with the range of materials expected. Acceptable limits for the level of active
substance in the source material should be set. Where the source material is milk,
specifications could be set in terms of product activity per unit of non fat dry solid. A single
batch of source material may involve pooling separate harvests and should be clearly
defined. While milk is a source material with a long history in which the safety issues are
generally well understood, pharmaceutical proteins may be given parenterally, not orally,
and it may not be possible to pasteurise or sterilise them in the ways which have been applied
to milk.
Limits for the microbiological status of the source material should be set. Milk is likely to be
contaminated with bacteria, although such contamination may be minimised by good
husbandry. Contamination by certain agents, such as zoonotic mycobacteria, would make
the material unacceptable. While bacteria may be removed by sterile filtration of the product,
mycoplasma may not and efforts should be made to exclude them from the source material.
5.3 P u r i t y of t h e a c t i v e s u b s t a n c e a n d v a l i d a t i o n of d o w n s t r e a m
processing
The purity of the active substance should be in accordance with criteria accepted for products
of rDNA technology. Most such products are currently manufactured by in vitro culture
methods involving either the fermentation of microorganisms or the large scale culture of
cells from higher organisms. A transgenic animal is unlikely to be free of pathogens to the
same degree as a well characterised cell bank. Validation of the purification process is
therefore important in ensuring the safety of the product. Guidelines on Virus Validation
Removal of Viruses have been prepared. Where the source material is milk or colostrum,
contamination with mycoplasma is possible, and the process should be validated for their
removal, as well as limits set for their levels in the starting material.
The source material, whether blood, milk, colostrum or other tissue will contain large
numbers of host derived proteins other than the desired product, some of which may be
present in large amounts which must be removed. Milk is known to contain proteases, and
the possible effect of these on the product should be addressed; if degradation occurs,
acceptable limits should be set for the products in the final material. Care should be taken to
document and if necessary eliminate host proteins homologous to the required product.
Limits should be set for contaminants which may copurify with the desired material.
Hypersensitivity to milk is common, and materials must therefore be of high purity.
Data on the carbohydrate components of the product should be presented. The non enzymic
glycosylation or glycation of proteins in the presence of free carbohydrate such as lactose
should be considered. This process is likely to be inevitable to some degree for a product
derived from milk but attempts should be made to reduce it to a minimum. Glycated proteins
can cause the activation of end stage macrophages to produce cytokines, and long term
exposure to a glycated product is likely to be harmful.
The attractions of transgenic animals as a means of production include the ability to
produce materials required on a scale which may otherwise be prohibitive because of the
large amounts required in therapy. This increases the concerns associated with the
immunogenicity of the proteins because of trace impurities or imperfect post translational
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modifications, and close attention should be given to the purity, quality and consistency of
the product.
6. CONCLUSION
Transgenic animals may have advantages over existing production methods with respect to
the quantity and quality of the source material, which may reduce production costs and
simplify downstream processing. Other than veterinary and environmental concerns,
which are outside the scope of this document, the issues they raise are principally those of
using a whole organism in production rather than a potentially more predictable cell culture
or fermentation system based on a seed lot. These include microbiological and virological
concerns, possible difficulties in purification and the consistency of the production and
purification process.
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VIRUS VALIDATION STUDIES: THE DESIGN,

CONTRIBUTION AND INTERPRETATION OF
STUDIES VALIDATING THE INACTIVATION AND
REMOVAL OF VIRUSES
Guideline Title Virus Validation Studies: The Design, Contribution and

Interpretation of Studies Validating the Inactivation and
Removal of Viruses
This version February 1996
force
Previous titles/other Validation of Virus Removal and Inactivation Procedures
references (III/8115/89) I CPMP/BWP/268/95
Additional Notes
This guideline discusses the need for, and the
contribution of, viral validation studies towards the viral
safety of biological products, providing guidance on the
design of a validation study including the choice of
viruses to be used and on the interpretation of the data.
This guideline was originally adopted in February 1991
under the title Validation of Virus removal and
Inactivation Procedures. It was revised to take into
consideration the ICH guideline Q5A Quality of
Biotechnological Products: Viral Safety Evaluation of
Biotechnological Products derived from Cell Lines of
Human or Animal Origin (CPMP/ICH/295/95)
CONTENTS
1. INTRODUCTION
2. SOURCES OF VIRAL CONTAMINATION
3. THE VALIDATION PROCESS
4. THE CHOICE OF VIRUSES FOR VALIDATION
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5. DESIGN O F VALIDATIO N STUDHS
6. INTERPRETATI
O N O F DATA
7. LIMITATI
O NS O F VALIDATIO N STUDHS
8. RE-EVALUATI
O N STUDIES
APPENDE* I: STATISTICAL EVALUATIO N O F VHIUS TITRES
APPENDED II: CALCULATI

O N O F REDUCTIO N FACTO RS
TABLE O F VIRUSES USED IN VALIDATIO N STUDIES
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VIRUS VALIDATION STUDIES: THE DESIGN,

CONTRIBUTION AND INTERPRETATION OF
STUDIES VALIDATING THE INACTIVATION AND
REMOVAL OF VIRUSES
L INTRODUCTION
1.1 This guideline discusses the need for and the contribution of viral validation studies
towards the viral safety of biological products. The principal aims of the guideline are to
provide guidance on the design of a validation study including the choice of viruses to be
used and on the interpretation of the ensuing data especially with respect to defining a
process step which can be considered to be effective in the inactivation and/or removal of
viruses.
1.2 The guideline concerns the validation of virus inactivation and/or removal
procedures for all categories of medicinal biological products for human use with the
exception of live viral vaccines including genetically engineered live vectors. The type of
products covered include:
products derived from in vitro culture of cell lines of human or animal origin,
products derived from in vivo culture of cell lines, or from organs or tissues of human
or animal origin,
products derived from blood or urine or other biological fluids of human or a n i m a l
origin.
1.3 The risk of viral contamination is a feature common to all biologicals whose
production involves the use of material of animal or human origin. Viral contamination of
a biological may arise from the source material, e.g. cell banks of animal origin, human
blood, human or animal tissues, or as adventitious agents introduced by the production
process, e.g. the use of animal sera in cell culture.
1.4 In the past, a number of biologicals administered to humans have been contaminated
with viruses. In several instances, the virus was only identified many years after the
product had been introduced into the market since contamination occurred prior to adequate
knowledge concerning the presence of the infectious agents. The primary cause of these
viral transmissions has been contamination of the starting or source materials. Examples
include Yellow Fever vaccine which was contaminated by avian leukosis virus by virtue of
its production in naturally infected hens eggs, whilst SV40 was a contaminant of poliovirus
and adenovirus vaccines prepared in the 1950's on primary cultures of kidney cells obtained
from Rhesus monkeys naturally harbouring a clinically inapparent infection with SV40. In
addition, viruses present in human plasma, e.g. and HCV, have contaminated blood
products whilst human growth hormone extracted from the pituitaries of cadavers has been
implicated in the transmission of the aetiological agent responsible for Creutzfeldt-Jakob
disease. Contamination of a biological can also arise from the use of infected material
during production or as an excipient. Perhaps the most notable was Yellow Fever vaccine
contaminated with HBV present in human serum used as a stabiliser in the 1940's.
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1.5 Three principal complementary approaches can be adopted to control potential viral
contamination of biologicals:
(i) selecting and testing source material for the absence of detectable viruses,
(ii) testing the capacity of the production processes to remove or inactivate viruses,
(iii) testing the product at appropriate stages of production for freedom from detectable
viruses.
No approach provides a sufficient level of assurance alone and this will only be achieved
using a combination of the above.
1.6 Testing of starting materials is essential to minimise viral contamination. While
tests may be able to detect one or more virus species, no single test will be able to
demonstrate the presence of all known viruses. Moreover all test systems require a
minimum level of viral contamination to record a positive and tests are also limited by
statistical considerations in sampling. Some tests, e.g. the test for antibody to HCV in
human plasma, may measure markers of infection which only become positive sometime
after infection. Similar considerations apply to testing of the final product.
1.7 Therefore establishing the freedom of a biological from infectious virus will in
many instances not derive solely from direct testing for their presence, but also from a
demonstration that the manufacturing process is capable of removing or inactivating them.
Validation of the process for viral inactivation/removal can play an essential and
important role in establishing the safety of biological products especially when there is a
high potential for the source material to be contaminated with a virus known to be pathogenic
for man, e.g. plasma derived products. Also, since many instances of contamination in the
past have occurred with agents whose presence was not known or even suspected at the time
of manufacture, an evaluation of the process can provide a measure of confidence that a
wide range of viruses including unknown, harmful viruses, may be eliminated.
1.8 The intention of this note for guidance is to provide a general framework for
validation studies and the virological approach which should be used in the design of virus
validation studies. Manufacturers should apply the recommendations presented here to their
specific product taking into consideration the nature of the source material, the procedures
used for production and purification and any other factors which can have consequences on
this safety issue. The approach used by manufacturers in studies for evaluating virus
elimination should be explained and justified.
2. SOURCES OF VIRAL CONTAMINATION

Viral contamination of biologicals can arise in the following ways:
2.1 Source material may be contaminated with a virus indigenous to the species of
origin. Blood can harbour many viruses and the use of products derived from human
plasma has caused infections by HBV, HCV, HTV, parvovirus B19 and occasionally HAV.
Murine viruses, some of which are pathogenic for man, may contaminate murine
hybridomas. Cell lines which are intended to be used for genetic manipulation may be
contaminated by viruses and, therefore, they should be chosen carefully and tested for
freedom from detectable adventitious agents even before genetic manipulation, in order to
start with a well characterised cell line.
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22 Cells may have a latent or persistent infection, for example, a herpesvirus or a

retrovirus, which may be transmitted vertically from one cell generation to the next as a
viral genome and which may be expressed intermittently as infectious virus.
2.3 The process of construction of a production cell line may introduce a contaminant
virus indigenous to another species, e.g. an EBV transformed human lymphoblastoid cell
line secreting a monoclonal antibody can be infected with a murine retrovirus after fusion
with a murine myeloma.
2.4 Adventitious viruses may be introduced by the use of contaminated animal products
in the production process e.g. cell cultures may be contaminated with bovine viruses through
the use of bovine sera or a murine monoclonal antibody used in affinity chromatography
may contaminate a product with a murine virus.
2.5 Other sources of contamination, e.g. operating personnel or raw materials of non-
biological origin, are possible.
3. THE VALIDATION PROCESS

3.1 The aim of viral validation studies is:
(i) to provide evidence that the production process will effectively inactivate/remove
viruses which are either known to contaminate the starting materials, or which could
conceivably do so, and
(ii) to provide indirect evidence that the production process might inactivate/remove novel
or unpredictable virus contamination.
This is achieved by deliberately adding ('spiking') a virus to material at various
production steps and measuring its removal or inactivation during the subsequent
individual step or steps. This will identify production steps which are effective i n
reducing the level of infectious virus and provide an estimate of the overall ability of
the process to eliminate contaminating viral infectivity.
3.2 Virus validation studies, as with direct testing of materials at appropriate steps,
contribute to confidence in the virological safety of the product. However, all virus
validation studies must be regarded as an approximation to the true capacity of the process
since it may be difficult or impossible to conduct a perfect validation study of a process
because of the large numbers of complex variables involved. Results have shown that even
small modifications in procedure or the particular laboratory strain of virus used can have
a large effect on virus removal or inactivation.
3.3 Where the starting or source material is less well characterised, such as blood,
tissues and organs of human or animal origin, or when cells have been cultured by in vivo
techniques, there is a higher possibility of viral contamination and the manufacturing
process will normally incorporate one or more effective virus inactivation/removal steps.
Products derived from human plasma raise particular viral safety concerns and specific
guidance is given in the guideline on Medicinal Products Derived From Human Plasma.
3.4 In the past, where the starting material posed a lower virological risk, such as a fully
characterised cell bank, the purification process often did not contain a specific virus
inactivation/removal step and a validated purification process was considered to give
sufficient levels of viral inactivation/removal. Clinical experience has not revealed any
problems with this approach. However, some manufacturers of monoclonal antibodies
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(mAbs) are introducing specific viral inactivation/removal steps into their production
process since mAb producing cell lines of murine origin inevitably secrete variable
quantities of retroviruses which may be infectious.
3.5 It should be borne in mind that cell culture systems inherently support virus
replication. Therefore, a distinct low level of risk of viral contamination of the culture
persists despite a high level of cell bank characterisation and occasional cases of
adventitious virus contamination have been reported.
3.6 The justification for, and the extent of, the required validation studies will vary
depending on the manufacturing process and type of product (e.g. species of origin of
starting material, whether source material is variable or defined, stability of the active
material, etc.). The appropriateness of the studies will be reviewed on a case by case basis.
4. THE CHOICE OF VIRUSES FOR VALIDATION

4.1 Viruses for validation should be chosen firstly to resemble viruses which may
contaminate the product as closely as possible and secondly to represent as wide a range of
physico-chemical properties as possible in order to test the ability of the system to eliminate
viruses in general.
4.2 Most validation studies employ laboratory strains of virus which can be produced
and assayed conveniently. However, experience has shown, and manufacturers should be
aware, that different laboratory strains of virus may have different properties from each
other and from naturally occurring viruses. Consequently, any virus used in a validation
study is actually a model virus. The manufacturer should justify the choice of viruses in
accordance with the aims of the validation study and the principles laid down in this
guideline. Unless otherwise justified, where two similar viruses could be used for
validation studies either because of their equal resemblance to possible contaminants or
similarities in their properties, the virus considered to be the more resistant should be used.
4.3 Examples of the choice of viruses are:
(i) Human plasma-derived clotting factor concentrates have been contaminated by HIV.
Thus the production process for such materials must be evaluated for its ability to
inactivate/remove infectious HIV.
(ii) Cell lines derived from rodents usually contain endogenous retroviral particles which
may be infectious (C-type particles) or non-infectious (A-type particles). Where the
source material is obtained from rodent cell lines, the production process should be
evaluated for its ability to inactivate/remove one of the closely related laboratory
murine retroviruses.
(iii) Examples of viruses representing a range of physico-chemical properties which have
been used to evaluate the general ability of a process to remove virus infectivity
include:
a) SV40, poliovirus or an animal parvovirus as small non-enveloped viruses,
b) a parainfluenza or a murine retrovirus as large enveloped RNA viruses,
c) a herpesvirus as a large DNA virus.
Examples of viruses which have been used in the past in validation studies are given i n
Table 1. However, since these and the viruses mentioned above are merely examples, the use
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of any of them is not mandatory and manufacturers are invited to consider other viruses
especially those which may be more appropriate for their individual processes. Further
guidance on the choice of viruses for the validation of manufacturing processes of plasma
derivatives is provided in the guideline Medicinal Products Derived From Human Plasma.
4.4 There should be an efficient, sensitive and reliable infectivity assay for the viruses
used. Viruses which can be grown to high titre will be desirable, although this may not
always be possible.
4.5 Products derived from ovine, caprine or bovine tissues raise the problem of
contamination by agents of transmissible spongiform encephalopathy, such as scrapie,
which accumulate in the central nervous system and lymphoid tissue. These agents are the
subject of a separate note for guidance on Minimising the Risk of Transmitting Agents causing
Spongiform Encephalopathy via Medicinal Products.
5. DESIGN OF VALIDATION STUDIES

5.1 Validation studies involve the deliberate addition of a virus at various production
steps and measuring the extent of its removal/inactivation during the subsequent individual
step or steps. It is not necessary to validate every individual step of a manufacturing process.
Only those steps which are likely to contribute to inactivation/removal of a virus need to be
subject to a validation study.
5.2 GMP restraints prevent the deliberate introduction of any virus into the production
facilities. Therefore, the validation should be conducted in a separate laboratory equipped for
virological work on a scaled-down version of the production process and performed by staff
with virological expertise in conjunction with the production bioengineers. Studies should be
carried out in accordance with the principles of GLP.
5.3 The comparability of the model and full scale procedures is the premise on which the
results obtained with the scaled-down system can be accepted in evaluating the virus safety
of the product. Therefore, the validity of the scaling down should be demonstrated, by
comparison of process parameters such as pH, temperature, concentration of protein and
other components, reaction time, column bed height, linear flow rate, flow rate to bed height
ratio, elution profile and step efficiency (e.g. yield, balance, specific activity, composition).
Deviations which cannot be avoided should be discussed with regard to any potential
influence on the results.
5.4 Whenever possible, it should be shown whether the reduction in virus infectivity is
accomplished by inactivation of virus or by removal of virus particles. This may be
achieved by establishing the kinetics of loss and/or a balance of infectivity, as appropriate.
Processes which reduce virus infectivity by inactivation are potentially more easily
modelled than those which physically remove particles. For a viral inactivation step, the
kinetics of inactivation should be studied and included in both tabular and graphical form
in reports. Where the inactivation is too rapid to plot the kinetics using process conditions,
further studies should be performed in order to prove that infectivity is indeed lost by
inactivation. Thus appropriate controls should be introduced to detect possible interference
with the assay from the sample or the matrix into which it is introduced and the limits of
detection should be established.
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5.5 Production parameters which influence the effectiveness of a process step to

inactivate/remove viruses should be explored and the results used in setting appropriate in-
process limits. Critical parameters include:
mechanical parameters such as flow rates, mixing rates, column dimensions, column
reuse, etc.
physico-chemical parameters such as protein content, pH, temperature, moisture
content, etc.
5.6 Antibodies present in the starting material may affect the behaviour of a virus i n
partition or inactivation steps. Validation studies should take this into account.
5.7 The validity of the log reduction achieved will be established from investigation of
the effects of variation in critical process parameters used to set in-process limits.
5.8 Published work concerning the ability of related or generic processes to
inactivate/remove viruses may provide an indication of which steps are likely to be
effective. However, the variability intrinsic to validation studies arising from the need to
model the process, choose viruses to be used and explore full scale production parameters on
a laboratory scale, means that validation data must be based on experimental studies
provided by the
5.9 The amount of virus added to the starting material for the production step which is to
be studied should be as high as possible in order to determine the capacity of the production
step to inactivate/remove viruses adequately. However, the virus spike should be added such
that the composition of the production material is not significantly altered (typically the
volume of the virus spike will be less than 10%). Whenever possible, calculated reduction
factors should be based on the virus which can be detected in the spiked starting material
and not on the amount of virus added.
5.10 If possible, virus in samples from model experiments should be titrated without
further manipulations such as ultra-centrifugation, dialysis or storage. Where further
treatments are unavoidable, e.g. to remove inhibitors or toxic substances, or storage for a
period to ensure that all samples are titrated together, appropriate controls should be included
to determine what effect the procedures have on the result of the study. Effects of the sample
on the detection system, including toxic effects, should be recorded as they influence the
limits of detection.
5.11 Quantitative infectivity assays should be performed according to the principles of
GLP and may involve plaque formation, detection of other cytopathic effects such as syncytia
or foci formation, end point titrations (e.g. TCID50 assays), detection of virus antigen
synthesis or other methods. The method should have adequate sensitivity and reproducibility
and should be performed with sufficient replicates and controls to ensure adequate statistical
accuracy of the result (see Appendix I).
5.12 Nucleic acid amplification methods, e.g. PCR, are a promising approach capable of
great sensitivity in detecting viral genomes and also can detect viruses such as hepatitis
and C for which culture systems are not available. However, an important limitation of the
technology is that inactivated virus may still score positive in a genome amplification assay
and thus may underestimate the degree of virus inactivation obtained by a potentially
effective step. On the other hand, PCR may be of value in studies of processes which depend
on virus removal. The use of this technology poses major problems in terms of
quantification, standardisation, quality control and interpretation of results. Validation and
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standardisation of these assays must be unambiguously demonstrated before they are

acceptable and extreme caution used in interpretation of both positive and negative results.
5.13 Assurance should be provided that any virus potentially retained by the production
system will be adequately destroyed prior to reuse of the system, e.g. by sanitisation of
columns, etc.
6. INTERPRETATION OF DATA
6.1 A combination of factors must be considered when judging the effectiveness of a
virus inactivation/removal step. Assessment of a step based solely on the quantity of virus
inactivated/removed can lead to the conclusion that a process meeting specified levels of
virus reduction will produce a safe product. This is not necessarily the case. The following
factors all contribute in defining the effectiveness of a step and the data must be carefully
evaluated in each case:
(i) The appropriateness of the test viruses used (see Section 4).
(ii) The design of the validation studies (see Section 5).
(iii) The lgn, reduction achieved. Log reductions of the order of 4 logs or more are
indicative of a clear effect with the particular test virus under investigation. However,
it is emphasised that log number reduction cannot be used as the single, absolute
measure of the effectiveness of a step.
(iv) The kinetics of inactivation. This will indicate whether or not the measured log
reduction is a conservative estimate. Virus inactivation is usually not a simple first
order reaction and often has a fast initial phase followed by a slower phase. However,
a dramatic reduction in the rate of inactivation with time may suggest a loss of
effectiveness of the inactivating agent or that a residual virus fraction is resistant to
the inactivating agent and implies that the step is neither highly effective nor robust.
(v) The nature of inactivation/removal and whether it is selective for only certain classes
of virus. A process step may be highly effective for some viruses but ineffective against
others, e.g. S/D treatment is effective against lipid-containing but not lipid-free
viruses.
(vi) The susceptibility of virus inactivation/removal to small variations in-process
parameters will affect the confidence placed in a step.
(vii) The limits of assay sensitivities.
It is the combined evaluation of the above factors that will lead to a decision on whether a
process step can be regarded as effective, moderately effective or ineffective in the
inactivation/removal of viruses.
6.2 The following examples are intended to illustrate some of these principles and are
neither definitive nor all encompassing:
(i) Where a process step is challenged with 6 logs of virus and 4 logs are recovered, the
step cannot be claimed to be effective, although it may contribute to overall removal.
(ii) Where a process step is challenged with 6 logs of virus, but because of the cytotoxicity of
the product the limit of assay sensitivity in the product is 4 logs, only 2 logs of removal
have been demonstrated, and the step cannot be claimed to be effective. The process step
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may in fact be able to remove far greater quantities of virus, which might be
demonstrated by a different experimental design.
(iii) Where a process step is challenged with 6 logs of virus and 2 logs are recovered,
substantial amounts of virus have been removed. The product is not virologically
sterile. However, if this reduction is reproducible and not influenced by process
variables, the step is of some efficacy. It contributes to overall reduction of virus load
and may be counted as such.
(iv) Where a process step is challenged with 6 logs of virus and no virus is detected in the
product with a limit of sensitivity of the order of 2 logs, approximately 4 logs of
removal have been demonstrated. This is substantial and the step may in fact remove
far greater quantities than can be quantified or claimed.
(v) Where virus is inactivated, the kinetics of loss of infectivity are important. If a
process step involves prolonged incubation, e.g. heating for ten hours, and infectivity
reaches the limits of detection rapidly, the process is likely to have a greater virucidal
effect than can often be demonstrated. On the other hand, if infectivity is lost slowly
and the limits of detection are reached towards the end of the treatment period, the step
provides less assurance of viral safety.
6.3 In general, partition processes are not considered to be effective viral removal steps
although it is recognised that they can contribute to virus removal. Partition processes
usually have a number of variables that are difficult to control and are often difficult to
scale down for validation purposes. Furthermore, partitioning is dependent on the extremely
specific physico-chemical properties of a virus which influence its interaction with gel
matrices and precipitation properties. Thus a model virus may be partitioned in a completely
different manner to a target virus because of relatively minor differences in surface
properties such as glycosylation. Even a relevant virus propagated in the laboratory may act
differently from the wild-type virus in this respect. However, if a partition process gives
reproducible reduction of virus load and if manufacturing parameters influencing the
partition can be properly defined and controlled and if the desired fraction can be reliably
separated from the putative virus-containing fraction, then it could fit the criteria of an
effective step.
6.4 The objective of the validation is to identify steps effective in the

inactivation/removal of viruses and to obtain an estimate of the overall capacity of the
manufacturing process to inactivate/remove them. An overall reduction factor is generally
expressed as the sum of individual factors (see Appendix II). However, a simple summing of
low individual reduction factors may be misleading. Reductions in virus titre of the order of
1 log or less are considered to be unreliable because of the limitations of virus validation
studies and should be ignored. Manufacturers should differentiate effective steps from
process steps which may contribute to removal but upon which less reliance can be placed.
Consideration should also be given to whether virus surviving one step would be resistant to
a subsequent step or alternatively have increased susceptibility. In general, a single step
having a large effect gives more assurance of viral safety than several steps having the
same overall effect.
6.5 If little reduction of infectivity is achieved by the production process, and the removal
of virus is considered to be a major factor in the safety of the product, a specific, additional
inactivation/removal step or steps should be introduced.
6.6 For all viruses, manufacturers will be expected to justify the acceptability of the
reduction factors obtained. Results will be considered on a case by case basis.
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6.7 The GMP principle that material subjected to an effective virus inactivation/removal
step should be segregated from untreated material should be rigorously applied.
7. LIMITATIONS OF VALIDATION STUDHSS

Validation studies are useful in contributing to the assurance that an acceptable level of
safety in the final product is established and do not by themselves establish safety. A number
of factors in the design and execution of virus validation experiments may lead to a n
incorrect estimate of the ability of the process to remove naturally occurring virus
infectivity. These factors include the following points.
7.1 Experience has shown that different laboratory strains of virus may differ in their
sensitivity to the same treatment. The particular virus chosen may therefore not resemble
the virus for which it has been chosen as a model. Native viruses may have unpredicted
properties, for example association with lipid, which may affect their properties. Virus
preparations used to validate a production process are likely to be produced in tissue culture.
The behaviour of tissue culture virus in a production step may be different from that of the
native virus for example if native and cultured viruses differ in purity or degree of
aggregation. The strains of virus, their cultivation and assay, and details of sampling and
storage should all be documented.
7.2 There are some situations in which it may not be valid to add logarithmic
reductions. For example, if a matrix is able to adsorb 104 infectious units of a virus and then
cannot adsorb further material with comparable affinity then it will remove all virus when
challenged with 104 infectious units, but only 1% when challenged with 106. The clearance
measured may therefore differ with the challenge titre.
7.3 Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid
initial phase is followed by a slower phase. It is possible that virus escaping a first
inactivation step may be more resistant to subsequent steps. As a consequence, the overall
reduction factor is not necessarily the sum of reduction factors calculated from each
individual step in which a fresh virus spike suspension is used. For example if the resistant
fraction takes the form of virus aggregates, infectivity may be resistant to a range of
different chemical treatments and to heating.
7.4 Model scale processing is likely to differ from full scale processing despite care
taken to design the scaled down process.
7.5 The presence of antibodies to a native virus may affect partition of the virus or its
susceptibility to chemical inactivation; but it may also complicate the design of the study by
neutralising infectivity. The appropriateness of the study design may be difficult to judge.
The level of antibody present may be considered a significant process variable.
7.6 Small differences in production parameters such as protein content or temperature
can produce large differences in the reduction of virus infectivity by whatever mechanism.
8. RE-EVALUATION STUDIES
8.1 Changes to the production process may necessitate a new validation study.
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8.2 As scientific experience accumulates, processes will require re-examination to

ensure that they remain of an acceptable standard.
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APPENDE* 1
STATISTICAL EVALUATION OF VIRUS TITRES AND REDUCTION FACTORS AND
ASSESSMENT OF THEIR VALIDITY
Virus titrations suffer the problems of variation in common to all biological assay systems.
Assessment of the accuracy of the virus titrations and reduction factors derived from them
and the validity of the assays are therefore necessary to define the reliability of a study. The
objective of statistical evaluation is to establish that the study has been carried out to an
acceptable level of virological competence.
1. Assay methods may be either quantal or quantitative. Quantal methods include
infectivity assays in animals or in tissue culture infectious dose (TCID) assays, i n
which the animal or cell culture is scored as either infected or not. Infectivity titres are
then measured by the proportion of animals or cultures infected. In quantitative
methods, the infectivity measured varies continuously with the virus input.
Quantitative methods include plaque assays where each plaque counted corresponds to
a single infectious unit. Both quantal and quantitative assays are amenable to
statistical evaluation.
2. Variation can arise within an assay as a result of dilution errors, statistical effects
and differences within the assay system which are either unknown or difficult to
control. These effects are likely to be greater when different assay runs are compared
(between assay variation) than when results within a single assay run are compared
(within assay variation).
3. The 95% confidence limits for within assay variation and for between assay variation
normally should be of the order 0.5 log10 or better. Between assay variation can be
monitored by the inclusion of a in-house reference preparation, the estimate of whose
potency should be within approximately 0.5 log10 of the mean estimate established in the
laboratory for the assay to be acceptable. Within assay variation can be assessed by
standard textbook methods. In any particular experiment, if the precision of the
titration is less than these target figures, the study may still be acceptable if justified.
4. The reduction in virus load should be calculated from the experimentally determined
virus titres. The 95% confidence limits of the reduction factors should be obtained
wherever possible. They can be approximated by +^](s +a ), where s is the 95%
confidence limits for the viral assays of the starting material, and for the viral
assays of the material after the step.
If after an inactivation/removal step no sample shows signs of infectivity, a reduction factor
cannot be estimated by statistical means. To obtain an estimate of a minimum reduction
factor, the titre should be expressed as less than or equal to one infectious unit in the volume
of the highest concentration tested. Especially after potent inactivation processes, it can be
expected that no sample shows signs of infectivity. To make the estimated m i n i m u m
reduction factor of an effective inactivation process as large as possible, as much processed
undiluted material as possible should be sampled.
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APPENDLX II
CALCULATION OF REDUCTION FACTORS
The virus reduction factor, R, for an individual inactivation or removal step is given by the
expression:
VI T\
R = log
V2 Tl
where, R = the reduction factor,
VI = volume of starting material,

T l = concentration of virus in starting material,
V2 = volume of material after the step, and
T2 = concentration of virus after the step.
This formula takes into account both the titre and the volume of the material before and after
the step.
Reduction factors are normally expressed on a logarithmic scale which implies that, while
residual virus infectivity may be greatly reduced, it will never be reduced to zero. The
European Pharmacopoeial convention* with respect to methods of sterilisation is that
processes which deliver a sterility assurance level (SAL) of 10'6 or better for bacteria, moulds
and yeasts are considered adequate. A SAL of 10"6 denotes a probability of not more than one
viable micro-organism in 1 IO"6 sterilised items of the final product.
"Methods of Preparation of Sterile Products" monograph of the European Pharmacopoeia
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TABLE 1
EXAMPLES OF VIRUSES WHICH HAVE BEEN USED IN VIRUS VALIDATION STUDIES
Resistance to
Natural Physico-
Virus Family Genus Genome Env Size Shape
Host chemical
Treatment*
Vesicular Rhabdo Vesiculovirus Equine RNA Yes 70x175 nm Bullet shaped Low
stomatitis virus Bovine
Parainfluenza Paramy Paramyxovirus Various RNA Yes 100-200nm Pleo/Spher Low

virus zo
Human imrauno- Retro Lentivirus Man RNA Yes 8)-100nm Spherical Low
deficiency virus
Murine leukaemia Retro TypeC Mouse RNA Yes 8)-110nm Spherical Low
virus (MuLV) oncovirus
Sindbis virus Toga Alpha virus Man? RNA Yes 60-70nm Spherical Low
Bovine viral Toga Pesti virus Bovine RNA Yes 50-70nm Pleo-Spher Low
diarrhoeal virus
(BVDV)
Pseudorabies virus Herpes Varicellovirin Swine DNA Yes 120-200nm Spherical Med
Poliovirus, Sabin Picorna Enterovirus Man RNA No 25-30nm Icosahedral Med

typel
Encephalomyocard Picorna Cardiovirus Mouse RNA No 25-30nm Icosahedral Med

itis virus (EMC)
Reovirus 3 Reo Orthoreovirus Various RNA No 60-80nm Spherical Med
Hepatitis A Picorna Hepato virus Man RNA No 25-30nm Icosahedral High
SV40 Papova Polyomavirus Monkey DNA No 40-50nm Icosahedral V.High
Parvoviruses Parvo Parvovirus Canine DNA No 18-24nm Icosahedral V.High

(canine, porcine) Porcine
This Table gives an Incomplete list of viruses which have been used in validation studies. Consequently, the use of any of the viruses in the Table is not
mandatory and manufacturers are invited to consider other viruses especially those which may be more appropriate for their individual production processes.
This general classification is based on validation studies of production processes
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VALIDATION OF VIRUS REMOVAL/INACTIVATION

PROCEDURES: CHOICE OF VIRUSES
Guideline Title Validation of Virus Removal and Inactivation

Procedures: Choice of Viruses
force
references
Additional Notes Data on the validation of processes for the removal or
inactivation of viruses are rapidly accumulating.
Validation studies should therefore be reviewed and
updated if necessary at intervals to ensure that they are
consistent with current scientific knowledge.
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VALIDATION OF VIRUS REMOVAL/INACTIVATION

PROCEDURES: CHOICE OF VIRUSES
Data on the validation of processes for the removal or inactivation of viruses are rapidly
accumulating. Validation studies should therefore be reviewed and updated if necessary at
intervals to ensure that they are consistent with current scientific knowledge.
Where possible studies should be performed with species of viruses which may be present i n
plasma, such as HIV. In some cases this will not be possible. For example hepatitis C virus
(HepCV) cannot be grown or assayed readily, so that model viruses must be used. The model
virus chosen should be as close in its relevant properties to HepCV as possible. In the past,
togaviruses such as Sindbis virus, flaviviruses such as yellow fever virus and pestiviruses
such as bovine viral diarrhoea virus have been used as models for HepCV. All have
properties in common with HepCV and the results have generally been consistent with the
safety of the product in clinical use. Further data on the behaviour of the viruses are needed
to identify the most appropriate model.
Hepatitis A virus is a non-enveloped virus of the Picornavirus family which is believed to
have been transmitted by clotting factors. Hepatitis A virus should be used whenever possible
as it is thought to be significantly more hardy than other picornaviruses. However plasma
pools can contain neutralising antibodies which may make validation studies difficult. In
addition, hepatitis A virus may be technically demanding to grow and assay. Alternative
viruses such as EMC or Theiler's virus or the use of pools screened for the absence of
antibodies to HAV in validation studies may be considered where relevant. The choice of
model viruses for hepatitis A, if any, will become clear with further studies.
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MINIMISING THE RISK OF TRANSMITTING AGENTS

CAUSING SPONGIFORM ENCEPHALOPATHY VIA
MEDICINAL PRODUCTS
Guideline Title Minimising the Risk of Transmitting Agents Causing

Spongiform Encephalopathy via Medicinal Products
force
Status Last revised 1991, revision in progress as of June 1997
Previous titles/other None/ III/3298/91
references
Additional Notes This note for guidance considers the implication of BSE
for medicinal products which contain materials of
bovine origin and methods for minimising the risk of
transmission by their use.
CONTENTS
1. GENERAL REMARKS
2. SCOPE OF THE NOTE FOR GUDDANCE
3. MANUFACTURE (INCLUDING COLLECTION OF SOURCE MATERIALS)
4. PROCEDURES WHICH REMOVE OR INACTrVATE AGENTS CAUSING

SPONGIFORM ENCEPHALOPATHIES
5. CONCLUDING REMARK
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MINIMISING THE RISK OF TRANSMITTING AGENTS

CAUSING SPONGIFORM ENCEPHALOPATHY
VIA MEDICINAL PRODUCTS
1 GENERAL REMARKS
Bovine spongiform encephalopathy (BSE) was first recognised in the United Kingdom i n
1986. Since then a large number of cattle and individual herds have been affected. This note
for guidance considers the implication of the disease for medicinal products and methods for
minimising the risk of transmission by their use.
The naturally occurring spongiform encephalopathies include scrapie (in sheep and goats),
chronic wasting disease (in mule deer and elk), bovine spongiform encephalopathy (BSE; i n
cattle) as well as Creutzfeldt-Jakob Disease (CJD) and Kuru (in humans). Agents causing
these diseases replicate in infected individuals without being detectable by diagnostic tests
applicable to the living organism. After incubation periods of up to several years the agents
cause disease and, finally, lead to a fatal outcome. No means of therapy are known.
Diagnosis is based on clinical signs with post mortem confirmation of characteristic brain
lesions by histopathology or immunological detection of the fibrillary proteins specific for
the spongiform encephalopathies. The demonstration of infectivity by the inoculation of
suspect tissue into target species or laboratory animals may also be used for confirmation
but with an incubation period of months or years. Iatrogenic transmission of spongiform
encephalopathies has been reported. In sheep scrapie has been accidentally transmitted via
the application of Louping III vaccine prepared from pooled, formaldehyde treated ovine
brain and spleen in which material from scrapie infected sheep had been inadvertently
incorporated. In humans cases of transmission of CJD have been reported which have been
attributed to the repeated parenteral administration of growth hormone and gonadotropin
derived from human cadaveric pituitary glands. Cases of CJD have also been attributed to
the use of contaminated instruments in brain surgery and with the transplantation of
human meninges and cornea.
There is no evidence that spongiform encephalopathies have been transmitted from animals
to humans. However, the possibility of such transmissions, although remote, cannot be
dismissed. Therefore due prudence is warranted if biological materials are used for the
manufacture of medicinal products from species affected via non-experimental routes by
those diseases, primarily ruminants and among these especially cattle, sheep and goats.
Information on the characteristics of the agents is limited. They are extremely resistant to
the chemical and physical procedures that inactivate conventional viruses. They do not
induce a detectable immune response. There are natural barriers which limit the
interspecies spread of infection, but they can be crossed under appropriate circumstances
usually involving efficient routes of administration and high doses of agent. Studies on
laboratory animals have shown that intracerebral inoculation is much more efficient than
any other route and is followed in decreasing order of efficiency, by intravenous,
intraperitoneal and subcutaneous administration. The oral route is less efficient than the
parenteral routes. In some cases species barriers can be crossed only after passage of the
agent through intermediary species.
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Human beings must have been naturally exposed to the scrapie agent for at least 200 years,
but despite extensive epidemiological studies no sign of transmission of scrapie to humans
has been detected. Insofar as BSE is different from scrapie, it is conceivable that also the
species barriers may be different. Therefore the recommendations below should be followed.
The acceptability of a particular medicinal product containing or derived from bovine
materials will be influenced by a number of factors including the selection and processing
of source materials, the age and geographical origin of the individual source animal, the
intended use of the product, its stipulated dose and route of administration, production
process and quality control. The state of science and technology must be taken into
consideration. All products will be considered on a case by case basis.

This note for guidance covers all medicinal products which contain active substances
and/or excipients derived from bovines, as well as medicinal products for which the
production process involves bovine materials.
The note also covers the use of such materials in procedures which are indirectly associated
with the manufacturing process, for example, in test media used in the validation of plant
and equipment to avoid cross-contamination.
3. MANUFACTURE (INCLUDING COLLECTION OF SOURCE

MATERIALS)
The safety of medicinal products can be further secured, and the risk of transmission of
infectious agents greatly reduced by combinations of the measures specified in this note for
guidance or other appropriate measures. The pharmaceutical manufacturers and the
producers of medicinal products of animal origin are responsible for the selection of
adequate measures.
3.1 Animals as source of materials

Careful selection of source materials is the most important criterion for the safety of
medicinal products. The use of source materials from countries where there is a high
incidence of BSE is to be avoided.
The following criteria should be taken into account when sourcing materials.
3.1.1 Materials may be sourced from countries which have not reported cases of BSE, if
they have an effective veterinary service capable of detecting a low incidence of disease and
if BSE is reportable. Official certification should be presented.
In addition, it should be ensured that there is no risk of BSE infection from the following
factors:
a) the feeding to ruminants of ruminant protein derived from the specified offal (brain,
spinal cord, spleen, thymus, tonsil and intestine from duodenum to rectum, placenta),
either produced in the country or imported from other countries;
b) the processes used in the rendering industry;
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3AB10a
c) scrapie-associated factors:
- the incidence and prevalence of scrapie;
- the ratio of sheep and goats to cattle;
- the relative geographical distribution of sheep and goats to cattle, where this
might have led to the use of sheep material in cattle feed in the past;
d) importation of cattle above the age of 6 months from countries where a high incidence
of BSE has occurred and/or importation of progeny of affected females.
3.1.2 Materials may also be sourced from countries where a low number of cases have
occurred, if in addition to the factors in paragraph 3.1.1:
- BSE has been made legally notifiable;
- the carcasses of all affected animals are destroyed;
- the progeny of affected females are not used.
3.1.3 Satisfactory source materials may be obtained from established and monitored herds,
where their feeding and breeding history is documented. This is possible even in countries
with a high incidence of BSE.
3.2 Age of animals

Natural scrapie or BSE has not been detected in animals under the age of 6 months.
Therefore, cattle yielding source materials should not be older than 6 months unless
otherwise justified.
3.3 Parts of animal bodies, body fluids and secretions as starting

materials
In the infected animal different organs and secretions contain different maximum
concentrations of infectivity. On the basis of experimental data on transmissible spongiform
encephalopathies, organs, tissues and fluids can be classified into four main groups bearing
different potential risks, as shown in the table below. These potential risks, amongst other
criteria, should be considered for the selection of source materials.
Although being based on studies of natural scrapie, the classification can be applied to the
related diseases in mule deer (CWD) and cattle (BSE), which have similar incubation
periods. However, the categories in the table are only indicative and it is important to note
the following points:
- the classification of tissues shown in the table is based on titration of infectivity i n
mice by the intracerebral route ( 1-3). In experimental models using agent strains
adapted to laboratory animals, higher titres and a slightly different classification of
tissues may occur (4-5). For experimental intraspecies transmission, titres of up to 10^
have been reported (4-5). Therefore, the risks could be higher when medicinal products
are manufactured from, and used in, the same species;
- the potential risks will be influenced by the circumstances in which tissues were
removed, especially by contact of material of a low-risk group with that of a high-risk
group. Thus the contamination of some tissues may be increased if infected a n i m a l s
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are slaughtered by penetrative brain stunning, or if the brain and/or spinal cord is
sawed;
- dura mater, hypophysis and pineal gland from animals older than six months should
be regarded as belonging to group 2 only if contamination with brain tissue can be
avoided;
- body fluids should be collected with minimal damage to tissue, and cellular
components should be removed; e.g. foetal blood should be collected without
contamination from placenta and amniotic fluids.
The information currently available suggests that, given assurances of adequate collection
and processing, certain materials and their derivatives are unlikely to present any risk of
contamination. These include: milk and its derivatives, for example, lactose and casein;
skin and its derivatives, for example, gelatine; hair and wool and their derivatives, for
example, wool alcohols and lanolin.
In addition, materials derived from rendered carcasses and subjected to rigorous processes
of extraction and purification (for example, triglycerides, glycerol, sorbitan esters, etc.
manufactured from tallow) may be considered unlikely to be contaminated.
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RELATIVE SCRAPIE INFECTIVITY TITRES IN TISSUES AND BODY FLUIDS FROM

NATURALLY INFECTED SHEEP AND GOATS WITH CLINICAL SCRAPIE *
CATEGORY I
High infectivity brain, spinal cord, (eye)
CATEGORY II
Medium infectivity ileum, lymph nodes, proximal colon, spleen, tonsil,
(dura mater, pineal gland, placenta), cerebrospinal
fluid, pituitary, adrenal
CATEGORY III
Low infectivity distal colon, nasal mucosa, sciatic nerve, bone marrow,
liver, lung, pancreas, thymus
CATEGORY IV
No detectable infectivity blood clot, faeces, heart, kidney, mammary gland,
milk, ovary, saliva, salivary gland, seminal vesicle,
serum, skeletal muscle, testis, thyroid, uterus, foetal
tissue, (bile, bone, cartilaginous tissue, connective
tissue, hair, skin, urine)
3.4 Cellular substrates

Cell lines known to be capable of concentrating or amplifying agents causing spongiform
encephalopathies must not be used in the manufacture of medicinal products, except for
reasoned exceptional cases.
4. PROCEDURES WHICH REMOVE OR INACTIVATE AGENTS

CAUSING SPONGIFORM ENCEPHALOPATHIES
Removal and inactivation procedures contribute to the reduction of the risk of infection.
Their effectiveness in removing infectivity during a given production process must be tested
and validated using appropriate model systems (presently: animal infection experiments).
Tissues in brackets were not titrated in the original studies1""', but their relative infectivity is indicated by
other data on spongiform encephalopathies. Materials not listed may be classified by analogy to those
mentioned on the basis of their composition.
No infectivity was transmitted in bioassays involving inoculation of up to 5 mg tissue into rodent trains.
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Whereas none of the following procedures may guarantee complete inactivation of the
infectious agents, the efficiency of the first three methods on this list is considered greatly
superior to that of the remaining ones:
- autoclaving at appropriate conditions (recommended parameters are 134-138C for 18
minutes for porous-load autoclaving, and 132C for one hour for gravity-displacement
autoclaving;
- treatment with sodium hydroxide (preferably: 1 N solution, for 1 h at 20C);
- treatment with sodium hypochlorite (preferably: solution containing at least 2%
available chlorine, for 1 h at 20C);
- autoclaving at shorter times and/or lower temperatures than those given above;
- extraction by organic solvents (use the organic phase);
- removal of protein by precipitation, ultracentrifugation or absorption;
- preparation of filtrates by passage through 10-nm-filters;
- passage through appropriate chromatographic columns (before reusing treat columns
for 4 h with at least 0.1 N sodium hydroxide);
- treatment with 6M urea (6).
5. CONCLUDING REMARK
Although this note for guidance relates particularly to BSE and materials of bovine origin,
similar considerations are also applicable to material from sheep, goats and other species
affected via non-experimental routes by agents causing spongiform encephalopathies.
Finally, while this note for guidance has general applicability, it may not be necessary to
fulfil all of the listed measures for all products. The potential risks associated with a given
medicinal product will have to be considered individually in the light of specific
circumstances and current knowledge.
1) Hadlow W J, Kennedy R C, Race R E, Eklund C M (1980) Vet Pathol 17, 187-199

2) Hadlow W J, Kennedy R C, Race R E (1982) J Infect Dis 146, 657-664
3) Kimberlin R H (1990) in Topley and Wilson's Principles of Bacteriology, Virology
and Immunity (L H Collier, M C Timbury, eds), Vol 4, pp 671-693, Edward Arnold,
London
4) Eklund C M, Kennedy R C, Hadlow W J (1967) J Infect Dis 117, 15-22
5) Diringer H, Hilmert H, Simon D, Werner E, Ehlers (1983) Eur J Biochem 134, 555-
560
6) Pocchiari M, Macchi G, Peano S, Conz A, Arch Virol (1988) 98, 131-135
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TESTS ON SAMPLES OF BIOLOGICAL ORIGIN
Guideline Title Test on Samples of Biological Origin


references
Additional Notes This document provides basic guidance on the
presentation of data validating test procedures carried out
for toxicological and pharmacological studies as well as
for clinical trials provided for by Directive 75/318/EEC as
amended with a view to the granting of a marketing
authorisation in respect of a medicinal product.
CONTENTS
1. INTRODUCTION
2. CRITERIA FOR VALIDATION OF TEST PROCEDURES
3. RECOMMENDATIONS
ANNEX
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TESTS ON SAMPLES OF BIOLOGICAL ORIGIN
L INTRODUCTION
The objective of analytical validation on samples of biological origin (plasma, urine, faeces
etc.) is to demonstrate the reliability of results for active substances and metabolites obtained
from pharmacokinetic, metabolic and bioavailability studies.
2. CRITERIA FOR VALIDATION OF TEST PROCEDURES

The validation criteria are those currently used in analytical chemistry (Good Laboratory
Practice) and consist of:
Specificity
Repeatability
Precision
Reproducibility
Accuracy
Linearity/Range/Sensitivity
Limit of detection
Limit of quantification
Each test procedure should be validated for each type of biological sample and each species
(animal, human).
If the same test procedure has been used during the development of the medicinal product (in
vitro) and during routine tests (in vivo), a revalidation is necessary.
The degree of validation depends, to a large extent, on the problem posed.
3. RECOMMENDATIONS
3.1 For assays on samples of biological origin, the following specific problems can arise,
which may influence both the validation and the interpretation of the results.
3.1.1 The test procedures (assays) carried out are not necessarily done in a single
laboratory, but in many and sometimes even outside of those of the manufacturer. Therefore,
it is very important - for the same test - to be able to compare the results between the two.
There are two cases to consider:
a) when the same test procedure is always used:
the quality control between laboratories is necessary (reproducibility);
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3AB10a
b) using different test procedures:

either:
it is necessary to have a reference test procedure (control, standard) developed
directly in the corresponding biological sample and used for either the assays of
substances to be analysed or as a test procedure to which other test procedures can
be referred to for validation (correlation between the two test procedures);
or:
it is recommended proper investigation of recovery in the individual methods
using the same reference material.
3.1.2 A significant time-lapse can pass between the moment of sampling and the moment
of analysis. For this reason, amongst others, it is necessary to know:
- the stability of the substances being examined in the biological fluid in the precise
storage conditions;
- the sorption of the substance by the sampling container and the stopper.
3.1.3 The test procedure may change over time according to the evolution of the problem
posed (clinical trials). In that case, a revalidation will be necessary.
3.2 Other recommendations:

3.2.1 A short description of the main principle of the test procedure should be indicated.
3.2.2 The test procedures, including the conditions of sampling, must be described
precisely, preferably in the form of a standard operation procedure.
This includes:
- the mode of sampling (type of container, anticoagulant, etc.);
- the conditions of storage before analysis;
- the exact description of the test conditions including precautions, methods of extraction,
reagents, reference substances and preparations;
- the exact description of the apparatus used;
- the verification of the test procedure under the defined operating conditions, for
example: verification of the separating power of a chromatographic system (system
suitability test);
- the detailed formulae of the calculation of results, including statistical evaluation as
appropriate.
3.2.3 The reference substances and preparations (in house standards) used for tests - if
pharmacopoeial or other official standards are not used- must be precisely described. Their
identity, purity and content must be fully established.
3.2.4 In all cases, the complete data which demonstrate validity should be indicated.
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ANNEX
Glossary
The annex is a glossary which should give the manufacturer a better understanding of the
different validation requirements and definitions. It should again be remembered here, that
the criteria which must be satisfied depend very much on the objective of the analysis.
1. TEST PROCEDURE
The test procedure is the total operation necessary to perform the analysis of an analyte:
preparation of the sample, of the reference substances or preparations, of the reagents, use of
the apparatus, calibration curve, formulae for the calculation, number of replicates and
operating procedure for the replicates etc.
2. SPECIFICITY
This means for:
IDENTIFICATION: to ensure the identity of an analyte
TESTS: to ensure that all the test procedures performed allow an evaluation
(Impurity content) of the content of impurities of an analyte i.e. related substances test,
heavy metals, organic solvent content etc.
ASSAY: to ensure that the signal measured with the test procedure comes
(Content or Potency) only from the substance being analysed i.e. no interferences from
excipients and/or degradation product and/or impurities.
A routine assay may not necessarily comply with the criterion of specificity. This can be
compensated by using one or more adequate related substances test(s) (applies mainly to
bulk material, see pharmacopoeia).
Specificity is assessed either by a single determination or by the total results of the test
procedures.
a) Specific test procedure:
a procedure to measure quantitatively a chemical-physical parameter or functional
group of one or even more but different analytes in the sample matrix;
for instance: titration of the carboxylic group of an acid, measure of the specific
absorbance, immunoassay.
b) Selective test procedure:
a procedure to detect qualitatively the analyte in the presence of components which
maybe expected to be present in the sample matrix;
for instance: chromatography, selective electrode.
c) Absolute test procedure:
a procedure which determines the molar purity of an analyte;
for instance: differential thermal analysis, phase solubility analysis.
Under the heading, the means of satisfying the criteria of specificity may be different:
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for instance; identification to ensure the identity of an analyte during:

- development: proof of the structure;
- quality control: comparison to a reference substance.
3. ACCURACY
The accuracy expresses the closeness of agreement between the value which is accepted
either as a conventional true value (in house standard) or an accepted reference
value(international standard, e.g. pharmacopoeial standard) and the value found (mean
value)obtained by applying the test procedure a number of times.
The accuracy provides an indication of systematic errors.
Several methods of determining accuracy are available of which the following are two
examples:
a) Comparing the proposed test procedure with a second test procedure, the accuracy of
which is stated and/or defined (for instance: pharmacopoeial method), (applies
normally to starting material).
b) Applying the test procedure to specimens or mixtures of excipients to which a known
quantity of the substance to be analysed has been added: the result maybe expressed as
percent recovery by the assay of known added amount of analyte (applies normally to
finished product).
4. PRECISION
The precision of a test procedure expresses the closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple sampling of the same
homogeneous sample under prescribed conditions.
Precision provides an indication of random errors.
4.1 Repeatability
Repeatability expresses the precision under same conditions:
- same analyst, . .
- same apparatus,
- short interval of time,
- identical reagents.
Results should be expressed as:
- repeatability standard deviation;
- repeatability coefficient of variation (relative standard deviation);
- the confidence interval of the mean value (n 2 6 = 0.05 or = 95%)
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4.2 Reproducibility
The reproducibility expresses the precision under different conditions:
for instance:
- laboratories,
- reagents from different sources,
- analysts,
days,
- apparatus from different manufacturers,
- etc.
Results should be expressed as:
- reproducibility standard deviation;
- reproducibility coefficient of variation (relative standard deviation);
- the confidence interval of the mean value (n > 6 = 0.05 or =95%).
5. LIMIT OF DETECTION (LOD)

The lowest amount of analyte in a sample which can be detected but not quantitated as an
exact value. The LOD is mostly a parameter of limit tests.
6. LIMIT OF QUANTITATION (LOQ)

The lowest amount of analyte in a sample which can be quantitatively determined with
defined precision and accuracy under the stated experimental conditions.
7. LINEARITY
The linearity of a test procedure is its ability (within a given range) to obtain test results
directly proportional to the concentration (amount) of analyte in the sample.
8. RANGE
The range of the test procedure is the interval between the upper and lower levels of analyte
(including these levels) for which the procedure has been demonstrated as suitable with
precision, accuracy and linearity using the method as written.
9. SENSITIVITY
Capacity of the test procedure to record small variations in concentration.
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TRANSLATION OF SOME IMPORTANT TERMS
FR DE EN
Exactitude Richtigkeit Accuracy

Fidlit Przision Precision
Spcificit Spezifizitt Specificity
Slectivit Selektivitt Selectivity
Linarit Linearitt Linearity
Intervalle linaire Linearer Bereich Linear range
Seuil de dtection Nachweisgrenze Limit of detection (LOD)
Seuil de quantification Bestimmungsgrenze Limit of quantitation (LOQ)
Sensibilit Empfindlichkeit Sensitivity
Valeur moyenne Mittelwert Mean value
Ecart type/Dviation standard Standardabweichung Standard deviation
Coefficient de variation Variationskoeffizient Coefficient of variation
Dviation standard relative Relative Standardabweichung Relative stand.deviation
Intervalle de confiance de la Vertrauensbereich des Confidence interval of the
valeur moyenne Mittelwertes mean value
Rptabilit Wiederholprzision Repeatability
Reproductibilit Vergleichsprzision Reproducibility
Mthode analytique Analysenmethode Analytical method
Procdure d'analyse Prfverfahren Test procedure
Rsultat Ermittlungsergebnis Result of determination
Erreur systmatique Systematische Systematic error of result
Ergebnisunsicherheit
Erreur due au hasard Zufllige Random error of result
Ergebnisunsicherheit
Valeur conventionnellement Richtiger Wert Conventional true value
vraie
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IT PT NL
Accuratezza Rigor, exactido Nauwkeurigheid

Precisione Preciso Precisie
Specificit Especificidade Specificiteit
Selettivit Selectividade Selectiviteit
Linearita Linearidade Lineariteit
Intervallo lineare Intervalo linear Lineair interval
Limite di rilevazione Limite de deteco Detectiegrens
Limite di determinazione Limite de quantificao Bepalingsgrens
Sensibilit Sensibilidade Gevoeligheid
Valore medio Valor mdio Gemiddelde waarde
Deviazione standard Desvio padro Standaardafwijking
Deviazione stand, relativa Coeficiente de variao Variatiecofficint
Coefficiente di variazione Desvio padro relativo Relat, standaardafwijking
Intervallo fiduciale del valore Intervalo de confiana do Betrouwbaarheidsinterval
medio valor mdio van de gemiddelde waarde
Ripetibilit Repetibilidade Herhaalbaarheid
Riproducibilit Reprodutibilidade Reproduceerbaarheid
Metodo di analisi Mtodo analitico Analysemethode
Procedimento Procedimento analitico Proefopzet
Risultato dell'analisi Resultado Resultaat van de bepaling
Errore sistematico Error sistematico do resultado Systematische afwijking
Errore casuale Errore aleatorio do resultado Toevallige afwijking
Valore reale Valor verdadeiro Conventioneel ware
convencional /Valor nominal waarde
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ES DA
Exactitud Njagtighed
Precisin Prcision
Especificidad Specificitet
Selectividad Selektivitet
Linealidad Linearitet
Intervalo lineal Lineari tetsomrde
Limite de deteccin Detektionsgrnse
Limite de cuantificacin Bestemmlsesgrnse (kvantitativ)
Sensibilidad Flsomhed
Valor medio Middelvrdi
Desviacin estndar Spredning
Coeficiente de variacin Variationskoefficient
Desviacin estndar relativa Relativ standardafvigelse
Intervalo de confianza del Konfidensinterval for
valor medio middelvrdi
Repetibilidad Repetrbarhed
Reproductibilidad Reproducerbarhed
Mtodo analtico Analysemetode
Procedimiento analtico Afprvningsmetode
Resultado de analisis Resultat
Error sistemtico del Systematisk fejl
resultado
Error aleatorio del resultado Tilfldig fejl
Valor verdadero Sand vrdi
convencional
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PLASMA DERIVED MEDICINAL PRODUCTS
Guideline Title Plasma derived Medicinal Products

Legislative basis Council Directive 89/381/EEC extending the scope of
Directives 65/65/EEC and 75/319/EEC to medicinal
products derived from human blood or human plasma
Date of first adoption First adopted December 1991
Revised version adopted March 1996
Date of entry into
September 1996
force
Status Last revised March 1996
Previous titles/other Medicinal Products derived from Human Blood and
references Plasma/ IH/8379/89
Additional Notes This guideline was originally published under the name
of Medicinal products derived from Human Blood and
Plasma in December 1991. The March 1996 revision
concerns the parts on viral validation (section 3, point 3.3,
section 5, and annexes I and II). This revision has been
done in parallel with the revision of the CPMP
guideline, on virus validation, now published as Virus
Validation Studies: The Design, Contribution and
Interpretation of Studies Validating the Inactivation and
Removal of Viruses. It applies to medicinal products
derived from plasma and focuses on specific aspects
relating to the manufacture and control of these products,
paying particular attention to the steps taken to minimise
the risks of microbial contamination of the finished
product. It does not cover cellular blood products.
CONTENTS
1. INTRODUCTION
2. SOURCE MATERIALS
3. MANUFACTURE
4. QUALITY CONTROL
5. VALIDATION STUDIES
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PLASMA DERIVED MEDICINAL PRODUCTS
L INTRODUCTION
Council Directive 89/381EC extends the scope of Directives 65/65/EEC and 75/319/EEC to
medicinal products derived from human blood or human plasma.
Human blood and plasma contain many proteins, the extraction and purification of which
are of great medical importance. The therapeutic use of blood and blood products goes back to
the turn of the century. Two important advances since then have led to a surge in the use of
blood products. These were the discovery of blood groups and the development of methods to
fractionate plasma into components of medical value.
Improvements in protein purification and molecular separation technology over recent years
have made available a wide variety of products with medical applications covering a large
and growing field. However, blood can harbour many viruses, and the use of medicinal
products derived from human blood or plasma has led to the transmission of severe viral
diseases, including hepatitis , and C and AIDS caused by the human immunodeficiency
virus (HIV). Other microbial contaminants have also been the cause of serious accidents.
Thus, measures designed to prevent the transmission of pathogens are essential to ensure
the general safety of products derived from human blood and plasma.
Products derived from human blood and plasma can roughly be divided into two groups:
The first group covers products derived from single donations or from small pools of source
material (< 12 donors). These products, for example, cell concentrates and cryoprecipitates
are commonly made and distributed by blood donor centres and used in transfusion
medicine. They are either subjected to one or a few separation procedures. Their quality and
safety are almost exclusively dependent on the careful selection and control of donors, on the
screening of donations and on measures taken to minimise contamination during
processing.
The second group is represented by derivatives of plasma produced on an industrial scale
from pools of source material through various manufacturing procedures. They may be used
as therapeutic medicines or as excipients. The quality and safety of these products rely both
on the selection and screening of source materials and on the choice of the manufacturing
processes, including processes which inactivate or remove microbial contaminants.
Directive 89/381/EEC covers only products belonging to the second group. These include:
albumin and plasma protein solutions;
immunoglobulins;
coagulation factors and antiproteases;
other isolated plasma fractions or combinations thereof.
This note for guidance covers medicinal products derived from plasma and focuses on
specific aspects relating to the manufacture and control of these products, paying particular
attention to the steps taken to minimise the risks of microbial contamination of the finished
product. It does not cover cellular blood products although many parts contained in this
document may be pertinent.
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The transmission of viruses is of particular concern. Measures taken to prevent infection by

the use of plasma products include selection of donors, screening of individual donations
and starting materials for markers of infection with known viruses and validation of the
production process for the inactivation or removal of viruses. Clinical follow up of recipients
is the final proof of the safety of a product.
Throughout this note for guidance it is assumed that the principles of Good Manufacturing
Practice are followed as laid down in Directive 91/356/EEC and the relevant guidelines.
Documents available stating the requirements and standards of blood products include:
1. the European Pharmacopoeia, Monograph # 853 (1995), "Human Plasma for
Fractionation", and specific monographs for plasma-derived products (see annex I and
annex II P. 18-19);
2. the document from the Council of Europe, entitled "Guide to the preparation, use and
quality assurance of blood components (1995)", which addresses the collection,
preparation and use of blood. This document deals primarily with the requirements for
blood and blood components for use in blood transfusion and in immunohaematology;
3. the forty-third report of the WHO Expert Committee on Biological Standardisation
(Technical Report Series 840, 1994) on the requirements for the collection, processing
and quality control of blood, blood components and plasma derivatives. This document
covers in five parts the requirements for the collection of source materials; single-
donor and small-pool products; the manufacture of blood products; the control of plasma
fractions; control of products by the competent authorities.
2. SOURCE MATERIALS
2.1 CLASSIFICATION
Several types of source materials currently used in the manufacture of medicinal products
derived from human plasma are specified by the European Pharmacopoeia and by the WHO
requirements. Their origin and means of collection differ in several respects.
Two types of these source materials are relevant in the context of this note for guidance:
a) Whole blood donations are collected at blood donor centres. This material is used to
prepare products made from single donations for direct transfusion while much of the
plasma is used for fractionation on an industrial scale;
b) Plasma obtained by plasmapheresis is collected in plasmapheresis centres and some
blood donor centres. It is used for products manufactured on an industrial scale.
2.2 RISK FACTORS

Many factors can affect the safety of blood donations in transfusion medicine. However, not
all of these are relevant to medicinal products derived from human plasma manufactured
on an industrial scale. Those which have implications include viruses found in plasma
which establish a viraemia such as HBV, HCV, HIV I and II, HAV and parvovirus B19. In
many cases such viruses can establish a persistent or latent infection. Other factors of equal
importance relate to the quality of the product, for example the integrity and biological
activity of immunoglobulins and the thrombogenicity, immunogenicity and activity of
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clotting factors, which can be affected by the treatment and preparation of the source
materials after collection.
2.3 COLLECTION AND CONTROL OF SOURCE MATERIALS

2.3.1 General
The means of collecting source materials and its control are major factors in the quality
assurance of the manufacture of biological medicinal products. Measures taken to reduce
risks include the meticulous control of source materials and their origin.
Collecting centres should be inspected and approved by a competent authority. Manufacturers
should provide documentary information on the acceptability of the centres, and on the
procedures for collecting, storage and shipping of source materials.
2.3.2 A Quality Assurance System for collection

Recommendations given in the document from the Council of Europe "Guide to the
preparation, use and quality assurance of blood components (1995)" and from the forty-third
report of the WHO Expert Committee on Biological Standardisation (WHO Technical Report
Series 840, 1994) with later amendments should be followed.
Each collection/transfusion centre should establish, document and maintain an effective
quality assurance system. The main requirements are summarised below:
the preparation of standard operating procedures;
the establishment of records so that donations can be traced, e.g. date of collection,
quality control tests undertaken, with results etc., should be included;
specifications for source plasma for further industrial processing into medicinal
products;
control of labelling, storage and transportation of donations;
establishment of quality audits/review;
appropriate premises.
a) Selection of donors and donations
The criteria of the Council of Europe, of WHO and of the European Pharmacopoeia
shall apply to the selection of blood donors and blood donations. Using a sensitive,
specific and validated test, each donation must be tested and found:
non-reactive for HBsAg, using an ELISA or RIA test which detects 0.5 IU per ml of HBs
antigen or less;
non-reactive for antibody to 1 and HTV 2;
non-reactive for antibody to hepatitis C.
b) Post-collection information system
A standard operating procedure must be set up so that the transfusion/collection centre
can inform the manufacturing/ fractionation centre, if, within six months of donation:
- it is found subsequent to donations that the donor did not meet the current donor
selection criteria;
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it is discovered that testing for viral markers has not been carried out according
to agreed procedures;
the donor develops an infectious disease caused by a transmissible agent (see
section 2.2);
the recipient develops post transfusion infection which implicates or can be
traced back to the donor.
3. MANUFACTURE
According to Directive 91/507/EEC, the preparation of plasma derivatives shall be defined
and justified in terms of strategy, and described with all relevant details regarding
procedures, in-process and final controls.
3.1 Risks arising during processing

In the manufacture of medicinal products derived from human plasma, consideration
should be given to the following factors:
a) bacterial contamination may lead to the accumulation of pyrogens;
b) viruses may be introduced by reagents during manufacture (e.g. enzymes from tissue
extracts or monoclonal antibodies used for affinity chromatography);
c) the methods of manufacture may introduce chemical contaminants such as enzymes,
solvents, detergents, and antibodies or other ligands from chromatography.
d) methods of manufacture may modify the product resulting in adverse consequences for
recipients, for example by the formation of neo-antigens or by compromising the
biological activity of the active component. This is true for steps introduced to
inactivate viral contamination which may affect the quality or yield of products.
3.2 The starting material

The manufacture of plasma derivatives should start from defined pools of source material.
For each source material, records allowing to trace back its origin and the controls to which
donor and donation were subjected should be kept and made available upon request to
manufacturers and competent authorities. Records as well as samples of each pool should be
stored for at least one year after the expiry date of the finished product with the longest shelf
life.
3.3 Manufacturing procedures

The strategies used in the manufacture of plasma derivatives are critical for product quality
and play an essential part in ensuring overall product safety. They vary according to
product and manufacturer, and usually include several fractionation/purification
procedures, some of which may also contribute to the inactivation and/or removal of
potential microbial contaminants. Additionally procedures specifically designated to
inactivate/remove viral contaminants are commonly part of the manufacturing strategy.
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It should be emphasised that a manufacturing process cannot be considered satisfactory

unless it is capable not only of generating a product of high quality but also effectively
inactivating and/or removing infectious agents.
Products derived from human plasma have been shown to transmit viruses to recipients
even where the starting material has been controlled for viral contamination in accordance
with state of the art procedures. This follows in part from the nature of the starting material,
which is obtained from a panel of heterogeneous human donors which cannot be
virologically characterised as thoroughly as other sources of biological materials, such as
cell banks. In addition any contaminating virus is able by definition to infect humans.
While selection of donors and testing of donations are essential safety measures, incidents
of viral transmission show that they are insufficient alone to ensure safety of the product.
The manufacturing process itself plays a central role and is of great significance for
products derived from plasma. Studies of a process for the ability to inactivate or remove
virus infectivity will be subject to particularly careful evaluation when products derived
from blood or plasma are considered. This will include consideration of the reduction i n
virus titre achieved, the rates of inactivation and the shape of inactivation curves, how robust
the step is to process variables, and whether virus inactivation or removal is selective for a
particular kind of virus.
The suitability of the various materials and procedures used in manufacture as well as the
selected operating conditions, parameters and tolerances should be validated by correctly
designed and interpreted studies.
3.3.1 Fractionation/purification procedures

a) Precipitation methods
Physical methods:
Cryoprecipitation is most often used as the initial step for the production of Factor VIII
concentrates.
Subsequent purification techniques, such as precipitation or chromatographic
separation as well as procedures for viral inactivation are used to obtain the finished
products. Cryoprecipitate-depleted plasma can be used for the preparation of other
coagulation factors or plasmaprotein solutions.
Physical/chemical methods:
Among these methods, the ethanol fractionation procedures derived from the Cohn
method are the most widely used for albumin and immunoglobulins. They commonly
incorporate several steps, in each of which compliance with specific requirements is
decisive for product quality; under proper conditions some of these steps may also
contribute to effective reduction of potential viral contaminants. Therefore, clear
specifications for ethanol and protein concentration, temperature, pH and ionic
strength, and time of treatment, with data on acceptable tolerance as well as the means
of controlling them should exist.
Appropriate data should also be provided for methods relying on other chemical agents
such as ethylacridin-lactate, methanol, ammonium sulphate, polyethylene glycol,
cationic detergents, which are sometimes used in the preparation of certain plasma
derivatives, as a rule in combination with other purification procedures. Some of these
substances may have an impact on viral safety, for others information is still scarce.
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b) Chromatographie methods
Three basic types of procedures play an increasing role in the processing of plasma
derivatives, as a rule in combination with precipitation procedures and often with each
other:
- gel filtration, mainly used for desalination or separation of components with
significantly different size;
- ion exchange and hydrophobic interaction chromatography;
- affinity chromatography based on specific interactions with immunological or
other ligands immobilised on the matrix.
The selectivity of the procedures and the yields depend critically on the quality of the
material as well as on factors like the capacity of the column, nature and
concentration of proteins in the product, ionic strength and the pH of buffers, flow rate
and temperature. Therefore, all appropriate specifications and accepted tolerances
should be stated, and control data documented.
Several other compounds like charcoal, bentonite, colloidal silica are sometimes used
for clearing various impurities like pigments, lipoproteins etc. Details on the
characteristics of the compounds, on their decontamination and on the operating
conditions should be provided.
The conditions of storage of the columns, preservation and elution of preservatives,
and methods of regeneration should also be described. Details should be given of
clarification and sterile, dia- or ultra-filtration procedures used.
c) Complementary procedures
Immunoglobulins intended for intramuscular administration may cause adverse
reactions upon intravenous administration. Therefore, the production process for
immunoglobulins intended for intravenous administration includes various
procedures which can substantially reduce such reactions. The materials and the
procedures used should be described and their suitability justified and documented.
3.32 Viral inactivation/removal procedures

Procedures specifically designed to inactivate/remove infectious viruses are included in the
manufacturing strategies for most plasma derivatives. The manufacturing process
conditions and in-process monitoring for viral inactivation/removal steps should be clearly
defined and justified.
It is essential that material that has been subjected to a viral inactivation/removal step
should be segregated from untreated material to prevent cross-contamination (as stated i n
the Guide to GMP, Annex 14).
The following is not a comprehensive account of available processes and points to consider
but identifies some common criteria that need to be considered for certain processes.
a) Heating in aqueous solution
Heating in aqueous solution at 60C for 10 hours in the final container is the
pharmacopoeial method for viral inactivation for albumin preparations. This method
of inactivation is also used for bulk preparations of some plasma-derived products. The
efficacy of such a treatment is dependent upon the composition of the solution.
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Stabilisation to protect proteins can also protect virus from inactivation. Careful
validation is, therefore, needed for each product ensuring that the validation includes
worst case conditions.
b) Heating of lyophilised products
The effectiveness may vary according to the conditions of lyophilisation. Upper and
lower limits of water activity or moisture, whichever is more appropriate, should be set
based on viral validation studies. Where such a treatment is applied to the product in
its final containers, the variation in water content between vials of product should be
within the limits set.
c) Solvent/detergent treatment
Treatment with a solvent such as tri-n-butyl-phosphate (TNBP) combined with' a non-
ionic detergent such as Triton X-100 or Tween 80 can inactivate enveloped viruses.
Prior to such treatment, in-process solutions should be free from gross aggregates that
may harbour virus and protect it from the treatment. This can be achieved by filtration
which should be done prior to addition of the solvent/detergent or if done after, the
filters should be demonstrated not to alter the levels of these additives in the incubation
solution. Physical validation must demonstrate that mixing achieves a homogeneous
mixture for the duration of the defined incubation time. In-process checks should be
carried out to confirm that the correct amount of solvent and detergent have been
added. Validation experiments should investigate the range of key process variables
and in-process limits should be set accordingly. Since lipid content can affect the
efficacy of inactivation, inactivation should be confirmed under worst case conditions
for lipid content. Residual levels of solvent and detergent should be minimised by
processing and carefully monitored in the final product. Non-enveloped viruses will
not be inactivated by this process.
d) Virus removal by filtration
This is a new and developing area of technology. There may be difficulties with
removing the smaller viruses by filtration while maintaining a satisfactory yield of
product, especially for material of high molecular weight such as Factor VIII. The
mode of action of the particular filter selected should be described and the parameters
critical for virus removal (e.g. volume, ionic strength, flow rate, pressure and
loading) should be identified. These critical parameters should be used to define
appropriate viral validation studies. Tests to confirm filter integrity are essential in-
process controls. In addition, the performance of filters used in virus validation
studies must be compared to that of the filters used in routine production. Aggregation
of viruses can affect the level of virus removal by filtration. This should be taken into
account when performing validation studies with viruses which will have been
propagated and concentrated under laboratory conditions and whose state of
aggregation may differ from that expected of a virus present in plasma. Information
on the characterisation of the filter material by the manufacturer should also be
provided.
e) Low pH
Low pH (approximately 4) can inactivate certain viruses. The reduction factors that
have been demonstrated depend on the exact conditions used in manufacturing (e.g.
pH value, time and temperature of treatment, composition of the solution, etc.). Each
process therefore has to be carefully validated.
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3.4 C o n s i s t e n c y of p r o d u c t i o n
The manufacturer should demonstrate consistency of the specifications of the product for at
least 3 full scale production batches.
4. QUALITY CONTROL
4.1 In-process controls
The procedures for production and equipment monitoring, the production steps where control
tests are carried out, the means of sampling and of storing the samples, as well as the
testing procedures should be described.
The pooling of source materials should be subject to careful control to avoid contamination
and introduction of foreign material.
Bulk purified material obtained from other producers should be retested according to the
recommendations of the WHO Requirements and samples should be stored as specified i n
section 3.2.
The testing of samples of starting and bulk material for specific viral markers should be i n
accordance with up to date methods validated for their intended use.
The monitoring of relevant parameters during fractionation, such as pH, temperature and
ethanol concentration where appropriate, as well as the results from bacterial counts and
endotoxin should be documented.
4.2 Quality control of products

All products should comply with the appropriate European Pharmacopoeia monographs. If
methods other than those specified by the European Pharmacopoeia are used, the alternative
procedures should be shown to give consistently equivalent results on several batches of
product.
5. VALIDATION STUDIES
Validation studies should be carried out by each manufacturer for the specific processes used
and, unless otherwise justified, for each production site. Moreover, if studies involve
modelling the process on a reduced scale, they should be capable of mimicking satisfactorily
the conditions of full scale production and the accuracy of the modelling should be
demonstrated.
5.1 Process Validation

The effectiveness of a given manufacturing process in consistently yielding a product with
expected quality, including biological activity, as well as freedom from undesirable
components, for example blood group substances or chemicals used for, or derived from,
fractionation/purification procedures, should be documented with data based on a broad set of
relevant analytical methods. Spiking experiments with certain potential contaminants may
be necessary to demonstrate the clearing efficiency of the process.
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The studies should be designed to justify the selected operating conditions and the acceptable
tolerances, including worst case conditions, and to document their adequacy in achieving
the expected process performances.
When chromatographic columns are used, conditions leading to overloading as well as
leaching from the gels, particularly in the case of affinity chromatography with potentially
harmful ligands, should be carefully investigated. Attention should also be paid to the
cleaning and regeneration of the columns and to the effective removal of residues from the
previous run or of preservatives added for storage, particularly if mild treatments are used.
5.2 V i r u s I n a c t i v a t i o n / R e m o v a l
52.1 Manufacturing process design
General principles concerning the incorporation of virus inactivation/removal steps in the
manufacture of biological products are outlined in the note for guidance Virus Validation
Removal of Viruses. This section contains further guidance relevant to plasma derivatives.
The principles in both guidelines should be taken into account when designing
manufacturing processes or modifying processes to give further assurance of viral safety.
a) Incorporation of effective steps for viral inactivation/removal in the manufacturing
process.
All production processes should incorporate effective validated steps for the
inactivation/removal of viruses. An effective step is defined in the note for guidance
Virus Validation Studies: The Design, Contribution and Interpretation of Studies
Validating the Inactivation and Removal of Viruses.
For all plasma derived medicinal products, it is an objective to incorporate effective
steps for inactivation/removal of a wide range of viruses of diverse physico-chemical
characteristics. In order to achieve this, it will be desirable in many cases to
incorporate two distinct effective steps which complement each other in their mode of
action such that any virus surviving the first step would be effectively
inactivated/removed by the second. At least one of the steps should be effective against
non-enveloped viruses. Where a process step is shown to be reliably effective in
inactivating/removing a wide range of viruses including enveloped and non-
enveloped viruses of diverse physico-chemical characteristics and the process contains
additional stages reliably contributing to the inactivation/removal of viruses, a second
effective step would not be required.
It is recognised that designing steps which will complement each other and also be
effective against a wide range of viruses including enveloped and non-enveloped
viruses of diverse physico-chemical characteristics, is not a straightforward task.
Viruses tend to fall into two groups in this respect, those susceptible to a wide range of
inactivation/removal procedures and those resistant. Also, there may be viruses
potentially present in plasma that are resistant to the inactivation/removal methods
that can currently be applied to a class of product, e.g. parvovirus B19 in coagulation
concentrates.
Manufacturers should apply their best efforts to develop methods to inactivate/ remove
viruses and this should be a continuing process. Previous experience clearly shows
that source material may contain unknown viruses and that new viruses may appear.
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This emphasises the need to design processes to inactivate/remove as wide a range of

viruses as possible. Even this may not preclude new or unknown infectious agents
breaking through a process.
b) Contribution of partition processes to virus removal.
It is recognised that partitioning through the fractionation process and through
purification procedures (e.g. immunoaffinity chromatography) can contribute to the
removal of viruses. However, cases of virus transmission have occurred clinically
with coagulation factors and intravenous immunoglobulins whose manufacture have
relied purely on partition processes. Furthermore, partition processes involve a large
number of variables that are difficult to control and are difficult to scale down for
validation purposes. Minor differences in physico-chemical properties of viruses can
have a major influence on partitioning which makes it difficult to extrapolate from
validation studies. Partitioning may also be affected by the presence or absence of
antibodies. Consequently, it may be difficult to demonstrate that partition processes are
reliably effective.
If a partition process gives reproducible reduction of virus load and if manufacturing
parameters influencing the partition can be properly defined and controlled and if the
desired fraction can be reliably separated from the putative virus-containing fraction,
then it could fit the criteria of an effective step.
Since fractionation can contribute to virus removal, particular attention needs to be
given to validation studies and clinical safety if novel manufacturing processes
depart from standard fractionation techniques.
c) Selection of specific virus inactivation/removal steps.
The rationale for the choice of specific virus inactivation/removal steps deliberately
introduced into the process should be given. Marketing authorisation holders are
expected to keep their processes under review in the light of developments in virus
inactivation/removal techniques and of any emergence of relevant, novel
contaminants of plasma and plasma-derived products.
d) Effect of virus inactivation/removal steps on the product.
It should be established that the virus inactivation/removal steps selected will
contribute to the net safety of the product. For example, a solvent/detergent step might
break up aggregates and allow more non-enveloped virus through a subsequent
filtration step intended to remove viruses. Consideration should be given to the
maintenance of the integrity of the components of the plasma derivative and clinical
efficacy, the potential for formation of neo-antigens and the possibility of toxic residues
from chemicals used in-process as well as to virological safety. Separate guidance is
available on the clinical studies that should be undertaken (Assessing the Efficacy and
Safety of Human Plasma Derived Factor VIILc and Factor LX:c Products in Clinical
Trials in Haemophiliacs Before and After Authorisation and Assessing the Efficacy and
Safety of Normal Intravenous Immunoglobulin Products for Marketing Authorisations).
e) Points to consider for specific product classes.
i) Coagulation factors Effective process steps for the inactivation/removal of
enveloped viruses are essential. Non-enveloped viruses such as hepatitis A and
parvovirus B19 have been transmitted by this class of products. Coagulation
factors are the highest priority class for the development of methods to exclude
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non-enveloped viruses from the product (see Addendum). However, it is

recognised that some viruses are very resistant to physico-chemical methods for
viral inactivation, e.g. parvovirus B19, and that development of an effective
inactivation/removal step may be difficult. This should be reflected in the
Summary of Product Characteristics.
ii) Immunoglobulins Certain intravenous immunoglobulins, without specific steps
for the elimination of enveloped viruses, have recently transmitted hepatitis C.
Effective process steps for the elimination of enveloped viruses are essential for
intravenous immunoglobulins.
Intramuscular immunoglobulins have a good safety record. However, the reason
for this is poorly understood. The virus challenge and the virus/antibody ratio
faced by processes are changing as a result of donor screening and changes i n
the epidemiology of virus infections in donor populations. Processes have been
developed to inactivate enveloped viruses in intravenous immunoglobulins
which may also be applicable to intramuscular products and these should be
introduced. It should also be established that the net safety of the product is not
adversely affected by the addition of such a process.
Immunoglobulins are used successfully to treat or prevent infection by non-
enveloped viruses, such as parvovirus B19 or hepatitis A. Provided the relevant
antibodies in immunoglobulin preparations are maintained at a protective level,
it is highly unlikely that infection will result from the presence of such viruses
in any immunoglobulin preparation. However, the possible transmission of a
novel non-enveloped virus or the decline of antibody to non-protective levels i n
donor pools cannot be totally excluded. The addition of a specific virus
inactivation/removal step for non-enveloped viruses is therefore an objective.
iii) Albumin manufactured by an established fractionation process that includes the
terminal pasteurisation specified in the European Pharmacopoeia monograph,
has an excellent viral safety record. However, further information is needed
from validation studies on the elimination of viruses. The effect of albumin
concentration on virus elimination should be considered. Since albumin has
additional uses as an excipient, attention should be given to the effect of any
proposed process change on other products that may include albumin in the
formulation. Albumin used as an excipient should be of the same quality as
albumin used as an active ingredient.
522 Choice of viruses for use in validation studies

General guidance on choice of viruses is given in the CPMP guideline Virus Validation
Removal of Viruses. Viruses to be used in validation studies on plasma-derived medicinal
products should include at least:
i) Hrv-i
It is not necessary to carry out additional studies with TTTV-2 as it is similarly affected
by inactivation procedures.
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ii) A model for hepatitis C virus

Biochemical characterisation of HCV classifies it in the Flaviviridae related to both
pestiviruses and flaviviruses. Currently, there are no methods available for
propagation of the virus. Various models have been used to validate viral inactivation
methods including togaviruses, e.g. Sindbis, flaviviruses, e.g. Yellow Fever virus, and
pestiviruses, e.g. bovine viral diarrhoea virus. These viruses have properties in
common with HCV. Minor differences in physico-chemical characteristics of viruses
can have major effects on how they partition. For example, there is evidence that
pestiviruses differ in their partition in the Cohn Oncley fractionation process from
togaviruses and that HCV resembles the pestiviruses more closely in this respect.
Currently there are insufficient data on HCV to identify the most appropriate model
virus for validation studies. Therefore, caution is required in the choice of a model
virus and in the interpretation of validation data.
iii) Non-enveloped viruses
The package of validation studies on non-enveloped viruses should establish the range
of viruses susceptible to the inactivation/removal processes and identify the limits of
the process. For example, a heat inactivation step used in the manufacture of a
coagulation factor might be effective against hepatitis A virus but ineffective against
another non-enveloped virus.
Hepatitis A transmission has been associated with certain coagulation factors.
However, there are insufficient data to identify appropriate models for hepatitis A at
the present time. HAV should be used for validation studies for coagulation factors as
it is thought to be significantly different to other picornaviruses. Consideration should
be given to the possible interfering effects of antibodies.
Validation studies for coagulation factors should also include an appropriate model for
the parvovirus B19. Models that have been used include canine, porcine, murine and
bovine parvoviruses. Studies using HAV and B19 are not required for
immunoglobulins if the presence of protective levels of antibodies in the product can be
assured. However, studies with non-enveloped viruses for which antibodies are
unlikely to be present should be performed to evaluate the ability of the process to
inactivate/remove possible unknown non-enveloped viruses.
iv) Enveloped DNA viruses
To date, there have been no recorded transmissions of a herpesvirus associated with
the use of non-cellular blood products. However, since novel herpesviruses continue to
be discovered, most of which are lymphotropic and some which may result in a
viraemia, a validation study should be performed with an appropriate enveloped DNA
virus, e.g. a herpesvirus such as Pseudorabies.
Currently, there is no practical test system for hepatitis virus validation.
5.2.3 Difficulties in the design and execution of virus validation studies.

Reliable experimental demonstration of the effectiveness of virus inactivation and removal
during the processing of plasma and the interpretation of data may be rendered difficult for
various reasons. The presence of antibodies may affect partition of viruses or their
susceptibility to chemical inactivation and may also complicate the design of the study by
neutralising infectivity. Furthermore, undiluted plasma or derived fractions are usually
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toxic for cell cultures used for virus detection as is the presence in intermediary products of
chemicals such as ethanol and ethylacridinlactate. Therefore, assays may have to be
preceded by procedures designed to counteract these effects, such as dilution, dialysis, etc. In
addition, the product itself or chemicals used to prepare or to treat it may change the
properties of viruses, for example leading to their coating and/or aggregation, which may
result in difficulties in reliable quantification of residual infectivity.
5.3 Revalidation
New validation studies are required when relevant changes in the manufacturing process or
in individual steps are being considered.
Validation experiments have many limitations. Any virus transmission seen in clinical
use should result in an evaluation of available data by manufacturers and regulatory
authorities so that appropriate action can be taken.
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ADDENDUM
STRATEGY FOR INTRODUCING ADDITIONAL PROCESS STEPS FOR THE
INACTIVATION/REMOVAL OF VIRUSES
Specific virus inactivation/removal steps are included in the manufacturing processes for
many plasma derivatives. However, recent transmissions of enveloped and non-enveloped
viruses by certain products have highlighted the need for a strategy to further increase the
assurance of viral safety of plasma derivatives. The objectives are set out in the guideline.
This Addendum sets out the priorities for action and the strategy to be followed.
Manufacturers should, as a matter of urgency, validate their processes for the
inactivation/removal of enveloped and non-enveloped viruses where they have not already
done so and, where the current process is not effective in inactivation/removal, develop and
validate additional virus inactivation/removal steps in order to improve safety. The priority
order is, starting from the highest: coagulation factors, intravenous immunoglobulins,
intramuscular immunoglobulins and albumin.
Marketing authorisation holders and applicants are required to set and justify timetables for
such developments; to submit a programme of process/product improvements to Member
States for their agreement and to commit themselves to providing 6 monthly reports to
Member States on their progress. Timescales for introduction of process changes should
reflect the manufacturer's best efforts. In the meantime, product literature needs to be looked
at critically and, where necessary, amended to provide relevant and specific information to
enable clinicians to make an informed choice of product.
Member States will keep the progress of process improvements under review. It will not be
acceptable to keep on the market products that have fallen behind general developments for
their class of products.
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ANNEX I
PUBLISHED MONOGRAPHS ON BLOOD PRODUCTS
Monograph Title and Serial Number Current version Implementation Revision Implementation
(date) date of revision
Albumin solution, human (255) 1995 1/1/96
Anti-D immunoglobulin, human (557) 1995 1/1/96
Antithrombin III concentrate, human 1994 1/V95
(878)
Factor VIII concentrate, human (275) 1994 1/1/95
Factor DC concentrate, human (554) 1987 1/1/88
fibrin sealant (903) 1994 1/1/95
Fibrinogen, freeze-dried human (24) 1996 1/1/97
Hepatitis A immunoglobulin (769) 1995 1/1/96
Hepatitis immunoglobulin (722) 1991 1/1/92
Hepatitis immunoglobulin for i.v. use 1995 1/V96
(1016)
Immunoglobulin, normal, human (338) 1994 1/7/94
Immunoglobulin. Normal, human, for 1994 1/7/94
intravenous use (918)
Measles immunoglobulin, human (397) 1995 1/1/96
Plasma for fractionation, human (853) 1995 1/V96
Rabies immunoglobulin, human (723) 1995 1/1/96
Rubella immunoglobulin, human (617) 1995 1/1/96
Tetanus immunoglobulin, human (398) 1995 1/1/96 1996 1/1/97
Vaccinia immunoglobulin, human (399) 1985 1/1/86
Varicella immunoglobulin, human 1995 1/1/96
(724)
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ANNEX II
GENERAL METHODS
Monograph Title and Serial Number Current version Implementation Revision Implementation
(date) date of revision
Factor VIII, assay (V.2.2.5) 1994 1/1/95
Anti- and anti-B haemagglutinins 1980
(VIII.5)
Haemolysin test for group 0 blood 1980
(VII. 12)
Fe function of immunoglobulin 1995 1/1/96
(V.2.2.10)
Anticomplementary activity (V.2.1.13) 1995 1/7/96
Prekallikrein activator (V.2.1.11) 1994 1/7/94
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PLASMA POOL TESTING
Guideline Title Plasma Pool Testing

Date of first adoption March 1994
force
Status Last revised March 1994
references
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PLASMA POOL TESTING
1. With regard to blood products as well as with other medicinal products, improvement
of testing is a continuous process, evolving with the state of the art. The introduction of a new
test or test method does not automatically mean that the formerly produced batches are
unsafe.
2. Following the specific requests for comments on plasma pool testing by

manufacturers and/or control authorities during the consultation period on the batch release
documents for medicinal products derived form human blood or plasma, plasma pool testing
was extensively discussed by the Biotechnology/Pharmacy Working Party.
3. Plasma pool testing is seen as one of a number of steps which, taken together, provide
assurance of the virological safety of blood products. Individual donations of blood/plasma
for manufacture of blood derivatives must be tested and found negative for viral markers
(anti-HrV 1 and 2, hepatitis surface antigen and anti-hepatitis C antibody). Because errors
in testing and/or pooling can occur, manufacturers of blood derivatives should, in addition,
introduce testing of their plasma pools for the above-mentioned viral markers.
4. For plasma pool testing by the manufacturers, the following applies:

- The pooled plasma or the pooled cryo-supernatent is the material to be tested;
- Test procedures must be the most up-to-date and must be validated for specificity and
sensitivity for testing plasma pools;
- Pools which are confirmed positive for any of the above markers must be rejected as
well as any intermediates or products produced therefrom;
- Manufacturers who are not already implementing plasma pool testing as described
above, should introduce plasma pool testing as from 1 November 1994.
5. Five Member States require batch release for some or all medicinal products derived
from human blood or plasma.
Currently the United Kingdom requires and operates plasma pool testing within the batch
release procedure. The same is envisaged in the near future in Germany.
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HARMONISATION OF REQUIREMENTS FOR

INFLUENZA VACCINES
Guideline Title Harmonisation of Requirements for Influenza Vaccines

Date of entry into April 1992
force
Status Last revised October 1991
Previous titles/other
references
Additional Notes The objective of this document is the harmonisation of
control tests carried out in the framework of batch
examination in order to achieve mutual recognition of
marketing authorisation applications for Influenza
vaccines.
CONTENTS
A. YEARLY CHOICE OF INFLUENZA VIRUS STRAINS FOR VACCINES
B. POTENCY OF INFLUENZA VACCINE
C. CONTROL AUTHORITY BATCH RELEASE OF INFLUENZA VACCINE
D. CLINICAL TRIAL RELATED TO YEARLY LICENSING OF INFLUENZA

VACCINE
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HARMONISATION OF REQUIREMENTS FOR

INFLUENZA VACCINES
A. YEARLY CHOICE OF INFLUENZA VIRUS STRAINS FOR

VACCINES
WHO has two international influenza centres (at the National Institute for Medical
Research in Mill Hill and at the Centre for Disease Control in Atlanta), which are assisted
by national laboratories, designated by WHO. The national laboratories isolate viruses and
then refer them to an international centre for detailed antigenic analysis. Reports are
regularly sent to WHO in Geneva.
Once a year, in mid-February, a meeting of WHO experts takes place in Geneva, leading to
a recommendation on the influenza A and virus variants which should be used for the
production of vaccine for the coming season, but there remains very broad flexibility within
this recommendation. The WHO recommendations are aimed worldwide and therefore need
to be adapted to the epidemiological situation of the European Community (EC). The
predominant influenza viruses are believed to be similar from one Member State of the EC to
another. There is thus little scientific justification for different composition of vaccines
throughout the EC.
Sincel992, an annual meeting of EC experts is convened after the WHO meeting, as soon as
practically possible, in order to take an EC wide decision regarding influenza virus strains
for vaccine production for the next season, taking into consideration the epidemiology of
influenza in the EC.
B. POTENCY OF INFLUENZA VACCINE

The potency of influenza vaccines used in the Member States is generally between 10 and
15 |jg HA per strain and per dose. There is concern at accepting antigen content below 15 pg
HA, especially because of poor antibody response in the elderly, who are one of the target
populations.
Thus for influenza vaccines to be acceptable throughout the EEC, they should contain 15 pg
HA per strain and per dose. The lower 95% confidence limits of the potency assay should
indicate a content of at least 13.5 ug HA per strain and per dose.
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C CONTROL AUTHORITY BATCH RELEASE OF INFLUENZA

VACCINE
L INTRODUCTION
1.1 Directive 89/342/EEC relating to immunological products (consisting of vaccines,
toxins or serums and allergens) provides in article 4.3 that, where a Member State considers
it necessary in the interests of public health, it may require that samples from each batch be
submitted for examination by a State laboratory or a designated laboratory for the following
medicinal products:
- live vaccines;
- immunological medicinal products used in the primary immunisation of infants or
other groups at risk;
- immunological medicinal products used in public health immunisation programmes;
- new immunological medicinal products or immunological medicinal products
manufactured using new or altered kinds of technology or new for a particular
manufacturer, during a transitional period normally specified in the marketing
authorisation.
Harmonisation of such examination by EC national authorities must be achieved to permit
effective batch release of vaccines within the EC.
The objective of this batch examination is the verification that the product is in conformity
with the approved specifications. The testing has to be completed within 60 days of receipt of
the samples.
1.2 Where a Member State has examined a batch of a product and declared it to be i n
conformity with the approved specifications, another Member State may not repeat this
examination for the purpose of release.
The objective of this document is the harmonisation of control tests carried out in the
framework of batch examination in order to achieve mutual recognition.
1.3 Batch release should be carried out by a control authority with recognised competence
in batch release of influenza vaccines. A vaccine batch released by one Member State must
be acceptable to other Member States. Batch release depends upon mutual confidence and
effective exchange of information between the Member States. The batch release procedures
outlined below are phased to deal with vaccine submissions under normal circumstances
(phase 1) and abnormal circumstances (phase 2). Phase 1 of batch release is necessary for
all vaccine batches whereas phase 2 of batch release is introduced under the special
circumstances described below. Test methods and results for phases 1 and 2 must comply
with the European Pharmacopoeia monograph on influenza vaccines.
1.4 Manufacturers are responsible for presenting release certificates delivered by the
competent authorities when required.
Records of batch release tests (phases 1 and 2) and the full documentation submitted by the
manufacturer should be kept for at least 10 years by the control authority. They should be
available to other EC control authorities upon request.
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2. PHASE 1 OF BATCH RELEASE: PROTOCOL SUBMISSION

AND BATCH RELEASE TESTS (BASIC EP TESTS)
2.1 Protocol submission
The manufacturer's detailed protocol of production and tests carried out according to the
European Pharmacopoeia monograph on influenza vaccines shall be approved by the control
authority for each vaccine batch. The protocol should be based upon the WHO summary
protocol for influenza vaccine (inactivated) (WHO Technical Report Series 638, 1979) an
example of which is illustrated in paragraph 5. Manufacturers should submit full details of
test results; it is insufficient to indicate only "pass" or "fail".
2.2 Basic EP tests

Tests to be performed by the control authority in accordance with the EP monograph as a
basis for batch release:
2.2.1 At least twenty doses of each vaccine batch (product supplied in final package) and 20
ml of bulk vaccine shall be submitted to the control authority. For purified subunit vaccines,
an additional 10 ml of monovalent vaccine shall be submitted for the first 5 lots of vaccine
produced from a new influenza strain;
2.2.2 Tests to be performed on each batch of vaccine prior to release:
a) haemagglutinin antigen concentration/identity test using reference materials supplied
currently by the National Institute for Biological Standards and Control, UK;
b) endotoxin content;
2.2.3 Tests to be performed on each lot of blended bulk vaccine:
a) none.
2.2.4 Tests to be performed on the first 5 lots of monovalent purified subunit vaccine
following the introduction of a new influenza strain:
a) test for purity.
3. PHASE 2 OF BATCH RELEASE: PROTOCOL SUBMISSION

AND ADDITIONAL EP TESTS
Additional tests from the EP monograph on influenza vaccines may be necessary for batch
release in special circumstances:
- a change in the vaccine production process has been approved;
- a change in the site of manufacture has been approved;
evidence of unexpected adverse clinical reactions or quality defects from previous
batches of a given vaccine;
- evidence of marked inconsistencies in the vaccine production process;
- a critical report from the inspector from the competent authority;
- changes in the manufacturer's testing procedures;
- identification of unexpected variability of the manufacturer's test results.
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Phase 2 batch release procedures:

3.1 The number of additional doses of each vaccine batch (product supplied in final
package) or the volume of trivalent or monovalent bulk vaccine to be submitted for testing to
the control authority will depend on the nature of the additional tests.
3.2 The nature of the additional batch release tests to be performed will depend on the
circumstances for introduction of phase 2 tests.
3.3 Information concerning failed batches may be required as part of phase 2 batch
release procedures.
4. RELEASE CERTIFICATE
A release certificate for each vaccine batch shall be presented to the manufacturer after
approval when the results of testing are satisfactory. The release certificate must give details
of:
4.1 Name and address of manufacturer
4.2 Trade name and proper name of product
4.3 Batch number
4.4 Number of containers
4.5 Number of doses per container
4.6 Type of container
4.7 Date of release and reference number
4.8 Date of expiry
5. SUMMARY PROTOCOL FOR INACTIVATED INFLUENZA

VACCINES
The following summary protocol is an example of the type of information required for batch
release. The data submitted should be in accordance with the current EP monograph on
influenza vaccines.
Name of product:
Marketing authorisation:
Name and address of manufacturer:
Batch number:
Filling lot number:
Date of manufacture:
Date of expiry:
Type of container:
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Number of doses:
Dose volume:
Composition:
e.g. strain 1 15 pg HA/0.5 ml

strain 2 15 pg HA/0.5 ml
strain 3 15 ug HA/0.5 ml
Statement of quality:
e.g. I certify that lot number of this product satisfies the requirements of the European
monograph on influenza vaccines.
Signature:
Name (typed):
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Production Flow Sheet
Primary seed Primary seed Primary seed

H3N2 HINI
0
Lotn : Lotn: Lotn:
Working seed Working seed Working seed

H3N2 HINI
Lotn: Lotn: Lotn:
Monovalent bulk Monovalent bulk Monovalent bulk

H3N2 HINI
0 Lotn:
Lotn : Lotn:
Trivalent bulk
Lotn 0 :
Final product
Filling lot N:
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Seed Virus
1. Information on manufacture
1.1 Virus strain:
1.2 Source and lot No of primary seed:
1.3 Passage history of receipt:
1.4 Date of receipt:
1.5 Comments:
1.6 Storage conditions:
1.7 Working seed lot No:
1.8 Passage history of seed lot(s):
1.9 Added antibiotics:
1.10 Storage conditions of working seed lot(s):
2. Tests on working seed virus

2.1 Sterility
method:
Date of test:
Volume tested:
Test results:
2.2 Test for mycoplasma

Method:
Date oftest:
Volume tested:
Test results:
2.3 Identity
a) Haemagglutinin
Date oftest:
Test results:
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e.g.
HI Titre
Antigen Antiserum
Shang/1 Sich/2/87 Taiw/1/86 B/Yam/16/88
1/87
A/Shang/11/87
(H3N2) Ref.
A/Sich/2/87
(H3N2) Ref.
A/Taiw/1/86
(HINI) Ref.
A/Sang/11/87
Working seed lot n
b) Neuraminidase
Date oftest:
Test results:
e.g.
NT Titre
Antigen Antiserum
anti-N2NA anti-NlNA anti-BNA
A/Shang/11/87
(H3N2) Ref.
A/Sich/2/87
(H3N2) Ref.
A/Taiw/1/86
(HINI) Ref.
B/Yam/16/88
Ref.
A/Sang/11/87
Working seed lot n ...
2.4 Infectivity titre:

Date of tests:
Test results:
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Monovalent Virus Pool
1.1 Virus strain:
12 Lot numbers):
1.3 Working seed lots used:
1.4 Date of inoculation:
1.5 Date of harvesting:
1.6 Method of inactivation:
1.7 Date of inactivation:
1.8 Concentration/purification procedure:
1.9 Added antibiotics:
1.10 Filtration details (if any):
2. Tests on monovalent virus pool

2.1 Test for inactivation
Date oftest:
Test result:
2.2 Test for haemagglutinin antigen content

Method:
Date of test:
Test results:
2.3 Identity of haemagglutinin

Method:
Date oftest:
Test results:
2.4 Purity (for surface antigen vaccines only)

Method:
(e.g. type of PAGE system, reducing/non reducing conditions)
Date oftest:
Test results:
(e.g. HA, M and NP bands must be identified. Comparison between whole virus and
surface antigen preparation must be made)
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Bulk Vaccine
Date of test:
Test results:
1.1 Lot number:
1.2 Lot number and volume of monovalent pools used to prepare bulk:
1.3 Other substances added and volumes:
1.4 Date of blending:
2. Tests on bulk vaccine

Analytical tests
Method(s):
Test results:
(include test for mercury, if appropriate)
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Finished Product
1.1 Lot number:
1.2 Date of filling:
1.3 Type of container:
1.4 Volume in container:
1.5 Number of doses filled:
2. Tests on finished product

2.1 Identity for haemagglutinin
Method:
Date of test:
Test results:
2.2 Sterility
Method:
Date of test:
Test results:
2.3 Haemagglutinin antigen content

Method:
Date oftest:
Test results:
2.4 Total protein (this test may be performed on bulk vaccine)

Method:
Date of test:
Test results:
2.5 Abnormal toxicity

Method:
Date of test:
Test results:
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2.6 Ovalbumin (this test may be performed on bulk vaccine)

Method:
Date oftest:
Test results:
2.7 Endotoxin
Method:
(e.g. type of limulus kit)
Date oftest:
Test results:
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D. CLINICAL TRIAL RELATED TO YEARLY LICENSING OF

INFLUENZA VACCINE
L INTRODUCTION
When a new application for marketing authorisation for an influenza vaccine is made, full
clinical trial data should be submitted with the application. Such clinical trials are outside
the scope of this note for guidance. However, the strain composition of influenza vaccines i s
modified periodically to take account of the changes in the prevalent viruses causing
influenza and manufacturers should apply for yearly licensing to accommodate strain
changes.
Vaccine manufacturers are required to be involved in ongoing clinical trials of influenza
vaccines and to present the results to the competent authorities. The results of the trials are
not part of the yearly licensing procedure. Guidance for performing these clinical trials is
given in this section.
The purpose of such trials is to verify:
- the tolerance or incidence of adverse reactions;
- the immunogenicity of the haemagglutinin of the vaccine strains, i.e. the titre and
frequency of anti-HA antibody responses;
Whenever the characteristics of a new strain incorporated into the vaccine or the
susceptibility of the population to the new strain requires adjustment of the doses, various
doses of antigens need to be tested to confirm the adequacy of 15 pg HA per strain and per
dose.
The yearly clinical trials on influenza vaccine shall be carried out in accordance with the
note for guidance on Good Clinical Practice.
The clinical trials are carried out by the manufacturers, who will forward the results, as
soon as they have been obtained, to the competent authorities and in any event before the next
influenza season.
2. GENERAL REQUIREMENTS
2.1 Vaccine used in the trial
The composition of the vaccine used in the trial shall be such as to fulfil the requirements of
the yearly EC recommendation with regard to vaccine strains. The batches of vaccine used
shall be representative of the product placed on the market.
2.2 Trial population

The tolerance and efficacy of the vaccine shall be evaluated separately in two groups of
healthy volunteers, aged between 18 and 60 and over 60; for the latter group, it is important
that the previous vaccination status of each subject be known and recorded.
Groups of at least 50 individuals shall be constituted.
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2.3 Exclusion criteria

The following subjects shall not be entered into a trial:
- subjects known to be allergic to chicken protein;
- subjects with fever or presenting an infectious episode;
- pregnant women.
2.4 Trial procedure

a) Just prior to vaccination, a 10 ml venous blood sample shall be taken from each trial
subject, for base-line titration of circulating anti-HA antibodies;
b) immediately thereafter, each subject shall receive 1 dose of vaccine (0.5 ml) by
intramuscular or subcutaneous injection into the upper arm. The injection shall be
given into the opposite arm from which blood was drawn;
c) approximately 3 weeks after vaccination, a 10 ml blood sample shall be taken from
each subject. Sera shall be separated and stored at -20C; samples shall be kept at the
disposal of the control laboratories for epidemiological studies and possible further
antibody titration;
d) in the event of intercurrent infection, nasal and/or pharyngeal swabs shall be
collected, in order to allow diagnosis of either influenza or another viral respiratory
infection.
2.5 Monitoring of adverse reactions

a) Trial subjects shall receive, at the time of vaccination, a standardised form to complete
and give to the investigator when they come for the post-vaccination blood sampling;
b) the form shall allow for collection of the following information:
- initials of the subject, with date or year of birth;
- previous anti-influenza vaccinations and previous side-effects, if any;
- previous influenza infections, with date, description of symptoms and virological
confirmation, if any;
- side-effects for the 3 days following vaccination, either local (induration,
erythema, ecchymosis, pain) or general (fever, shivering, malaise, other side-
effects);
- other side-effects lasting 2 days beyond vaccination should be noted.
2.6 Antibody titration

All sera shall be assayed for anti-haemagglutinin antibody against the prototype strains by
HI (Palmer et al., 1975) or SRH (Schild et al., 1975, Aymard et al., 1980) tests.
Positive and negative sera as well as reference preparations may be obtained from a
reference laboratory.
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2.7 Interpretation of results and statistics

Antibody titrations shall be done in duplicate; pre- and post-vaccination sera shall be titrated
simultaneously.
The titre assigned to each sample shall be the geometric mean of two independent
determinations:
a) for the purposes of calculation, any HI result < 10 (= undetectable) shall be expressed as
5 and any negative SRH result shall be expressed as 4 mm 2 * ;
b) in HI tests, seroconversion corresponds to:
- negative pre vaccination serum/postvaccination serum 2 40;
- a significant increase in antibody titre, i.e. at least a fourfold increase in' titre;
c) in SHR tests, seroconversion corresponds to: (*)
- negative prevaccination serum/postvaccination serum: area 2 25 mm 2 ;
- a significant increase in antibody titre, i.e. at least a 50% increase in area;
d) statistical parameters to be determined:
- geometric mean of prevaccination serum anti-HA antibody titres;
- increase in the geometric mean of antibody titre;
- number of seroconversions;
- proportion of subjects with a titre of antibodies before vaccination;
- proportion of subjects with a titre of antibodies after vaccination;
e) clinical tolerance: frequency, mean time of appearance and duration of all local and
general side-effects shall be calculated.
3. CRITERIA FOR ASSESSMENT OF VACCINES

3.1 Serological data
a) The following serological assessments should be considered for each strain in adult
subjects, aged between 18 and 60:
- number of seroconversions or significant increase in antihaemagglutinin
antibody titre > 40%;
- mean geometric increase > 2.5;
the proportion of subjects achieving an HI titre 2 40 or SRH titre 2 25 mm 2 ( )

should be > 70%.
In most SRH test systems, a zone area of 25 mm 2 is approximately equivalent to an HI titre of 1:40. However,
this relationship can be affected by experimental conditions and should be re-examined in each laboratory so
as to calibrate the test system adequately.
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b) The following serological assessments should be considered for each strain in the
group of subjects aged over 60:
- number of seroconversions or significant increase in antihaemagglutinin
antibody titre > 30%;
- mean geometric increase > 2;
- the proportion of subjects achieving an HI titre 2 40 or SRH titre 2 25 mm2* should
be > 60%.
3.2 Clinical data

The frequency of the following symptoms should be assessed:
a) local reactions:
- indurations larger than 50 mm diameter and persisting for more than 3 days;
- ecchymosis;
b) general symptoms:
- temperature above 38 C for 24 hours or more;
- malaise;
- shivering.
References
Aymard, M., Million, J., Kessier, N.: Diagnostic srologique rapide de la grippe par la
mthode d'hmolyse radiale modifie et volution des anticorps. Path. Biol. 1980, 28, n 8,
535-539.
Palmer D.F., Dowie, W.R., Coleman M.T. et Schild G.C.: Advanced laboratory technicals for
immunological diagnostic. U.S. Dept. Hith. Ed. Welfare, P.H.S. Atlanta. Immunology ser.
Nr. 6, Procedural guide. Part 2: haemagglutination - inhibition test, 1975, 25-62.
Schild G.C., Pereira, M.S. Chakraverty, P.: Single radial haemolysis: a new method for the
assay of antibody to influenza haemagglutinin. Bull. WHO. 1975, 52, 43-50.
In most SRH test systems, a zone area of 25 mm 2 is approximately equivalent to an HI titre of 1:40. However,
this relationship can be affected by experimental conditions and should be re-examined in each laboratory so
as to calibrate the test system adequately.
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ALLERGEN PRODUCTS
Guideline Title Allergen Products

Legislative basis Directive 89/342/EEC, Directive 75/318 EEC as amended
This version revised May 1996
force
Status Last revised May 1996
references
Additional Notes This revision consists of a clarification related to chapter
4, control of starting material, first paragraph, last
sentence of point 4.2.1.
This note for guidance only refers to industrially
produced allergen products placed on the market as
medicinal products for the purpose of in vivo diagnosis
or for treatment of allergic disease.
CONTENTS
1. INTRODUCTION
2. QUALITATIVE AND QUANTITATIVE PARTICULARS OF THE

CONSTITUENTS
3. DESCRIPTION OF METHOD OF PREPARATION
4. CONTROL OF STARTING MATERIAL
5. CONTROL TESTS CARRIED OUT AT AN INTERMEDIATE STAGE OF THE

MANUFACTURING PROCESS OF THE FINISHED PRODUCT
6. CONTROL TESTS CARRDED OUT ON THE FINISHED PRODUCT
7. STABILITY
8. SAFETY TESTING
9. EFFICACY TESTING
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ALLERGEN PRODUCTS
L INTRODUCTION
Directive 89/342/EEC extends the scope of Directive 65/65/EEC and 75/319/EEC to
immunological medicinal products consisting of vaccines, toxins or serums and allergens.
For this purpose, Article 1, paragraph 2 of Directive 89/342/EEC defines 'allergen product' as
any product which is intended to identify or induce a specific acquired alteration in the
immunological response to an allergising agent. Thus, the adoption of Directive 89/342/EEC
implies that any allergen's product is now subject to the requirements of European
pharmaceutical legislation, namely as regards the quality, safety and efficacy testing and
marketing authorisation. However, Directive 89/341/EEC also lays down exemptions from
the general requirements of the European pharmaceutical legislation. Under Article 1,
paragraph 4 of this directive, a Member State may, in accordance with legislation in force
and to fulfil special needs, exclude from Chapters II to V of Directive 65/65/EEC medicinal
products supplied In response to a bona fide unsolicited order, formulated in accordance with
the specifications of an authorised health care professional and for use by his individual
patients on his direct personal responsibility ('named patient exemption').
Therefore, for the purpose of this note for guidance, allergen products are divided into two
categories:
a) industrially produced allergen products containing either a single allergen or defined
mixtures placed on the market as medicinal products either for:
the purposes of in vivo diagnosis or for
treatment of allergic disease;
b) allergen product prepared on the basis of an individual prescription and intended to be
used on a 'named patient' basis.
This note for guidance only refers to industrially produced allergen products placed on the
market as medicinal products for the purpose of in vivo diagnosis or for treatment of
allergic disease (point a) above).
2. QUALITATIVE AND QUANTITATIVE PARTICULARS OF

THE CONSTITUENTS
Qualitative Particulars
The name (scientific name, e.g. genus and species as well as any common name), and type
(e.g. pelt, dander, saliva) of the allergenic source material(s) should be stated. In the case of
modified or adsorbed allergen products, this and the agents used in the modification
procedures should be described. For all the excipients, the names, grades and quantities
should be given. This also includes the composition of any separate packaged dilution or
reconstitution fluid to be used with the product.
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Quantitative Particulars
Whenever possible, the potency of the active ingredient should be expressed in units of
biological activity, and the unit system used should be unambiguously indicated, in order to
avoid confusion with similar unit systems currently in use on the market.
3. DESCRIPTION OF METHOD OF PREPARATION

The manufacturing process within the meaning of this section is the preparation of the
finished product from the starting materials. The description should include details of any
process employed, in particular sterilisation, filling, freeze-drying, addition of any
preservative and of any stabiliser. Whenever materials of human or animal origin are
present in the finished product as excipients, particular attention should be given to safety
from transmission of infectious diseases. The use of materials and/or excipients known to
give rise to sensitisation should be avoided or justified.
4. CONTROL OF STARTING MATERIAL

General Requirements
The allergenic source material should be described in as much detail as possible. Details
concerning collection, pre-treatment and storage should be supplied for each separate
allergen. The name and the address of the supplier shall be stated. Specifications and
control methods for the source material(s), applied by the supplier, should be included. The
specifications should ensure that the qualitative and quantitative composition of the material
is as uniform as possible from one delivery to another. They should encompass
requirements and control methods relating to identity and purity. Quality control of source
materials should be documented. The source materials should be stored under controlled
conditions.
Additional Requirements for Specific Source Materials

42.1 Pollens
Nature of the fields, seeds used, field characteristics and treatment, visual control, manner
of collection and random sampling should be described. The manner of collection of pollens
should be stated. Tests for content of foreign pollens, spores, extraneous plant material from
the same species and non-related contamination should be included. The content of
representative pesticides and lead should be measured on a limited number of pollen
batches, in order to demonstrate that their level is kept at a minimum.
The pollen content from other species should be limited to 1% of mixed pollens and 0.5% of a
single pollen as determined by a microscopic particle count. Detectable spores should not
exceed 1%. Contamination by particles of plant origin, other than pollen, should not exceed a
total of 10% in terms of microscopic count. Justification should be given if these standards
cannot be compiled with.
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4.2.2 Moulds
The strain or strains of moulds should be specified. The cultivation method should be
described. Details of the composition of the cultivation medium should be submitted.
Strains which produce mycotoxins such as anatoxins or ochratoxins should not be used
unless justified. In this case, the source material should be tested for mycotoxins; the source
materials should be tested for mutagenicity before processing unless the removal of
mycotoxins has been validated.
Synthetic and consequently allergen-free media shall preferably be used. Morphological
characteristics (mycelium and spores / spores only / mycelium only) as well as the
cultivation method in the isolated raw material should be specified. Conditions of culture
must be validated to provide evidence that mycotoxins are not produced.
42.3 Mites
The cultivation method and the composition of the cultivation medium should be described.
When substrates of human origin are used in the culture medium, the absence of risk of
transmission of infectious diseases should be demonstrated. To this end, the manner of
collection of these substrates should be described in detail. Any allergenicity of the medium
should be as low as possible in order not to produce any unspecific reactions of the finished
product. Therefore, the use of animal dander or any animal protein in the culture medium
should be avoided. It should be indicated whether for further processing, mites only or the
whole mite culture is used.
4.2.4 Animal allergens

The species of the animal should be stated. An account should be given of the health
examination of the animals from which the raw materials are collected. Materials should be
collected from animals that do not exhibit overt infections at the time of collection. The
collector should certify that the animals used are overtly healthy and have not recently been
treated with antiparasitics or other medicinal products.
When killed animals are used, the epidermals should be collected within a few hours. The
animal must be stored in conditions ensuring that post-mortem decomposition processes do
not affect the epithelium; these storage conditions should be described. Collection of hair and
dander must take place using methods which provide a good epithelial harvest without
injuring the skin of the animal. Methods employing the grinding of whole skin/pelts must
not be used. The composition of the final source material (e.g. hair, dander, pelt, saliva,
urinary fluid) should be indicated.
4.2.5 Hymenoptera venom

The method of collection of venom from the venom sacs of hymenoptera species should be
described and should be such as to ensure that the raw material is of a proper quality.
Description of the Production Process

The production process should be described, step by step with a diagram (flow-chart)
indicating the principles of the process, accompanied by an explanatory text. The different
stages of the production process, such as grinding, extraction, filtration, clarification,
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dialysis, concentration, fractionation, sterilisation, lyophilisation etc. should be clearly

defined. The description should state the stage at which aseptic precautions are introduced.
Intermediate or bulk products in the process should be identified and the in-process controls
performed at these or other stages of production reported. The principle of the purification
and fractionation methods should be defined, and it should be clearly apparent at which step
in the process special biochemical techniques are used.
The manufacturer should demonstrate his capability of obtaining batch to batch consistency.
Batch to batch consistency

Since allergen products are generally represented by a complex mixture of allergenic and
non-allergenic components, they cannot be easily standardised and each component cannot
be defined, with a few exceptions, in a quantitative way. Although some international
reference preparations or standards are available, It is commonly accepted that allergenic
extracts cannot all be standardised in the same manner as the other biological products.
An extract will be different from one company to another and possibly, within a single
company, from one batch to another. These characteristics represent a real problem as
regards any future harmonisation of the extracts between companies. It must be stressed that
at least a good batch to batch consistency has to be reached by a company within its
production runs by introducing in House Reference preparations (IHR) which should be used
as internal reference preparation and using a number of biological and analytical methods.
The IHR is derived from a production run following the manufacturing process defined i n
the dossier; it establishes a reference point against which extracts from all future production
runs will be compared. Thus the qualitative composition of regular production batches should
match the IHR.
4.4.1 Characterisation of the in house reference preparations (IHR)

The IHR shall be characterised using available relevant methods and its specific allergenic
activity shall be established, and data should be provided on protein and, whenever possible,
carbohydrate composition. Some of the following methods should be applied: Crossed-
immunoelectrophoresis (CIE), isolectricfocusing, electrophoresis in Polyacrylamide gel,
determination of the distribution of molecular weight by SDS-PAGE analysis, HPLC, gel
electrophoresis and quantitative determination of total protein. Information regarding the
allergenic specificity of the proteins in the IHR may be obtained from experiments
involving combinations of electrophoretic methods and immunoblotting techniques or
Crossed-Radio-immuno-Electrophoresis (CRIE). Sensitivity spectra (allergograms) derived
from such basic documentation, thus identifying the major, intermediate and minor
allergens. The presence of all relevant allergens in the IHR shall have been demonstrated
in comparative studies involving several batches of extract. As far as possible, the
individual allergens should be identified using internationally accepted nomenclature or
the correspondence with allergens described in the scientific literature should be given,
including literature references. The potency of this IHR should be judged by immuno-assays
(e.g. IgE-inhibition, ELISA-techniques, immunofluorescence techniques) or/and Skin Prick
Test and expressed in terms of Units of Biological Activity. When a product consists of one
or a few well characterised allergenic components, potency can be assayed by means of
alternative relevant techniques, such as single radial immunodiffusion, quantitative
Immunoelectrophoresis or other quantitative techniques. All these methods and the
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immunological reagents mentioned above should be in accordance with the scientific

knowledge at the time of application.
The stability of the IHR and storage conditions should be documented.
4.42 Use of the IHR

The characterised and standardised IHR for a given allergen product should be used to
prove batch to batch consistency, by using relevant methods already employed in the
characterisation and standardisation of the IHR. The choice of the methods used must be
justified and limits for variations of the method should be defined and documented.
5. CONTROL TESTS CARRIED OUT AT AN INTERMEDIATE

STAGE OF THE MANUFACTURING PROCESS OF THE
FINISHED PRODUCT
Control tests carried out at intermediate stages of manufacture should be defined. When
certain control tests cannot be applied to the finished product, for instance in the case of
chemically modified, precipitated or adsorbed allergen preparations, quality specifications
should be defined for the product just prior to the modification, dilution, etc.
6. CONTROL TESTS CARRIED OUT ON THE FINISHED

PRODUCT
Measurements of the total allergenic activity of individual batches of an allergen extract
should be undertaken preferably by IgE Inhibition or by direct IgE-binding or other immuno-
assays, all methods having to be suitably validated.
The estimated potency derived from the assay of total allergenic activity should be not less
than 50% and not more than 200% of the stated potency.
The characteristics of the finished product should ideally be documented for all strengths
(dilutions). Where appropriate testing is not possible due to methodological limitations, this
should be justified.
For adsorbed/modified products, where measurement of allergenic activity is not possible on
the finished product, documentation of adsorption/modification should be provided.
7. STABILITY
The note for guidance Stability tests of Active Ingredients and Finished Products should be
followed. However, in some circumstances, It is impossible or difficult to fully evaluate the
allergenic potency and other characteristics of the finished product. In these cases, such as
adsorbed-modified or adsorbed-unmodified allergens, it would be acceptable to carry out
stability tests just before applying the modifying treatment.
In addition, the stability of adsorption should be monitored over the proposed shelf life, since
free allergens can cause immediate (anaphylactic) reaction.
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For stability data, the concept of taxonomie family may be applied and data obtained on one
member of such a family may be extrapolated within that same family. This extrapolation
should be discussed and justified. In the case of mixtures of members of different taxonomie
families, extrapolation is not acceptable.
No less than 30% of the stated allergenic activity should be maintained at the end of shelf
life.
8. SAFETY TESTING
Details of the safety testing undertaken should be provided. Safety testing may have to be
adapted to individual products and, if so, any omissions with regard to the requirements
laid down in Part 3 of the Annex to Directive 91/507/EEC should be justified.
For safety testing, the concept of taxonomie family may be applied and data obtained on one
member of the family may be extrapolated to another member of that family providing the
manufacturing procedures applied are the same. In the case of mixtures of members of
different taxonomie families, extrapolation is not acceptable.
9. EFFICACY TESTING
Details of clinical trials performed should be provided.
For the performance of clinical trials, the concept of taxonomie family may be applied and
data obtained on one member of the family may be extrapolated to another member of that
family providing the manufacturing procedures applied are the same. In the case of
mixtures of members of different taxonomie families, extrapolation is not acceptable.
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ASSESSING THE EFFICACY AND SAFETY OF

HUMAN PLASMA DERIVED FACTOR VllhC AND
FACTOR IX:C PRODUCTS IN CLINICAL TRIALS IN
HAEMOPHILIACS BEFORE AND AFTER
AUTHORISATION
Guideline Title Assessing the Efficacy and Safety of Human Plasma

Derived Factor Vlllrc and Factor IX:c Products in Clinical
Trials in Haemophiliacs Before and After Authorisation
Date of entry i n t o February 1996
force
Previous titles/other Guidelines to Assess Efficacy and Safety of Human Plasma
references Derived Factor VIII:c and Factor IX:c Products in Clinical
Trials in Haemophiliacs Before and After Authorisation
IH/5769/94, CPMP/BPWP/198/95
Additional Notes This note for guidance describes the clinical trials
required for authorisation with respect to two different
human plasma derived factor VIII and Factor IX products:
1) Products for which a marketing authorisation is to be
submitted and 2) Authorised products in which a change
in the manufacturing process has been made.
CONTENTS
1. INTRODUCTION
2. PRODUCTS FOR WHICH A MARKETING AUTHORISATION IS APPLDED
3. AUTHORISED PRODUCTS IN WHICH A CHANGE IN THE

MANUFACTURING PROCESS HAS BEEN MADE (E.G. ADDITIONAL VHIAL
INACTrVATION STEP)
APPENDIX 1
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HUMAN PLASMA DERIVED FACTOR VIII:C AND
FACTOR IX:C PRODUCTS IN CLINICAL TRIALS IN
HAEMOPHILIACS BEFORE AND AFTER
AUTHORISATION
1 INTRODUCTION
Before, 1960, plasma was the only agent generally available for the treatment of the
hereditary coagulation disorders. Several plasma product concentrates are now available for
this purpose which have been purified and virally inactivated in various ways. With respect
to factor VIII deficiency the replacement therapy consists of factor VIII concentrates of
different purity. Random reports of a relatively high incidence of inhibitors after
administration of factor VIII products together with reports of viral infections (hepatitis C,
hepatitis A and 7) after administration of human plasma derived coagulation factors,
has made the regulatory authorities more aware of the potential risks of these products. As
one of several measures aimed at improving viral safety of blood products, it is necessary to
demand adequate clinical investigation before a marketing approval is granted. Clinical
trials are necessary addressing efficacy, safety with respect to transmission of viral
infections and immunogenicity for FVIII and FDC concentrates. In addition in applications
for FDC concentrates thrombogenicity should be addressed.
In view of outbreaks of hepatitis A among haemophiliacs treated with a solvent detergent

factor VIII in 1992 the Committee on Proprietary Medicinal Products (CPMP) approved the
position paper of the biotechnology working party (IH/5830/93 Final) on blood products and
non-enveloped viruses. It was recommended that the manufacturing process should include
viral inactivation/removal which is also effective against non-enveloped virus. As a virus
inactivation/removal step (or any change in the purification process) may alter the structure
of the coagulation factor and/or induce loss of activity, efficacy and immunogenicity data
are clearly needed. This guideline describes the clinical trials required for authorisation
with respect to two different human plasma derived factor VIII and Factor TX. products:
1) Products for which a marketing authorisation is to be submitted.
2) Authorised products in which a change in the manufacturing process has been made
(e.g. additional viral inactivation step).
The clinical trials should be performed according to the guidelines on Good Clinical
Practice.
Recombinant DNA products are excluded from this guideline.
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2. PRODUCTS FOR WHICH A MARKETING AUTHORISATION

IS APPLffiD
2.1 Efficacy
In clinically evaluating a new human plasma derived coagulation factor for the treatment
of haemophilia A or patients, the initial trial is typically one that examines the
pharmacokinetics of the principal active factor. Half life and recovery data are the most
important (surrogate) endpoints for efficacy of a new factor VIII or Factor DC product. The
guideline below is according to the guidelines proposed by the International Society of
Thrombosis and Haemostasis (ISTH) (Thrombosis and Haemostasis 1991; 66 (3):384-386).
2.2 Safety
Safety aspects of a new factor VIII or Factor DC product refer to viral safety, immunogenicity
and any other adverse events. For factor DC products thrombogenicity should also be
considered as a safety issue.
22.1 Adverse events

All adverse events after infusion of the new product should be reported.
22.2 Viral Safety

Manufacturers of coagulation factor concentrates are obliged to optimise viral safety by
rigorous selection of donors, screening of donations, including testing for HBsAg, antibody
to hepatitis C virus, antibody to 1+2 and by using appropriate viral
elimination/inactivation methods according to the requirements in CPMP guideline
Medicinal Products derived from Human Blood and Plasma and Virus Validation Studies:
The Design, Contribution and Interpretation of Studies Validating the Inactivation and
Removal of Viruses. Three principal complementary approaches are adopted to control
potential viral contamination of coagulation factor products: selecting and testing source
material, testing the capacity of the production process to remove or inactivate viruses, and
testing the product at appropriate stages of production for absence from contaminating
viruses.
When medicinal products prepared from human blood or plasma are administered,
infectious diseases due to transmission of ineffective agents cannot be totally excluded. This
applies also to hitherto unknown pathogens. The current procedures applied in the
manufacture of medicinal products derived from human blood or plasma are effective
against enveloped viruses such as HIV and Hepatitis and C. These procedures are of
limited value against non-enveloped viruses such as Hepatitis A and Parvovirus 19.
All patients included in the trials should be followed up for safety issues according to a
protocol described in the appendix.
22.3 Immunogenicity
2.2.3.1 Factor VIIIproducts
The occurrence of antibodies against factor VIII is one of the major possible complications of
haemophilia treatment. The risk of inhibitor occurrence is higher in patients with severe
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haemophilia A than in patients with moderate and mild disease. Inhibitor risk may be
associated with commencing or changing treatment or where the antigenicity of the product
has been altered due to changes in the manufacturing process. Prior to authorisation
immunogenicity of a new factor VIII product should be investigated first in previously
treated patients (PTPs) and then, depending on the claimed indication, in previously
untreated patients (PUPs). The Summary of Product Characteristics (SPC) should include a
section stating the experience with respect to immunogenicity in PUPs or state that there is
no such experience.
2.2.32 Factor IX products
Haemophilia is from 4 to 8 times less common than haemophilia A. The incidence of
inhibitors in these patients after administration of factor DC is rarer than in Haemophilia A
patients. Inhibitors to factor DC have been demonstrated in approximately 4% of patients with
severe haemophilia B. Nevertheless with the development of purified factor DC concentrates
the immunogenicity should be investigated prior to authorisation of factor DC products. The
SPC should include a section stating the experience with respect to immunogenicity i n
PUP's or state that there is no such experience.
2.2.4 Thrombogenicity (factor IX products)

Treatment with Factor DC concentrates containing Factors II, VII and X has been associated
with a risk of thrombosis. Purified Factor DC products have been shown to be associated with
a lower thrombogenic risk. For all new factor DC concentrates testing for markers of
activation of coagulation should be carried out in the non-bleeding state.
2.3 Clinical trials w i t h factor VIII p r o d u c t s

2.3.1 Pre-Authorisation
2.3.1.1 Efficacy
A pharmacokinetic trial, measuring half-life and recovery, should be performed in at least
12 subjects with haemophilia A (factor VIII < 2%) without inhibitor and not actively bleeding.
Patients should be at least 12 years of age and should not have received an infusion of
concentrate in at least the past 3 days (if possible 7 days). Samples for factor VIII activity
determination should be taken before injection of 25-50 IU/kg of the new factor VIII product
and between 15 and 30 minutes, 1, 3, 6, 9, 12 24 and 30-36 hours after the infusion. At least 3
different lots should be employed in the trial. Recovery should be determined from the peak
factor VIII activity in the first four sample time periods post-infusion.
Patients who participated in the pharmacokinetic trial should continue treatment with the
product for 6 months and at least 5 patients should be tested for half-life and recovery after 3-
6 months.
Clinical efficacy and tolerability after administration should be assessed from the clinical
response as reported by patients in the safety trials (see 2.3.2) at the regular visits. Response
should be assessed as "none", "moderate", "good" or "excellent" by the physician for those
patients who were treated with the product for major bleeds. In addition, response will be
determined by the physician in a minimum of 5 patients undergoing at least 10 surgical
procedures, including achievement of haemostasis, loss of blood and requirement for
transfusion.
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23.12 Safety
Clinical safety will be assessed in all patients receiving the new factor VIII product.
In hospitalised patients, such as patients included in the pharmacokinetic trial, for
blood pressure, heart rate, temperature, respiratory rate and adverse events.
In out-patients for adverse events.
2.3.1.2.1 FTP (Previously treated patient) study
A minimum of 30 immunocompetent (CD4 lymphocytes > 400/ml) previously treated patients
with severe haemophilia A with at least 10 exposure days to the new factor VIII product and a
follow up of at least 6 months for all patients must be included prospectively.
Immunogenicity
The factor VIII inhibitor titre will be determined every 3 months. An interim analysis will
be performed when 30 patients not having undergone surgery have been treated for 6 months
with a minimum of 10 exposure days each. The titre of the inhibitor should be reported i n
Bethesda Units (BU) as well as the clinical relevance the cumulative incidence and the
number of exposure days (also in relation to development of inhibitors).
Viral safety
According to EC Good Clinical Practice the PTP patients should be followed up for viral
safety markers. Full baseline data for markers of viral infection (aminotransferase, HIV
1+2 ab, HCV ab, HBV antigen and ab and HAV ab) should be provided according to the table
in Appendix 1. All patients negative for these markers should have regular testing
according to the schedule in the Table. In patients who are Parvovirus 19 antibody negative
at entry a sample should be tested using gene amplification methods at one week after the
first treatment. Serum samples should be stored at -70C whenever the patient is sampled, for
possible future testing. No claims can be made in the SPC on viral safety of the product with
respect to parvovirus 19 transmission.
2.3.1.2.2 PUP (previously untreated patient) study
PUP studies should be carried out, or at least initiated. The SPC should include a section
stating the experience with respect to inhibitor development in PUPs or state that there is no
such experience. A PUP study should be done only after results of the PTP trial are available
and according to the guideline.
An open label uncontrolled multicentre trial in previously untreated severe haemophilia A
patients should include at least 20 patients. These patients should be tested for viral safety
and inhibitor development.
Viral safety
Viral safety data (serum aminotransferases, 7, hepatitis C) should be monitored at 3
month intervals for at least 2 years and be reported at 6 month intervals. In patients who are
Parvovirus B19 antibody negative at entry a sample should be tested using gene
amplification methods at one week after the first treatment. Serum samples should be stored
at -70C whenever the patient is sampled, for possible future testing.
Immunogenicity
The patients should be tested every 3 months for at least 100 exposure days or 5 years
whichever comes first. The titre of the inhibitors should be reported (Bethesda Units = BU) as
well as the clinical relevance, the cumulative incidence and the number of exposure days.
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2.3.2 Post-Authorisation
2.3.2.1 PTP
After authorisation laboratory parameters (according to the table in the appendix) from at
least 50 PTPs treated for at least 2 years should be included in the Periodic Safety Update.
2.3.22 PUP
Immunogenicity and viral safety data (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update as described
in the note for guidance Clinical Safety Data Management: Periodic Safety Update Reports
for Marketed Medicinal Products.
2.4 C l i n i c a l t r i a l s w i t h f a c t o r LX p r o d u c t s
2.4.1.1 Efficacy
A pharmacokinetic trial, measuring half-life and recovery, should be performed in at least
10 subjects with haemophilia (factor DC <2%). Patients should be at least 12 years of age
and should not have received an infusion of concentrate in at least the past 4 days (if
possible 7 days). Samples for factor DC activity determination should be taken before
injection of 50-75 IU/kg of the new factor DC product and 30 minutes, 1, 3, 6, 9, 12, 24, 30, 36
and 50 hours after the infusion. At least 3 different lots should be employed in the trial.
Recovery should be determined from the peak factor IX activity in the first four sample time
periods post-infusion.
product for 6 months and at least 5 patients should be tested again for half life and recovery
after 3-6 months.
Clinical efficacy and tolerability after administration should be assessed from the clinical
response as reported by patients in the safety trials (see 2.4.2) at the regular visits. Response
should be assessed as "none", "moderate", "good" or "excellent" by the physician for those
patients who were treated with the product for major bleeds. In addition, response will be
determined by the physician in a minimum of 5 patients undergoing at least 10 surgical
procedures, including achievement of haemostasis, loss of blood and requirement of
transfusions.
2.4.12 Safety
In addition to the requirements for factor VIII products, tests for activation of coagulation
after administration of the product should be carried out. This should be determined in the
patients of the pharmacokinetic trial as well in a minimum of 5 patients undergoing at least
10 surgical procedures. In the surgical patients clinical evaluation of thrombosis should be
undertaken by safe, objective means. Due to the lower incidence of haemophilia as
compared to haemophilia A, the number of previously treated patients followed up for viral
safety and immunogenicity should be lower than for factor VIII products: 12 for viral safety
and 20 patients for immunogenicity. If a PUP study is done, it should include 15 patients.
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2.4.2.1 PTP
After authorisation, laboratory parameters (according to the table in the appendix in the
appendix) from at least 30 PTPs treated for at least 2 years should be included in the
Periodic Safety Update.
2.42.2 PUP
Immunogenicity and viral safety data (including parvovirus 19 seroconversion) on any
PUPs treated with the produce should be included in the Periodic Safety Update.
3. AUTHORISED PRODUCTS IN WHICH A CHANGE IN THE

MANUFACTURING PROCESS HAS BEEN MADE (E.G.
ADDITIONAL VIRAL INACTWATION STEP)
3.1 Efficacy
As a virus inactivation/removal step (or any change in the purification process) may alter
the structure of the coagulation factor and/or induce loss of activity, pharmacokinetic
studies, measuring half-life and recovery are clearly needed.
3.2 Safety
Viral safety
If the additional viral reduction process concerns reduction of non-enveloped viruses only,
and the original manufacturing process has already been validated for the removal of
enveloped viruses, then it may be assumed that the original factor VIII or Factor DC product
is safe with respect to removal/inactivation of hepatitis virus, H P / 1+2 and hepatitis C
virus and that the additional viral removal/inactivation step is designed to remove non-
enveloped viruses such as hepatitis A and parvovirus 19. As most haemophilia patients are
vaccinated against hepatitis A, clinical follow-up of these patients is difficult. The high
incidence of parvovirus B19 seroconversion in all age groups makes it very difficult to
assess the infectivity of the product with respect to parvovirus 19. No clinical trials testing
parvovirus 19 seroconversion are mandatory before authorisation. After authorisation it is
recommended that parvovirus 19 seronegative patients should be tested by gene
amplification methods one week after the first treatment. Serum should be stored at -70C for
future testing.
Immunogenicity
The currently available factor VIII preparations differ with respect to purity and method of
viral removal/inactivation. Until recently, the evidence supporting the notion that a
particular production process is associated with a higher than normal risk of inhibitor
induction has been very limited. Nevertheless some products do cause a higher incidence
inhibitors than others. The clusters of inhibitors after infusion of factor VIII CPS-P in
previously treated patients in 1991, illustrate the necessity to perform immunogenicity trials.
Such inhibitors will be demonstrable in previously treated patients.
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3.3 Clinical trials w i t h factor VIII p r o d u c t s

3.3.1.1 Efficacy
A comparative pharmacokinetic trial, measuring half-life and recovery, should be
performed in at least 12 subjects with haemophilia A (factor VIII < 2%). Patients should be at
least 12 years of age without inhibitor and not actively bleeding and should not have
received an infusion of concentrate in at least the past 3 days (if possible 7 days). As
comparative product the currently authorised factor VIII product should be used. Samples for
factor VIII activity determination should be taken before injection of 25-50 IU/kg of the new
factor VIII product and between 15 and 30 minutes, 1, 3, 6, 9, 12, 24, 30-36 hours after the
infusion. A minimum of 3 days (if possible 7 days) should be maintained between infusions.
At least 3 different lots should be employed in the trial. Recovery should be determined from
the peak factor VTII activity in the first four samples time periods post-infusion.
product for 6 months and at least 5 patients should be tested again for half-life and recovery
after 3-6 months.
After administration of the factor VIII product in any patients treated with surgical
procedures, response will be determined by the physician, including achievement of
haemostasis, loss of blood and requirement of transfusions.
3.3.12 Safety
3.3.1.2.1 PTP Study
with severe haemophilia A with at least 10 exposure days to the new factor VIII product and a
follow up of at least 6 months for all patients must be included prospectively. The factor VIII
inhibitor titre will be determined every 3 months. The titre of the inhibitor should be reported
(BU) as well as the clinical relevance, the cumulative incidence and the number of exposure
days (also in relation to development of inhibitors). The follow up of these patients should be
in accordance with EC Good Clinical Practice. The SPC should include a section stating the
experience with respect to immunogenicity in PUPs or state that there is no such experience.
3.3.1.2.2 PUP Study
PUP studies should be carried out, or at least initiated. At the date of implementation of the
modified manufacturing process, the SPC should state the experience with respect to
immunogenicity in PUPs or state that there is no such experience. The PUP study should be
done only after results of the PTP trials are available and according to the guideline (see
2.3.1.2.1) including at least 20 patients.
3.32.1 PTP
3.322 PUP
Immunogenicity and viral safety data (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update.
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3.4 Clinical Trials w i t h F a c t o r LX P r o d u c t s

3.4.1.1 Efficacy
A comparative pharmacokinetic trial, measuring half-life and recovery, should be
performed in at least 10 subjects with severe haemophilia (factor DC < 2%) patients should
be at least 12 years of age and should not have received an infusion of concentrate in at least
the past 4 days (if possible 7 days). As comparative product the currently authorised factor DC
product should be used. Samples for factor DC activity determination should be taken before
injection of 50-75 IU/kg of the new factor DC product and 30 minutes, 1, 3, 6, 9, 12, 24, 30, 36
and 50 hours after the infusion. A minimum period of 4 days (if possible 7 days) should be
maintained between infusions. At least 3 different lots should be employed in the trial.
Recovery should be determined from the peak factor DC activity in the first four samples time
periods post-infusion.
product for 6 months and at least 5 patients should be tested again for half life and recovery
after 3-6 months.
After administration of the factor DC product in any patient treated with surgical procedures,
adverse events and response will be determined by the physician, including achievement of
haemostasis, loss of blood and requirement for transfusions and occurrence of
thromboembolic episodes.
3.4.12 Safety
3.4.1.2.1 PTP Study
with severe haemophilia with at least 10 exposure days to the new factor DC product and a
follow up of at least 6 months for all patients must be included prospectively. The factor DC
inhibitor titre will be determined every 3 months. The titre of the inhibitor should be reported
(BU) as well as the clinical relevance, the cumulative incidence and the number of exposure
days (also in relation to the development of inhibitors). The follow up of these patients should
be in accordance with EC Good Clinical Practice. In addition, appropriate tests for activation
of coagulation after administration of the product should be carried out in the patients
included in the pharmacokinetic trial (see 2.4.2.). If they undergo surgery, clinical
evaluation of thrombosis should be undertaken by safe, objective means.
3.4.1.2.2 PUP study
PUP studies should be carried out or at least initiated, however due to the lower incidence of
haemophilia as compared to haemophilia A, the study should include at least 15 patients.
At the date of the implementation of the modified manufacturing process, the SPC should
include a section stating the experience with respect to immunogenicity in PUPs or state that
there is no such experience. The PUP study should be done only after the results of the PTP
trial are available.
3.4.2.1 PTP
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3.4.22 PUP
Immunogenicity and viral safety date (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update report.
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APPENDLX 1
BIOCHEMICAL AND SEROLOGIC SURVEBLLANCE EV
HAEMOPHBLIAC PATB3NTS COMMENCING TREATMENT WITH THE
FACTOR VIH AND FACTOR LX CONCENTRATE UNDER STUDY
Month after commencing new concentrate 0 3 6 12 18 24 36 48

Recovery of Factor VIII or Factor IX *
ALT * * * * * * * *
HIV I + II * * * * * * * *
HCV * * * * * * * *
HBV * * * * * * * *
HAV * * * * * * * *
Parvo Virus B 19 * * * * * * * *
In patients with a previously detected coagulation inhibitory antibody, surveillance will be

carried out at monthly intervals for the first three months following by 3 monthly intervals
for one year. In the second year controls will be sampled every 6 months, after which 12
month intervals will be employed.
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NORMAL INTRAVENOUS IMMUNOGLOBULIN
PRODUCTS FOR MARKETING AUTHORISATIONS
Guideline Title Assessing the Efficacy and Safety of Normal Intravenous

Immunoglobulin Products for Marketing Authorisations
Date of entry i n t o August 1996
force
Previous titles/other Guidelines to Assess Efficacy and Safety of Normal
references Intravenous Immunoglobulin Products for Marketing
Authorisations/
HI/5819/94, CPMP/BPWP/3 88/95
Additional Notes This note for guidance describes the information to b e
documented for polyvalent IV immunoglobulin
preparations (IVIg), including biological data, c l i n i c a l
trials and patient follow-up. These data are necessary for
both new applications and variations to marketing
authorisations.
CONTENTS
1. INTRODUCTION
2. rVlg PRODUCTS FOR WHICH AN APPLICATION FOR A MARKETING

AUTHORISATION IS TO BE SUBMITTED
3. CLINICAL TRIALS WITH AUTHORISED PRODUCTS WHERE A

SIGNIFICANT CHANGE IN THE MANUFACTURING PROCESS HAS BEEN
MADE (E.G. ADDITIONAL VTRAL REMOVAL/INACTrVATION STEP)
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NORMAL INTRAVENOUS IMMUNOGLOBULIN
PRODUCTS FOR MARKETING AUTHORISATIONS
L INTRODUCTION
These Guidelines describe the information to be documented for polyvalent IV
immunoglobulin preparations (IVIg), including biological data, clinical trials and patient
follow-up. These data are necessary for:
i) products for which an application for a marketing authorisation is to be submitted,
referred to as "New products" in the text
ii) variations to authorised products where a significant change in the manufacturing
process has been made (e.g. additional viral removal/inactivation step), referred to as
"modified product" in the text.
The clinical trials described in these Guidelines should be performed according to the note
for guidance on Good Clinical Practice.
11 Efficacy
Currently, a number of indications are considered as "well established". These Guidelines
outline the general principles for design of clinical trials in the following claimed
indications:
i) Replacement therapy in:
Primary immunodeficiency syndromes:
- congenital agammaglobulinemia and hypogammaglobulinemia
- common variable immunodeficiency
- severe combined immunodeficiencies
- Wiskott Aldrich syndrome.
Myeloma and chronic lymphatic leukaemia with severe secondary
hypogammaglobulinemia and recurrent infections.
Congenital AIDS with recurrent infections.
ii) Immunomodulatory effect in:
Idiopathic Thrombocytopenic Purpura (ITP) in children or adults, at high risk of
bleeding or prior to surgery to correct the platelet count.
iii) Kawasaki disease
iv) Bone marrow transplantation
Biological data and clinical evidence of efficacy and safety in primary/secondary
humoral immunodeficiencies and ITP are the key elements required.
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3AB17a
) Other indications
Where the mechanism of action is essentially unknown, relevant clinical data are
required. The trials should be carried out with reference to the Notice to Applicants and
all relevant EC Guidelines for clinical studies of medicinal products.
1.2 Safety
1.2.1 Immediate adverse events
All adverse events in clinical studies must be recorded and reported.
Safety data from trials in indications not claimed in the application can be used as
supportive data.
12.2 Viral safety

It is mandatory for manufacturers of blood products to improve viral safety by rigorous
selection of the donors and screening of the donations, including testing for antibodies to
HIV type I and II, to hepatitis C (HCV), as well as HBsAg, and by using in the
manufacturing process viral removal/inactivation steps, validated according to the EC
Guidelines.
Despite selection criteria for donors and testing of the product at appropriate stages in the
manufacturing process (described in Part II-V of the dossier) it is still necessary to follow-up
patients treated with the product in clinical trials. For new products, viral safety data must
be provided as part of the marketing application dossier. These data should be derived from
all patients having entered clinical trials and any other patients having received the
product. The company should continue to follow-up patients treated with the product in the
long term as a post-marketing surveillance for viral markers.
In view of the difficulty in establishing seroconversion in immunodeficient patients,
regular testing for liver function should be carried out; search for viral antigens and use of
nucleic acid amplification methods could also be considered.
1.2.3 Other safety issues

The effect of passive transmission of isoglutinins (anti-A/anti-B), and anti-D should be
evaluated in patients receiving high doses of P/Ig.
2. rvlg PRODUCTS FOR WHICH AN APPLICATION FOR A

MARKETING AUTHORISATION IS TO BE SUBMITTED
Biological and pharmacokinetic data are the key elements to evaluate activity and safety of
IVIg preparations with reference to physiological immunoglobulins.
2.1 Biological and pharmacokinetic data

2.1.1 Biological (cross-reference to relevant Part IT)
Adequate documentation should be provided regarding batch to batch consistency.
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Most of the data are provided in Part II of the dossier and follow the European
Pharmacopoeia monograph 918, Xth of January l8t, 95. However, specific data are needed to
support the pharmacodynamic and therapeutic activities as well as the safety profile of the
P7Ig preparation. These data should thus appear along with the cross-reference to Part II, i n
Part TV of the dossier.
For the values not defined in the European Pharmacopoeia 918, ranges and/or limits are to
be defined.
i) Biological characteristics
- General:
Molecular size distribution: quantification of monomers + dimers, fragments
and polymers + aggregates
Impurities (proteins - IgE, IgM - other)
- Of interest for pharmacodynamic and therapeutic activity
Distribution of IgG subclasses
Antibody content:
Bacteria:
Diphtheria
Haemophilus
Pneumococcus
Streptococcus
Virus:
Hepatitis A range or limit to be defined
Hbs range or limit to be defined
CMV range or limit to be defined
Herpes-zoster range or limit to be defined
Measles
Parvovirus 19
Poliovirus type I
- Of interest for safety profile
IgA content range or limit to be defined
Anti-complementary activity
Anti- and anti-B isoglutinins range or limit to be defined
Anti-D antibodies absence thereof
Prekallikrein activator,
ii) Biological activity
- In vivo and/or in vitro quantification of neutralising antibodies (depending on
the claimed neutralising activities)
- Fab and Fc functions (functional integrity): ability to fix complement,
opsonisation, phagocytosis, antibody-dependent complement cytotoxicity (ADCC).
Immunomodulatory and anti-inflammatory activities for auto-immune diseases,
depending on the claimed indications and the relevance of in vitro and/or in
vivo models such as:
ability to inhibit auto-antibody activity in vitro,
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experimental auto-immune models.
2.1.2 Pharmacokinetics
Pharmacokinetic data are essential to support the pharmacological activity and efficacy of a
particular PTIg preparation, and may differentiate one from another. Therefore, they must be
provided in each application dossier. Pharmacokinetic data should be derived from patients
with hypo- or agammaglobulinemia.
Half-life should be studied in 15 patients with primary immunodeficiency due to primary
immunodeficiency syndromes and possibly in myeloma or chronic lymphatic leukaemia
with severe secondary hypogammaglobulinemia and recurrent infections. Patients, of whom
at least 10 should have primary immunodeficiency, may already be stabilised. The
pharmacokinetics should be assessed over a period of 6 months (6.5 times the expected half-
life). No cross-over study is necessary.
The IgG concentration should be determined before injection of the recommended dose of the
DTIg. Pharmacokinetic profile should be assessed by repeated sampling during the first
infusion, and followed by trough levels (measured before the next injection). In patients
nave to PTIg, the Time to reach Steady State (Tss) could be determined.
2.2 Efficacy
22.1 Replacement therapy in primary immunodeficiency syndromes
Clinical data should include an open study comparing historical data with reference IVIg in
at least 15 patients, whatever the primary immunodeficiency syndrome. Evaluation criteria
would be: number of days out of school/work, number of days of hospitalisation. Trough
levels of IgG and T s s when possible, should be documented over 6 months. Trough levels
should be no less than 4 to 6g/L.
The results regarding efficacy would apply to all types of primary immunodeficiency
syndrome due to deficiency of functional IgG.
2.2.2 Replacement therapy in myeloma or chronic lymphatic leukaemia with

severe secondary hypogammaglobulinemia and recurrent infections
Indication in myeloma or chronic lymphatic leukaemia with severe secondary
hypogammaglobulinemia and recurrent infections would be granted, as long as efficacy has
been proven in primary immunodeficiency syndromes.
2.2.3 Replacement therapy in congenital AIDS with recurrent infections

Indication in congenital AIDS with recurrent infections would be granted, as long as
efficacy has been proven in primary immunodeficiency syndromes.
22.4 ITP
TVLg is used for the treatment of ITP in children or adults, at high risk of bleeding, or prior
to surgery to correct the platelet count.
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There are no data to support the equivalence of different P7Ig preparations, especially with
regard to immunomodulatory activities. Thus a clinical efficacy study is required to
establish efficacy of the preparation in this indication.
- Clinical efficacy data should include an open study comparing historical data with
reference rVIg, performed over a few days in acute phase on at least 15 adult chronic
ITP patients, with a platelets count below 20 109/L.
- Information required would be:
response of platelet count > 50 109/L.
regression of haemorrhages
duration of platelet response.
- Standard doses should be studied (lg/kg b.w./day for 2 days, or 0.4g/kg b.wVday for 5
days) Other dosage regimens should be documented.
22.5 Kawasaki disease

It is no longer ethical to conduct placebo controlled trial in this indication and it can be
granted by reference to the literature, providing that efficacy in primary immunodeficiency
syndromes and in ITP has been established for the relevant PTIg.
2.2.6 A l l o g e n i c B o n e M a r r o w T r a n s p l a n t a t i o n
rVIg effect in BMT requires both substitution and immunomodulatory activities. In
reference to this indication, specific data are not required as long as efficacy has been
proven in primary immunodeficiency syndromes and in ITP for the relevant rVIg.
22.7 Other indications

Other possible indications can not be granted without relevant clinical data. Biological and
pharmacokinetic data alone are not sufficient to support clinical efficacy.
Controlled clinical trials comparing the DTIg preparation with placebo or with an established
therapy are thus required to substantiate marketing authorisation in other indications. These
trials should be carried out with reference to the Notice to Applicants and all relevant EC
Guidelines for clinical studies of medicinal products.
2.3. Safety
2.3.1 Immediate safety events
All adverse events in clinical studies should be recorded in all patients treated, whatever the
indication, and reported in reference with the note for guidance on Structure and Content of
Clinical Study Reports. Data from at least 30 patients or 180 infusions are required.
Safety evaluation should include monitoring of short term tolerance (blood pressure, heart
rate, temperature, respiratory rate, and monitoring of other adverse events) at 30 minutes
intervals for 4 hours and at 12 and 24 hours following the injection of the new product i
patients, such as patients included in the pharmacokinetic studies or patients included i n
clinical studies for efficacy. Renal function should be monitored.
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2.32 Viral safety

For new products, some viral safety data should be provided as part of the marketing
application. These data should be derived from all patients having entered efficacy clinical
trials and for a minimum of 3 batches of the P7Ig preparation. Each patient should preferably
be treated with one batch only. The company should continue to follow-up patients treated with
the product in the long term as a post-marketing surveillance for viral markers. Updated
data should be provided on a yearly basis after the marketing authorisation has been
granted.
Blood sampling for the viral safety study should include: one pre-infusion sample for every
parameter, and appropriately thereafter for up to 6 months following exposure to any new
batch of the preparation. Sera from these points should be stored at -70C as well as being
tested as set out below for each patient group.
The monitoring of viral safety depends on the category of patients receiving the new product
(immunocompetent or non-immunocompetent patients). Non-immunocompetent patients who
are primary recipients of rVIg may not have formed detectable antibodies against human
viruses which may be transmitted through rVIg. All safety data obtained in various
populations receiving the new product should be presented together but identifying
immunocompetent and non-immunocompetent patients. This will provide a basis for an
overall safety assessment for all clinical studies.
Passive transmission of HBs, HBc, HAV and parvovirus B19 antibodies by the IVIg
preparation may be difficult to differentiate from transmission of infection, and this should
be discussed.
i) Immunocompetent patients
- Transaminases: the baseline should be properly established before the first
infusion and all samples taken before each infusion for the 6 months after the
first infusion.
- Seroconversion for:
HAV Ab (IgM); HIV 1-2 Ab: at weeks 12 and 24
HBsAg; HBc Ab and HCV Ab: at weeks 16 and 24
Parvovirus 19: a sample before and at week 1 after the first infusion must
be taken.
If the pre-treatment sample has no anti-B19 Ab (IgM or IgG), the week 1-sample
should be tested with gene amplification method.
In patients parvovirus 19 seropositive in the pre-treatment sample, no further
investigation is required.
ii) Non-immunocompetent patients
- Transaminases: the baseline should be properly established before the first
infusion and all samples taken before each infusion for the 6 months after the
first infusion.
- Antigen detection for
HfV and HBs: at week 8 after the first infusion.
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HCV: nucleic acid amplification method before the first infusion and at
weeks 8 and 16 after the first infusion.
Parvovirus B19: before the first infusion and at week 1 after the first
infusion using either nucleic acid amplification method or Ag detection.
3. CLINICAL TRIALS WITH AUTHORISED PRODUCTS

WHERE A SIGNIFICANT CHANGE IN THE
MANUFACTURING PROCESS HAS BEEN M ADE (E.G.
ADDITIONAL VIRAL REM OVAL/INACTIVATION STEP)
Appropriate viral removal/inactivation steps in the manufacturing process for PTIg are
mandatory, to increase the viral safety of these products.
Little data exist on the effect of some viral inactivation steps or other purification steps on
IgG integrity and function, or on PTIg immunomodulatory activity. Thus, it is important to
include full data on antibody integrity and function, in Part II and crossrefer to this in Part
TV of the dossier as for new products. Data on pharmacokinetics and on immediate safety
should also be provided with the application.
Pharmacokinetic data must be provided in each application dossier, from patients with
primary immunodeficiency syndromes
Halflife should be studied in 15 patients with primary immunodeficiency due to primary
immunodeficiency syndromes and possibly in myeloma or chronic lymphatic leukaemia
with severe secondary hypogammaglobulinemia and recurrent infections. Patients, of whom
at least 10 should have primary immunodeficiency, may already be stabilised. The
pharmacokinetics should be assessed over a period of 6 months (6.5 expected halflife). No
crossover study is necessary.
The IgG concentration should be determined before injection of the recommended dose of the
DTIg. Pharmacokinetic profile should be assessed by repeated sampling during the first
infusion, and followed by trough levels (measured before the next injection). In patients
nave to P7Ig, the Time to reach Steady State (T ss ) could be determined.
3.2 I m m e d i a t e s a f e t y
Immediate safety for modified products should be the same as required for a new product,
that is: all adverse events in clinical studies should be recorded in all patients treated,
whatever the indication, and reported in reference with the note for guidance on Structure
and Conte nt of Clinical Study Re ports. Data from at least 30 patients or 180 infusions are
required.
Safety evaluation should include monitoring of short term tolerance (blood pressure, heart
rate, temperature, respiratory rate, and other adverse events) at 30 minutes intervals for 4
hours and at 12 and 24 hours following the injection of the modified product in patients, such
as patients included in the pharmacokinetic studies or patients included in clinical studies
for efficacy. Renal function should be monitored.
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3.3 Efficacy
3.3.1 Products for which biological, pharmacokinetic and immediate safety data
are demonstrative of identity to the parent product
Where biological data, pharmacokinetics and immediate safety profile show identity to the
parent product, further efficacy data will be required with the application in order to verify
efficacy of the modified preparation.
Requirements on efficacy data will be as follow:
3.3.1.1 Replacement therapy in primary immunodeficiency due to Primary
immunodeficiency syndromes
No further clinical trial would be required, as long as biological data, pharmacokinetics
and immediate safety data have been provided and show identity to the parent product.
3.3.1.2 Replacement therapy in myeloma or chronic lymphatic leukaemia with severe
secondary hypogammaglobulinemia and recurrent infections.
3.3.1.3 Replacement therapy in congenital AIDS with recurrent infections
3.3.1.4 ITP
Clinical efficacy data should include an open study comparing historical data with
reference PVIg, performed over a few days in acute phase on at least 15 adult chronic ITP
patients, with a platelets count below 20 109/L.
Information required would be:
- response of platelet count > 50 109/L.
- regression of haemorrhages
- duration of platelet response.
3.3.1.5 Kawasaki disease
This indication can be granted by reference to the literature, providing that biological data,
pharmacokinetics and immediate safety data have been provided and show identity to the
parent product, and that efficacy has been established in ITP for the modified product.
3.3.1.6 Bone Marrow Transplantation
No further clinical trial would be required, providing biological data, pharmacokinetics and
immediate safety data have been provided and show identity to the parent product, and that
efficacy has been established in ITP for the modified product.
3.3.1.7 Other indications
For modified preparations only the four established indications, foreseen in the core SPC,
would be granted, unless relevant clinical data was submitted, either with the modified or
with the parent preparation.
Providing that the modified product satisfies the above requirements, no further clinical
trial would be required for the other indications.
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3.3.2 Products for which biological data, pharmacokinetics or immediate safety

profile are different from the parent product
If the biological data, pharmacokinetics or immediate safety data are different from the
parent preparation, the product is then considered as a new product and, as such, should
comply with the requirements defined in section 2.2.
Any new indication would have to be supported by full efficacy and safety data, as for a new
product.
403
INDEX
consistency, 50; 53; 74; 177; 188; 189; 208; 211; 214;
216; 219; 225; 226; 229; 231; 244; 247; 248; 251;
252; 261; 280; 283; 284; 291; 292; 294; 342; 378;
379; 396
abnormal toxicity, 208; 226; 241 control of starting mate rials, 34; 179
absorbed dose, 26; 27; 28; 29; 183; 193 cross-contamination, 188; 209; 211; 229; 242; 243;
accelerated te sting, 131; 134; 137; 166 282; 318; 340
accuracy, 36; 55; 109; 111; 112; 115; 124; 125; 173; cytokine, 223; 225; 226; 227; 230; 231; 232; 233; 234;
291; 292; 302; 307; 328; 329; 342 235; 246; 265; 281; 293
active moiety, 33; 121
additive, 6; 7; 8; 71; 73; 79; 80; 81; 82; 233; 247; 273;
341 D
adventitious age nts, 73; 188; 208; 210; 228; 239; 245;
247; 256; 280; 297; 298 delayed re le ase , 170
allergen, 13; 266; 358; 373; 375; 376; 377; 378; 379; Development che mistry, 52
380 Development Pharmaceutics, 3; 14; 15; 16; 69; 87; 90;
analytical validation, 52; 86; 91; 325 178
dissolution te st, 7; 111; 121; 170; 171; 172; 173
Dosage form, 80
Dose Mapping, 29
Dosimeter, 29
batch, 5; 7; 14; 15; 25; 26; 27; 29; 34; 35; 37; 38; 42; Drug Master File, 41; 44; 47; 49; 50; 51; 53; 54
50; 53; 59; 60; 61; 62; 69; 71; 81; 86; 87; 88; 89; 90 DMF, 41; 44; 47; 49; 50; 51; 53; 54
92; 93; 94; 98; 99; 100; 102; 130; 132; 133; 134;
135; 136; 138; 139; 140; 141; 159; 162; 163; 166,
170; 171; 172; 173; 179; 183; 184; 188; 189; 193 E
194; 210; 213; 214; 215; 216; 229; 231; 232; 234
235; 247; 248; 250; 251; 252; 262; 266; 267; 269 electrophoresis, 213; 230; 250; 269; 378
283; 284; 285; 293; 342; 353; 355; 358; 359; 360 end-of-shelf life , 16; 17; 18
369; 376; 378; 379; 396; 400 endotoxins, 208; 226
batch analysis, 215; 252 enhancers, 220
batch to batch consiste ncy, 378 evidence of structure, 35; 52
bioburden, 16; 17; 26; 27; 28 excipient, 5; 6; 13; 14; 67; 69; 70; 71; 72; 73; 85; 90; 97;
bracketing, 135; 137; 267 98; 100; 101; 102; 110; 137; 171; 173; 174; 201;
BSE, 291; 317; 318; 319; 322 234; 269; 273; 297; 318; 327; 328; 335; 345; 375;
bulk harvest, 216; 248; 250; 251; 257; 262 376
bulk product, 25; 30; 87; 88; 93; 94; 214; 231; 250; Excipients, 5; 67; 69; 70; 73; 90
284; 378 Expert Report, 35; 37; 53
expiry date, 183; 193; 245; 338
expression construct, 209; 210; 217; 219; 220; 221;
222; 277; 279
chemical de ve lopme nt studie s, 62

Clinical, 169; 181; 182; 190; 191; 192; 225; 253; 299;
321; 336; 344; 369; 372; 381; 383; 385; 386; 387;
389; 390; 395; 398; 399; 401; 402 factorisation, 14; 90
colouring matte rs, 74 Finished product, 173; 215; 216; 253
composition, 6; 8; 14; 25; 26; 69; 71; 73; 74; 77; 79; 80; flavouring age nts, 69
81; 82; 90; 101; 116; 122; 137; 140; 162; 171; 174;
177; 83; 189; 193; 198; 200; 214; 215; 220; 230;
231; 232; 234; 246; 247; 250; 252; 284; 293:301;
302; 321; 340; 341; 357:361; 369; 375; 376; 377;
378 gene therapy, 275; 277; 278; 279; 285; 291
405
Index
genetic founder, 289; 291 murine, 187; 188; 228; 237; 239; 240; 241; 242; 245;
genetic instability, 208 248; 255; 298; 299; 300; 346
genetically-modified, 280; 283
glycoproteins, 213; 214; 225
N
H new active substance, 6; 31; 33; 35; 41; 57; 59; 71; 72;
88; 97; 99; 127; 129; 143; 155; 159
harvest, 199; 210; 211; 214; 216; 222; 227; 229; 234; nomenclature, 33; 378
246; 247; 248; 250; 251; 257:261; 262; 273; 283; Notice to Applicants, 50; 51; 69; 77; 92; 169; 290; 396;
293; 365; 377 399
herbal remedies, 197 nucleic acid analysis, 219; 221
nucleic acid techniques, 219
O
Immediate packaging, 75; 78; 137; 166
impurities, 31; 34; 36; 37; 42; 43; 49; 50; 51; 52; 55; overage, 5; 13; 86; 90
57; 59; 6; 61; 62; 63; 64; 65; 70; 73; 85; 86; 97; 98;
99; 100; 101; 109; 110; 112; 113; 121; 123; 124;
125; 179; 189; 190; 208; 212; 214; 226; 230; 231;
249; 251; 252; 280; 284; 293; 327; 340
impurity profile, 43; 55; 60; 62; 63; 64; 65; 98; 99; 101; packaging material, 18; 25; 29; 30; 75; 77; 78; 79; 80
102;110 81; 135; 184; 194
in vitro, 7; 66; 105; 169; 170; 171; 172; 173; 174; 190; parametric release, 16; 179
220; 221; 233; 239; 246; 253; 258; 268; 279; 280; periodic tests, 88; 94
284; 291; 293; 297; 325; 397 pharmaceutical development studies, 6
in vivo, 7; 170; 171; 172; 173; 174; 180; 182; 187; 190; physical characteristics, 33; 37; 162; 163; 213; 232
192; 213; 237; 239; 240; 247; 248; 253; 268; 277; physico-chemical properties, 35; 72; 212; 230; 246;
285; 297; 299; 325; 373; 375; 397 250; 300; 304; 344
inducer, 227 pilot plant scale, 130
in-process controls, 14; 15; 16; 17; 18; 88 pollen, 376
integration site, 221; 280; 292 polymorphism, 292
interactions, 162; 164; 182; 192; 201; 271; 340 post translational modifications, 294
Intermediate product, 172 post-translational modifications, 213; 219; 230
isoelectric focusing, 213; 250 potency, 37; 93; 123; 124; 134; 189; 190; 208; 214;
isomer, 34; 35; 36; 52 215; 216; 226; 231; 232; 233; 241; 251; 252; 262;
265; 268; 271; 272; 284; 285; 307; 327; 357; 376;
378; 379
potential toxicology, 52
precision, 36; 55; 90; 109; 111; 112; 113; 114; 115;
lactation, 292 123; 124; 125; 138; 173; 307; 328; 329
letter of access, 53; 54 preservative
ligand, 180; 212; 338; 340; 343 preservatives, 6; 7; 80; 85; 87; 91; 133; 134; 202;
linearity, 109; 111; 112; 125; 173; 329 233; 269; 340; 343; 376
process validation, 9; 16; 17; 18; 49; 69; 77
production, 14; 15; 16; 18; 25; 29; 30; 37; 86; 87; 88,
M 89; 90; 92; 93; 130; 132; 133; 135; 139; 172; 173
177; 178; 181; 187; 188; 189; 202; 207; 208; 209
manufacturing formula, 13; 14 210; 211; 212; 213; 214; 216; 217; 219; 220; 221
markers, 200; 210; 221; 222; 228; 243; 244; 245; 298; 222; 225; 226; 227; 228; 229; 230; 231; 234; 235.
336; 338; 342; 353; 385; 386; 396; 400 239; 240; 241; 242; 243; 244; 245; 246; 247; 248,
master cell bank, 188; 216; 228; 261 249; 250; 251; 252; 253; 254; 255; 256; 257; 261
matrixing, 135 262; 266; 273; 278; 279; 280; 281; 282; 283; 284,
Mean Kinetic Temperature, 138 285; 289; 290; 291; 292; 293; 294; 297; 298; 299
modified release, 7; 9; 88; 155; 170 300; 301; 302; 303; 304; 305; 309; 318; 321; 336,
mould, 29; 282; 283; 308; 377 339; 340; 341; 342; 343; 357; 359; 377; 378; 384,
388
406
Index
prolonged release, 167; 169; 170 specificity, 109; 110; 112; 123; 124; 138; 173; 178;
proof of structure, 33; 35; 41; 43 192; 239; 240; 243; 246; 252; 253; 260; 279; 327;
purification, 34; 60; 71; 189; 190; 202; 208; 210; 212; 353; 378
213; 214; 216; 222; 226; 228; 230; 231; 234; 239; spongiform encephalopathy, 290; 301; 317
240; 249; 250; 251; 252; 253; 266; 273; 280; 283; stability indicating, 130
284; 293; 294; 298; 299; 320; 335; 338; 339; 342; starting material, 8; 25; 29; 34; 36; 49; 51; 65; 69; 71;
344; 365; 378; 383; 388; 401 72; 73; 179; 197; 199; 202; 289; 290; 293; 298; 299;
purity, 35; 36; 37; 38; 43; 55; 59; 60; 61; 62; 63; 64; 65; 300; 302; 307; 308; 319; 328; 336; 339; 376
66; 71; 74; 85; 98; 99; 100; 101; 102; 105; 109; 110; stereochemistry, 33
111; 112; 121; 163; 164; 178; 179; 184; 187; 189; sterility, 9; 16; 17; 70; 91; 178; 179; 190; 208; 226; 308
190; 194; 199; 213; 214; 215; 231; 232; 250; 251; storage conditions, 60; 70; 86; 98; 99; 129; 130; 131;
252; 265; 268; 269; 271; 272; 273; 283; 284; 290; 134; 137; 139; 141; 183; 193; 199; 221; 248; 267;
291; 293; 294; 305; 326; 327; 359; 376; 383; 388 268; 270; 326; 377; 379
pyrogenicity, 179; 208; 226 stress testing, 135; 140; 159; 163
Summary of product characteristics, 181; 345; 385
synthesis, 34; 35; 36; 37; 60; 65; 71; 74; 97; 98; 122;
Q 207; 220; 226; 239; 242; 243; 278; 302
qualification, 13; 59; 63; 64; 97; 99; 100; 215

quality assurance, 13; 14; 336; 337
quality control, 13; 34; 37; 49; 51; 73; 178; 187; 189;
190; 205; 207; 208; 209; 210; 226; 228; 229; 237; trace elements, 36
240; 241; 245; 252; 254; 278; 279; 280; 290; 302; transgenic animal, 287; 289; 290; 291; 293
318; 325; 328; 336; 337 transgenic animals, 289; 291
R
reagent, 34; 36; 124; 189; 190; 207; 220; 226; 241; vaccines, 13; 208; 266; 279; 297; 355; 357; 358; 359;
279; 326; 327; 328; 329; 338; 379 360; 361; 365; 369; 375
reference material, 79; 109; 110; 112; 190; 215; 232; validated limit of quantitation, 61; 65
235; 252; 268; 326; 359 validation, 9; 16; 17; 18; 27; 42; 49; 51; 52; 61; 69; 77;
reference standards, 215 85; 86; 87; 88; 91; 109; 112; 121; 122; 130; 133;
reprocessing, 211; 249 162; 173; 178; 179; 188; 189; 199; 211; 212; 220;
re-test, 129; 130; 131; 132; 138; 139; 141; 215; 252 246; 249; 252; 254; 270; 290; 293; 295; 296; 297;
routine tests, 52; 53; 69; 70; 78; 86; 87; 325 298; 299; 300; 301; 302; 303; 304; 305; 309; 311;
313; 318; 325; 326; 327; 333; 336; 341; 343; 344;
345; 346; 347; 384
variations, 15; 63; 113; 116; 125; 129; 171; 172; 303;
329; 379; 393; 395
scrapie, 290; 301; 317; 318; 319 vector, 207; 208; 209; 210; 211; 214; 216; 219; 220;
solvent, 14; 18; 34; 36; 61; 65; 71; 79; 80; 81; 85; 88; 222; 226; 227; 228; 229; 231; 244; 251; 261; 275;
124; 198; 200; 202; 322; 327; 338; 341; 344; 383 277; 278; 279; 280; 281; 282; 283; 284; 285; 291;
sorption, 7; 8; 29; 78; 79; 81; 169; 181; 191; 322; 326; 297
379 vegetable substance, 41; 42; 197; 198; 199; 200; 202
specifications, 14; 15; 16; 17; 18; 50; 54; 55; 59; 61; virus removal, 290; 299; 302; 304; 311; 341; 344
62; 63; 65; 69; 70; 71; 73; 77; 78; 80; 85; 86; 87; 88;
89; 90; 91; 92; 93; 99; 102; 111; 134; 164; 165; 173;
177; 184; 187; 189; 190; 207; 226; 250; 267; 268;
272; 273; 279; 293; 337; 339; 340; 342; 358; 375;
w
376; 379 working cell bank, 188; 210; 222; 228; 245; 261; 282;
283
407
European Commission
The rules governing medicinal products in the European Union

Guidelines - Medicinal products for human use
Quality and biotechnology
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EN

The rules governing medicinal products

Cataloguing data can be found at the end of this publication.

Luxembourg: Office for Official Publications of the European Communities, 1998

European Communities, 1998

Quality and biotechnology

Volume 1 Pharmaceutical legislation

Volume 2 Notice to applicants

Volume 4 Good manufacturing practices

Volume 5 Pharmaceutical legislation

Volume 6 Notice to applicants

Volume 8 Maximum residue limits

Volume 3B - Safety and the environment

Volume 3C - Efficacy and information on the medicinal product

BIOTECHNOLOGY GUIDELINES 203

DEVELOPMENT PHARMACEUTICS AND PROCESS

Guideline Title Development P h a r m a c e u t i c s a n d Process Validation

DEVELOPMENT PHARMACEUTICS AND PROCESS

2.1.2 Physical characteristics

3.1.3 Compatibility with other products

3.2 Semi-solid dosage forms

medicinal products containing active substances with a narrow therapeutic window. A

4.1 Sorption to container

4.3 Dose reproducibility

II. PROCESS VALIDATION

MANUFACTURE OF THE FINISHED DOSAGE FORM

Guideline Title Manufacture of t h e Finished Dosage F o r m

Date of entry i n t o May 1996

2. THE APPLICATION FOR MARKETING AUTHORISATION AND GMP

4. DESCRIPTION OF THE MANUFACTURING PROCESS

5. DESCRIPTION OF THE MANUFACTURING CHAIN

6. VALIDATION OF THE MANUFACTURING PROCESS

MANUFACTURE OF THE FINISHED DOSAGE FORM

For this purpose it shall include at least:

2. THE APPLICATION FOR MARKETING AUTHORISATION

4. DESCRIPTION OF THE MANUFACTURING PROCESS

5. DESCRIPTION OF THE MANUFACTURING CHAIN

6. VALIDATION DATA OF THE MANUFACTURING PROCESS

Examples are: new dosage forms, the manufacturing of liposomes, etc.

7.2 Re-processing of residual product

7.3 Removal of solvents or gases

7.4 Cleaning of primary packaging material

7.5 Sterilisation of primary packaging material

7.6 Production areas

LIMITATIONS TO THE USE OF ETHYLENE OXIDE IN

Guideline Title Limitations to the use of Ethylene Oxide in the

2 CATEGORIES OF USE OF ETHYLENE OXIDE

LIMITATIONS TO THE USE OF ETHYLENE OXIDE IN

2. CATEGORIES OF USE OF ETHYLENE OXIDE

3.1 Raw materials

3.2 Finished product

THE USE OF IONISING RADIATION IN THE

Guideline Title The use of Ionising R a d i a t i o n in t h e Manufacture of

4. VALIDATION OF THE IRRADIATION PROCEDURE

THE USE OF IONISING RADIATION IN THE

3.1 Description of the irradiation plant

3.2 Description of the irradiation process

c) the material and dimensions of the irradiation container should be described;

h) a written standard operating procedure should be established including the following

- the loading pattern of product(s) within the irradiation container;

- any adjustments to be applied to the routine dosimeter measurements to convert

4. VALIDATION OF THE IRRADIATION PROCEDURE

4.2 Validation with regard to the purpose of irradiation (see section