Vous êtes sur la page 1sur 15

9/28/2014

Definitions related to the topic


Parenteral Products:-Definition,
Types and Importance of parenteral products,
Different air filtration units,
Temperature, Pressure, Humidity control,
Protocol for personnel and goods entry into the clean
PARENTERAL PREPARATIONS room, protective clothing,
Routine monitoring tests,
Sterilization and Validation,
Department of Applied Chemistry & Chemical Engineering Formulation of parenteral products.
University of Dhaka Water for parenteral, Vehicles and additives,
Dhaka 1000, BANGLADESH Containers,
Manufacture,
Quality Control (Pyrogen test, LAL test. etc).

28-Sep-14 2

Parenteral Products Why Parenteral?

Parenteral Route Is Used bcoz


para: outside
enteron: intestine (i.e. beside the intestine) 1) Rapid action
These are the preparations which are given other than oral 2) Oral route can not be used
routes.
Injections: 3) Not effective except as injection
These are 4) Many new drugs particularly those derived from new
Sterile,
Pyrogen free preparations intended to be administered development in biotechnologically can only be given by
parenterally (outside alimentary tract). parenteral bcoz they are inactivated in GIT if given orally.
(Tubelar passage of mucous membrane and muscle extending
about 8.3 m from mouth to anus: function in digestion) 5) New drugs require to maintain potency & specificity so
that they are given by parenteral
28-Sep-14 3 28-Sep-14 4

Advantages Disadvantages

Quick onset of action. Once injected cannot be controlled (retreat)


Suitable for the drugs which are not administered Injections may cause pain at the site of injection
by oral route Only trained person is required
Useful for unconscious or vomiting patients. If given by wrong route, difficult to control adverse effect
Duration of action can be prolonged by modifying Difficult to save patient if overdose
formulation. Sensitivity or allergic reaction at the site of injection
Suitable for nutritive like glucose & electrolyte. Requires strict control of sterility & non pyrogenicity
Suitable for the drugs which are inactivated in GIT than other formulation.
or HCl (GI fluid)
28-Sep-14 5 28-Sep-14 6

1
9/28/2014

Necessities of Parenteral preparations Routes of Parenteral Administration

Sterility (must)
Pyrogen (must) Most Common: 1. Subcutaneous (SC; SQ ;Sub Q)
Free from particulate matter (must) 2. Intramuscular (IM)
Clarity (must) 3. Intravenous (IV)
Stability (must)
Isotonicity (should) Others: 4. Intra-arterial (IA)
Solvents or vehicles used must meet special purity and other 5. Intrathecal
standards. 6. Intraarticular
Restrictions on buffers, stabilizers, antimicrobial preservative. Do 7. Intrapleural
not use coloring agents. 8. Intracardial
Must be prepared under aseptic conditions. 9. Intradermal (Diagnostic)
Specific and high quality packaging.

28-Sep-14 7 28-Sep-14 8

Routes of Parenteral Administration


Subcutaneous Injections
Subcutaneous (SC; SQ ;Sub Q)
The injection is given under the skin
Need to be isotonic Given at a 45-degree
Upto 2 ml is given angle
Using to 1 inch 23 gauge needle or smaller 25- or 26-gauge needle,
needle 3/8 to 5/8 inch length
No more then 1.5 mL
Given should be injected into
Vaccines
Insulin
the site
Scopolamine to avoid pressure on
Epinephrine sensory nerves causing
pain and discomfort
28-Sep-14 9

Routes of Parenteral Administration


Intramuscular Injections
Intramuscular (IM)
Striated muscle fibre
0.5 to 2 ml sometimes upto 4 ml
1 to 1.5 inch & 19 to 22 gauge needle is used
Preferably isotonic Typical needle is 22- to 25-
gauge - to 1-inch needle
Principle sites:
Gluteal (buttocks) Intramuscular (IM) injections
Deltoid (upper arms) are administered at a 90-
Vastus lateralis (lateral thigh)
degree angle
Given: volume limited to less than 3
Solutions mL
Emulsions
Oils
Suspension

28-Sep-14 11

2
9/28/2014

Routes of Parenteral Administration


Intravenous Injections or Infusions
Intravenous (IV)
Into the vein Intravenous (IV) injections are administered at a
1 to 1000 ml 15- to 20-degree angle
1 inch ,19 to 20 gauge needle with injection rate 1ml/ 10
sec. for volume upto 5 ml & 1 ml/ 20 sec. for volume more
than 5 ml.
Given:
Aqueous solutions
Hydro alcoholic solutions
Emulsions
Liposome

28-Sep-14 13

Routes of Parenteral Administration Routes of Parenteral Administration

IV infusion of large volume fluids (100- 1000 ml)


has become increasingly popular. This technique Intra-arterial (IA)
is called as Venoclysis. Direct into the artery
This is used to supply electrolytes & nutrients to 2 to 20 ml
restore blood volume & to prevent tissue 20 to 22 gauge
dehydration. Solutions & emulsions can be administered
Combination of parenteral dosage forms for
administration as a unit product is known as an IV Given:
admixture. Radio opaque media
Lactated Ringer Injection USP Antineoplastic
NaCl Injection USP (0.9 %) (replenish fluid & Antibiotics
electrolyte)
Dextrose Injection USP (fluid & electrolyte)

28-Sep-14 15 28-Sep-14 16

Routes of Parenteral Administration Routes of Parenteral Administration

Intrathecal: Intraarticular:
Also called intra-spinal Given directly into the joints
Directly given into the spinal cord 2 to 20 ml
1 to 4 ml 5 inch 22 gauge
24 to 28 gauge Must be isotonic
Must be isotonic
Given:
Given: Morphine
LA (Local anaesthetic) LA
Analgesics Steroids
Neuroleptics NSAIDs
Antibiotics

28-Sep-14 17 28-Sep-14 18

3
9/28/2014

Routes of Parenteral Administration Routes of Parenteral Administration

Intrapleural: Intracardial:
Given directly into the pleural cavity or lung Directly given into the heart
Used for fluid withdrawal 0.2 to 1 ml
2 to 30 ml
2 to 5 inch, 16 to 22 gauge needle 5 inch , 22 gauge needle

Given: Given:
LA
Narcotics Cadiotonics
Chemotherapeutic agents Calcium salts as a calcium channel blockers

28-Sep-14 19 28-Sep-14 20

Routes of Parenteral Administration


Intradermal Injections
Intradermal: Given into capillary-rich layer just below epidermis for
Also called as diagnostic testing
0.05 ml local anesthesia
inch, 25 to 26 gauge needle diagnostic tests
Should be isotonic immunizations

Given:
Diagnostic agents

28-Sep-14 21

Official Types of Injections Formulation of Parenteral Products


1. Solutions of Medicinal
Example: Codeine Phosphate Injection 1. Therapeutic agents
2. Vehicles
Insulin Injection i. Water
2. Dry solids or liquid concentrate does not contain diluents etc. ii. Water miscible vehicles
Example: Sterile Ampicillin Sodium iii. Non- aqueous vehicles
3. If diluents present, referred to as.....for injection 3. Added substances (Additives)
i. Antimicrobials
Example: Methicillin Sodium for injection ii. Antioxidants
4. Suspensions iii. Buffers
"Sterile....Suspension" iv. Bulking agents
Example: Sterile Dexamethasone Acetate Suspension v. Chelating agents
5.Dry solids, which upon the addition of suitable vehicles yield vi. Protectants
preparations containing in all respects to the requirements for vii.Solubilizing agents
sterile suspensions. viii.Surfactants
Title: Sterile....for Suspension ix. Tonicity- adjusting agents
Example: Sterile Ampicillin for Suspension
6. Injectable Emulsions:
Example: Propofol injection
28-Sep-14 23 28-Sep-14 24

4
9/28/2014

General steps involved Formulation of Parenteral


1. Cleaning 1.Therapeutic ingredients:
2. Preparation of bulk products
Insulin
Antibiotics
3. Filtration
Anticancer
Steroids
4. Filling of solution in or product in ampoule or vial Vaccines
Antipyretic
5.Sealing Analgesics
Anti- inflammatory
6. Sterilization LVPs like Dextrose, NaCl or combination
etc.
7. Tests for Quality control
28-Sep-14 25 28-Sep-14 26

Formulation of Parenteral Formulation of Parenteral


2.Solvents:
Water 3. Added substances (Additives)
oShould meet compendial requirements i. Antimicrobials:
Water miscible vehicles
Added for fungistatic or bacteriostat action or
concentration.
oEthyl alcohol
Used to prevent the multiplication of micro-organisms.
oPEG
oPG Example
Non aqueous vehicles Benzyl alcohol ------ 0.5 10 %
oFixed oils Benzethonium chloride -- 0.01 %
Solvents used must be: Methyl paraben ---- 0.01 0.18 %
Non-irritating Propyl paraben --- 0.005 0.035 %
Non-toxic Phenol --- 0.065 0.5 %
Non-sensitizing
No pharmacological activity of its own
Not affect activity of medicinal
28-Sep-14 27 28-Sep-14 28

Formulation of Parenteral Formulation of Parenteral

iii. Antioxidants:
Used to protect product from oxidation.
Acts as reducing agent or prevents oxidation.
ii. Preservatives:
Examples
Multidose containers must have preservatives unless A) Reducing agent:
Ascorbic acid -- 0.02 0.1 %
prohibited by monograph. Sodium bisulphite-- 0.1 0.15 %
Large volume parenteral must not contain preservative Sodium metabisulphite-- 0.1 0.15 %
Thiourea - 0.005 %
becoz it may be dangerous to human body if it contain B) Blocking agents:
in high doses. Ascorbic acid esters- 0.01 0.015%
BHT- 0.005 0.02 %
C) Synergistic:
Ascorbic acid , Citric acid , Tartaric acid
D) Chelating agent:
EDTA- 0.01- 0.075 %

28-Sep-14 29 28-Sep-14 30

5
9/28/2014

Formulation of Parenteral Formulation of Parenteral

iv. Buffers:
Added to maintain pH, v. Chelating agents:
Change in pH may causes degradation of the products Used to form the complex with the metallic ions present in the
Acetates, citrates, phosphates are generally used. formulation so that the ions will not interfere during mfg. of
Factors affecting selection of buffers: formulation.
Effective range, They form a complex which gets dissolved in the solvents.
Concentration
Chemical effect on the total product
Examples:
EXAMPLES: Disodium edetate 0.00368 - .05 %
Acetic acid ,adipic acid, benzoic acid, citric acid, lactic acid Disodium calcium edetate - 0.04 %
Used in the conc. of 0.1 to 5.0 % Tetrasodium edetate 0.01 %

28-Sep-14 31 28-Sep-14 32

Formulation of Parenteral Formulation of Parenteral

vii. Solubilizing agents:


Used to increase solubility of slightly soluble
vi. Stabilizers: drugs
As parenterals are available in solution form they are most they acts by any one of the following:
prone to unstabilize
Used to stabilize the formulation solubilizers,
Maintain stable emulsifiers or
wetting agents.
Examples:
Creatinine 0.5- 0.8 %
Glycerin 1.5 2.25 % Examples:
Niacinamide 1.25 -2.5 % Dimethylacetamide,
Sodium saccharin 0.03 % Ethyl alcohol
Sodium caprylate 0.4 % Glycerin
Lecithin
PEG 40 castor oil
PEG 300
Polysorbate 20, 40, 80
28-Sep-14 33 28-Sep-14 34

Formulation of Parenteral Production facilities

viii. Tonicity- adjusting agents:


Used to reduce the pain of injection.
Buffers may acts as tonicity contributor as well as
stabilizers for the pH. Clean- up area
Isotonicity depends on permeability of a living
semipermaeable membrane. Preparation area
Hypotonic : swelling of cells (enlargement)
Hypertonic: shrinking of cells (reduction) Aseptic area

Example: Quarantine area


Glycerin
Lactose Finishing and packaging area
Mannitol
Dextrose
Sodium chloride
Sorbitol
28-Sep-14 35 28-Sep-14 36

6
9/28/2014

LAY OUT OF PARENTERAL MANUFACTURING AREA Production facilities

Clean area:
Non aseptic area.
S COMPOUNDING ASEPTIC QUARANTINE
Free from dust ,fibres & micro-organisms.
T AREA AREA AREA
Constructed in such a way that should withstand moisture,
O
C STORAGE steam & detergent.
K
AND Ceiling & walls are coated with material to prevent.
R TRANSPORT accumulation of dust & micro-organisms.
O
O PACKING Exhaust fans are fitted to remove heat & humidity.
M CLEAN UP
AREA
STERILIZATION AND The area should be kept clean so that to avoid
LABELLING
contamination to aseptic area.
The containers & closures are washed & dried in this area.

28-Sep-14 37 28-Sep-14 38

Production facilities Production facilities

Aseptic area:
Preparation area: Filtration & filling into final containers & sealing is done
The entry of outside person is strictly prohibited
The ingredients are mixed & prepared for filling
To maintain sterility, special trained persons are only
Not essential that the area is aseptic allowed to enter & work
Strict precaution is taken to prevent contamination from Person who worked should wear sterile cloths
outside Should be subjected for physical examination to ensure
the fitness
Cabinets & counters: SS
Minimum movement should be there in this area
Ceiling & walls : sealed & painted Ceiling & walls & floors : sealed & painted or treated with
aseptic solution and there should not be any toxic effect
of this treatment

28-Sep-14 39 28-Sep-14 40

Production facilities Production facilities


Protocol for Personel entrance in clean area
Cabinets & counters: SS
Mechanical equipments : SS
AIR: Autoclave
Free from fibres, dust & micro organisms
HEPA filters are used which removes particles upto 0.3
micron
Fitted in laminar air flow system, in which air is free
Grey White
from dust & micro organisms flows with uniform Area Area Aseptic area
velocity 30pa
Black
Air supplied is under positive pressure which prevents Area
particulate contamination from sweeping
UV lamps are fitted to maintain sterility

28-Sep-14 41 28-Sep-14 42

7
9/28/2014

Production facilities Production facilities


Protocol for Personel entrance in clean area
Personal entrance area

Black area Mask


Change room for clothing, jewellery and shoe.
Grey area Dress
Sticky materials to remove loose soiling from the shoe. Sequence to wear and hood Sequence to put
Preliminary hand washing is done using detergant (- sterile dress off sterile dress
chloro hexadiene solution). Foot
Grey area is seperated from white area by a low cover

threshold bench.
White area gloves
Aseptic area where the preparation takes place.

28-Sep-14 43 28-Sep-14 44

Production facilities Production facilities


Protocol for goods entry in clean area
Quarantine area:
Entry of goods must be through a cabinet type hatch or air After filling, sealing & sterilization the product or batch is
lock.
kept in this area.
Entry of sterile products may be double wrapped or tripple
wrapped. The random samples are chosen and given for analysis to
The outmost wrappers can be left behind in stage during
QC dept.
entry.
The batch is send to packing after issuing satisfactory
reports of analysis from QC.
Cabinet type hatch has two windows-
If one is opened, then the other is automatically locked and thus preventing If any problem is observed in above analysis the decision
any contamination.
is to be taken for reprocessing or others.

28-Sep-14 45 28-Sep-14 46

Production facilities Containers


Plastic containers
Finishing and packaging area:
Glass containers
After proper labelling, the product is given for packaging. Plastic containers
Packaging is done to protect the product from external Thermoplastic polymers
Contain relatively low amount of added substances-fillers,
environment. plasticizers.
The ideal Packing is that which protects the product Ex- LDPE (-CH2-CH2-)-n, HDPE, Polypropylene, etc.
Adv.-
during transportation, storage, shipping & handling. Light in weight.
The labeled container should be packed in cardboard or Non brittle.
Low toxicity and low reactivity with products.
plastic containers Disadv-
Ampoules should be packed in partitioned boxes. Tissue toxicity can occur.
Sterilization temperature adversely effect most polymers.
Relatively high permeability for water vapor.
28-Sep-14 47 28-Sep-14 48

8
9/28/2014

Glass containers Glass containers


The USP determine 3 types of glass containers
Type I: (Borosilicate glass) Commonly known as neutral glass. It has
a high resistance to hydrolysis and withstands autoclaving, Adv.-
weathering and solution of pH of up to 8. Contain less leachable constituents.
Amber glass containers can filter out UV rays completely.
Type II (treated soda lime glass): Containers may be treated with Low thermal expansion coefficient.
moist sulphur dioxide at high temperature to create a neutral Withstand the pressure differential that develop during
surface film with high hydrolytic resistance. Lower resistance to autoclaving.
autoclaving than for type I glass. Their transparency facilitates the inspection of the content.
Physically strong and rigid.
Type III (soda-lime glass): This offers very little resistance to Disadv.-
hydrolysis and should only be used for powders for reconstitution Glass containers are basically fragile.
prior to injection and for non aqueous preparations. Contain higher weight than plastic material.
Type II and Type III glass containers should be used once only
for parenteral preparations.
28-Sep-14 49 28-Sep-14 50

Types of containers Production facilities

Glass ampoules are the most commonly used single dose


containers and can range from sizes of 1 to 50mL.
For aqueous solutions, neutral glass is used.
After filling, glass ampoules are sealed by fusion of the
glass and hence there is no danger of entry of micro-
organisms. Amber glass ampoules are available for light
sensitive products. Clear ampoules may be used provided that
the ampoules are packaged in a light-resistant box.
Glass vials sealed by rubber closures are commonly used as
multi-dose containers. Figure. 1 (a) Figure. 1 (b) Figure. 1 (c)
The rubber closure is held in place by an aluminum sealing ring. Glass ampoule Multi dose glass Syringe with membrane filter
The rubber closure permits the penetration of a syringe needle vial and needle attachment
to allow the withdrawal of a dose of injection.

28-Sep-14 51 28-Sep-14 52

Units of concentration Production facilities


Filling and hand sealing of glass ampoules
The concentration of the components in parenteral products may
be expressed in various ways: rinsed out using water for injections to remove any dust, particulate matter and/ or
glass fragments.
Using a syringe, gently draw the volume required to be filled plus an excess volume.
Percentage weight/volume Invert the syringe to allow the air to rise towards the needle and push up the
Magnesium sulphate injection 50%w/v, plunger to remove all air.
sodium chloride intravenous infusion 0.9% w/v Attach the membrane filter and needle to the syringe . Discard the first 0.5-1mL of
filtrate.
Weight per unit volume Invert the ampoule over the needle and expel the required volume of liquid into the
Atropine sulphate 600g/ml ampoule taking care not to splash the liquid into the neck of the ampoule. Each
ephedrine hydrochloride injection 30mg/ml ampoule must also contain a slight excess volume of product.
Ampoules are sealed in the flame.
Millimoles per unit volume
Filling and hand sealing of glass vials
Potassium chloride solution, strong (sterile) contains 2mmol
each of K + and Cl - per ml; Glass vials are filled using the same rinsing and filling procedure as for
Calcium chloride injection BP contains 2.5 mmol of Ca 2+ ampoules.
and 10mmol of Cl - in 5ml. Vials containing a fixed number of dose units, an excess volume is required.
Vials are sealed using rubber closures which are held in place by an
28-Sep-14 53 aluminum sealing.
28-Sep-14 54

9
9/28/2014

Production facilities Production facilities


Cleaning of glass ampoules Cleaning of glass vial

Batch type- Vials are dipped in the netted blanket where washing and sterilization is done.
This process is carried out in aseptic area under laminar air flow
Continuous type-
Utilize rotary washing machine, one after another with cleaning
agents.
Cleaning agents-
Hot detergent water under high pressure
Tap water
Deionized water
Distilled water
WFI

28-Sep-14 55 28-Sep-14 56

Parenteral manufacture Parenteral manufacture

Parenteral Dosages Forms Solutions


Active ingredients are soluble in water.
According to their presentation Isotonic aqueous solution
Solutions-aqueous/oily PH close to body fluid (PH-7.4)
Suspensions Ex-
Emulsions Infusion fluid-IV route
Dry Powders Local anesthetics-spinal route

Other types- Suspensions


Large volume parenteral ( vol >25ml ) Medicament is insoluble in water or
Ex-saline Ingredient adsorbed onto an insoluble matrix.
Small volume parenteral (vol <25 ml) Ex-
Ex- ampoule, vials Insulin Zinc suspension BP

28-Sep-14 57 28-Sep-14 58

Parenteral manufacture Parenteral manufacture


Different type of vehicles/solvents
Emulsions
Active ingredients are lipophilic. Sterile water
Form O/W emulsion Used for aqueous preparation
Medicament is dissolved in oily phase (vehicle) and Are safe and easier to administer
emulsified water phase. o Quality of water vehicles for injection
Ex- Free from solid content
IV Infusion fluid-carry fat soluble vitamins. Free from pyrogen.
Dry Powders Chemically pure
Medicament is not stable in solution Conductivity of water < 1micro mho.
Solution is prepared before administration. Oily vehicles
Ex- Medicament is insoluble in water or
Penicillins Slightly soluble in water
Cephalosporins Prolong the action of drug and stability.
Ex- Cotton seed oil, peanut oil, olive oil.
28-Sep-14 59 28-Sep-14 60

10
9/28/2014

Parenteral manufacture Parenteral manufacture

Requirements
Non aqueous oil based vehicle. 1) Medicament
Solvent which are miscible in water are usually used in 2) Excipients
combination with water as the vehicle. Buffer
Solution must not irritating, toxic and sensitive. Antioxidants
Dont have adverse effect. Preservatives
Ex- Solubilizing agent
Dioxalane Suspending agent
Ethyl acetate. 3) Deionized water
4) Containers

28-Sep-14 61 28-Sep-14 62

Parenteral manufacture Parenteral manufacture


Steps involved in industrial manufacture 3) Sterilization validation
Product stability
1) Preparation of solution/solid mass. Efficiency of sterilization
Solution are filtered through 0.45 micro meter filter to remove fine I. Sterility test
particles. II. Pyrogen test
Pyrogen are endotoxin are produced from outer membrane of
2) Sterilization of solution or powder. gram negative bacteria.
Heat of sterilization Test is done by-
o 180 C---30 min I. Invivo test/ rabbit test
o 170 C---1 hr II. Limulus amebocyte lysate (LAL) test
o 160 C---2 hr
4) Sterilization of containers and its validation
Powers are sometime unstable to sterilization. Glass containers are sterilized at 250 C for 2 hr.
Powder-- solution sterilization-filtration (0.2 micro meter filter)-freeze drying. Validation is carried out with bacterial endotoxin and followed
by LAL test.
28-Sep-14 63 28-Sep-14 64

Parenteral manufacture LABELING


5) Filling, purging (air) and sealing
Name of product
Automated nozzle id used for filling operation and air above the Quantity of the product
product is purged with N2. containers are sealed under aseptic % of drug or amount of drug in specified volume of
conditions. amount of drug and volume of liquid to be added
Name and quantity of all added substances
6) Leak testing for ampoules and vials Mfg. license no.
Batch no.
Dye bath test
Manufacturer/Distributor
Liquid loss test Mfg. & Expiration date
High voltage detection Retail price (incl. of all taxes)
Visual technique. Mfger. address
Veterinary product should be so labeled
7) Labelling of parenteral products

28-Sep-14 65 28-Sep-14 66

11
9/28/2014

Sterilization methods Sterilization methods


Sterilization means destruction of all living organisms and their 2. Moist heat (steam) method-in autoclave
spores or their complete removal from the preparation. Steam sterilization is conducted in an autoclave and employs
Thermal methods steam under pressure.
1. Dry heat method-oven This method is preferred to other sterilization methods if the
Substances that withstand degradation at temp 140C (284F) product and container can withstand it.
may be rendered sterile by this method. Process
Kill spores and negative form of micro-organism. Temperature required 121 C for 15 to 30 minutes, depending on
the penetration time of moist heat into the load.
Process Adv.
2 hrs exposure at temp 180C (356F) or More effective than dry heat method for thermal sterilization.
45 min exposure at temp 260C (500F) Steam under pressure applied,
The object is heated much more promptly by quickly changing
Sterilization cycle time- fresh vapor.
log time = sterilization period + cooling period It cause the coagulation of cell protein at a much low temp.
28-Sep-14 67 28-Sep-14 68

Sterilization methods QC check for parenteral products (IPC)


Filtration For solution in ampoule
Sterilization by filtration depends on the physical removal of Filled volume
microorganisms by adsorption on the filter medium or by a sieving Room condition
mechanism. Temperature
For heat-sensitive solutions. Relative humidity
2 types Active constituent
Depth filters PH
Membrane filters (0.22 m)
For powder filling in vials
The filtration process might be affected by adsorption. Filled volume
The integrity of the filters has to be proven Bulk density
Avoid filters that cause particles. Room condition, Temp, R/H, etc
Test the filters so that they do not give extractable. Flow properties

28-Sep-14
69 28-Sep-14 70

QC check for finished products QC check for finished products

Standard specification Assay


1) Appearance a) Active ingredient identification and content
2) Identification
3) Average filled volume Packed
4) PH
a) Pack size
5) Uniformity of filled volume
b) Appearance (packed)
6) Loss on drying
c) Unit pack description
7) Particulate matter
d) Shelf life
8) Sterility test
9) Bacterial endotoxin test

28-Sep-14 71 28-Sep-14 72

12
9/28/2014

Sterility test Sterility test- Membrane Filtration


Apparatus
A sterility test may be defined as a test that critically A membrane essentially bears a nominal pore size not more than
assesses whether a sterilized pharmaceutical product is free from 0.45 m, and diameter of nearly 47 mm.
contaminating microorganisms. Salient Features
1) filtered via a hydrophobic-edged membrane filter that would
precisely retain any possible microorganisms.
2) The membrane is duly washed in situ to get rid of any possible
Two well-recognized universally accepted methods traces of antibiotic
3) Finally, the segregated microorganisms are meticulously
Membrane Filtration, and transferred to the suitable culture media
Direct Inoculation. 4) Once the filtration is completed, 1/2 of the membrane, in 100 mL
of the Fluid Soyabean-Casein Digest Medium, and incubate at
2025C for a duration of seven days.
5) Likewise, other half of the membrane, in 100 mL of Fluid
Thioglycollate Medium and incubate duly at 3035 C for not less
28-Sep-14 73 than seven days.
28-Sep-14 74

Sterility test- Direct Inoculation Sterility test- Direct Inoculation

Direct Inoculation of Culture Media Methodology


The various sequential steps involved are as given under
Three types of medium are commomly employed-
a) Liquid from the test containers must be removed carefully
Nutrient Broth-aerobic microorganisms with a sterile pipette or with a sterile syringe or a needle.
Thioglycollate Medium- anaerobic microbes. b) Transfer aseptically the requisite prescribed volume of the
Sabouraud Medium-fungal species substance from each container to a vessel of the culture
medium.
c) Mix the liquid with the medium carefully taking care not to
aerate excessively.
d) Incubate the inoculated media for not less than 14 days
at 3035C for Fluid Thioglycollate Medium, and 2025C
for Soyabean-Casein Digest Medium.

28-Sep-14 75 28-Sep-14 76

Sterility test- Direct Inoculation Pyrogens and pyrogen testing

Observation and Interpretation of Results Pyrogenic - means producing fever


Pyrogens - fever inducing substances-endotoxin
a) Both at intervals during the incubation period, and at its
completion, the media may be examined thoroughly for the Having nature
critical macroscopic evidence of the bacterial growth. Endogenous (inside body)
Exogenous (outside body)
b) In the event of a negative evidence, the sample under
examination passes the tests for sterility. Exogenous pyrogens
mainly lipopolysaccharides
bacterial origin, but not necessary

28-Sep-14 77 28-Sep-14 78

13
9/28/2014

Pyrogens and pyrogen testing Pyrogens and pyrogen testing

Structure of endotoxins

Produced mostly by gram-negative bacteria.


Endotoxin - complex of pyrogenic lipopolysaccharide, a protein
and inert lipid;
lipid part of the lipopolysaccharide is the main pyrogenic agent;
polysaccharide part increases solubility.

28-Sep-14 79 28-Sep-14 80

Pyrogens and pyrogen testing Pyrogens and pyrogen testing


Sources of pyrogen contamination
Solvent - possibly the most important source Tests for pyrogenic activity
The medicament
The apparatus Test for pyrogens = Rabbit test/Invivo test
The method of storage between preparation and sterilization Bacterial endotoxins/ LAL test

The endotoxin characteristics


Thermostable
water-soluble
unaffected by the common bactericides
non-volatile

These are the reasons why pyrogens are difficult to destroy once
produced in a product
28-Sep-14 81 28-Sep-14 82

Pyrogens and pyrogen testing Pyrogens and pyrogen testing


Test for pyrogens = Rabbit test/Invivo test
Test consists of
The test consists of measuring the rise in body temperature in
healthy rabbits by the intravenous injection of a sterile solution 1) Group of 3 rabbits
of the substance under the test. 2) Preparation and injection of the product
warming the product
Why the Rabbit? dissolving or dilution
Reproducible pyrogenic response. duration of injection: not more than 4 min
Other species not predictable. 3) Determination of the initial and maximum temperature
Similar threshold pyrogenic response to humans. All rabbits should have initial T: from 38.0 to 39.8C
Rabbit vs. dog as model? After injection, an elevation of temp. occurs within 3 hrs.
Rabbits: false positives Temp. is recoded by rectal thermocouple connected to an
Dogs: false negatives electric thermometer.

28-Sep-14 83 28-Sep-14 84

14
9/28/2014

LAL test LAL test


Bacterial endotoxins
Media
To detect or quantify endotoxins of gram-negative bacterial origin. LAL reagent
CSE (control standard Endotoxin)
Reagent: LRW (LAL reagent water)
amoebocyte lysate from Buffer
horseshoe crab (Limulus
polyphemus or Tachypleus Procedure
tridentatus). Equal Vol. of LAL reagent and test solution (usually 0.1 ml of
each) are mixed in a depyrogenated test-tube
The name of the test is also Mixing in a vortex mixer and
Limulus amebocyte lysate incubation at 37C, 1 hour
(LAL) test. Observation

28-Sep-14 85 28-Sep-14 86

LAL test LAL test


Advantages
Observation Greater sensitivity
Less variability
In the presence of bacterial endotoxin, gel formation may Much less false positives
occurs and degree of gelling can be quantified and related to Less expensive
the amount of endotoxin present. Alternative to animal model
Gelling can be measured by Cheaper,
More accurate than other
Performed in the pharmaceutical laboratory
I. Gel-clot technique - gel formation Specific for endotoxins of gram-negative origin
II. Turbidimetric technique - the development of turbidity Particularly useful for:
after cleavage of an endogenous substrate Radiopharmaceuticals and cytotoxic agents
Products with marked pharmacological or toxicological
III. Chromogenic technique - the development of color activity in the rabbit (e.g. insulin)
after cleavage of a synthetic peptide-chromogen Blood products which sometime give misleading results in
complex the rabbit
Water for injection where LAL test is potentially more
stringent and readily applied.
28-Sep-14 87 28-Sep-14 88

Depyrogenation Clearity test


Inactivation For solution in ampoule
Chemical reactions as oxidation, hydrolysis or alkylation of the
endotoxins.
By autoclaving at 121C, which has limited success.
Incineration, i.e. by dry heating at 170-350 C. ( usually at 250C
for 30 mins.)

Removal
Distillation-effective for water as pyrogens are non volatile.
Ultrafiltration-combination of ultra filter (0.1 ) and sterilization
filter (0.2 ).
Ion exchanger-using resin.
Gamma irradiation.
Ringing container/equipment-with pyrogen free water.

28-Sep-14 89 28-Sep-14 90

15