BASIC PRINCIPLES IN THE VALIDATION OF STERILE PRODUCTS Theoretical Approaches

Generally, five basic steps are necessary to validate any manufacturing process:

1. Written documentation

2. Manufacturing parameters

3. Testing parameters

4. In-process controls

5. Final product testing

In sterile product manufacturing, five major steps are involved in approaching the validation of a sterile process.

These are outlined below using thermal sterilization as the example process.

1. Select or define the desired attributes of the product. Example: The product will be sterile.

2. Determine specifications for the desired attributes. Example: The product will be sterilized by a sterilization

process sufficient to produce a probability of nonsterility of one out of 1 million containers (10−6).

3. Select the appropriate processes and equipment. Example: Use microbial kinetic equations such as Eq. (11) to

determine the probability of nonsterility. Select cleaning equipment and container component procedures designed

and validated to reduce the product bioburden to the lowest practical level. Select an autoclave that can be

validated in terms of correct operation of all mechanical controls. Use the appropriate types of thermocouples,

thermal sensing devices, biological indicators, integrated chemical indicators, and culture media to conduct the

validation tests.

4. Develop and conduct tests that evaluate and monitor the processes, equipment, and personnel.

Examples:

a. Determine microbial load counts prior to container filling.

b. Determine D and Z values of biological indicator organism.

c. Perform heat distribution studies of empty and loaded autoclave.

d. Perform heat penetration studies of product at various locations in the batch.

5. Examine the test procedures themselves to ensure their accuracy and reliability.

Examples:

a. Accuracy of thermocouples as a function of variances in time and temperature.

b. Repeatability of the autoclave cycle in terms of temperature and F value consistency.

c. A challenge of the sterilization cycle with varying levels of bioindicator organisms.

d. Reliability of cleaning processes to produce consistent low-level product bioburdens. Each validation process

should have a documented protocol of the steps to follow and the data to collect during the experimentation. As an

example,

App. I presents a protocol for the validation of a steam sterilization process.Upon completion of the experimental
phase of validation, the data are compiled and evaluated by qualified scientific personnel. The results may be

summarized on a summary sheet, an example of which is shown in Table 2. Once a process has been validated, it

must be controlled to assure that the process consistently produces a product within the specifications established

by

the validation studies. As shown in Table 2, documentation should present original validation records, a schedule of

revalidation dates, and data from the revalidation studies. The interval between validation studies strictly depends

on the judgment of the validation team based on the experience and history of the consistency of the process.

Table 3 lists the sterilization methods used for sterile products. There are five basic methods—heat, gas, radiation,

light, and filtration. The first four methods destroy microbial life, while filtration removes micro-organisms. Vali-

dation approaches and procedures used for most of these methods will be addressed in the remainder of this

chapter. Gaseous validation and radiation validation approaches will be focused on ethylene oxide and gamma

radiation,

respectively. The other gaseous and radiation methods, however, generally will follow the same principles as those

discussed for ethylene oxide and gamma radiation. Some extra coverage will be given to vapor phase hydrogen

peroxide because of its increased application, particularly in the sterilization of barrier isolators.
VALIDATION OF STERILIZATION

JM Tech.

Do-Young Ahn

Moist Heat Sterilization

Definition

 Sterilization

“The act or process, physical or chemical, that destroys or eliminates all viable microbes including resistant
bacterial spores from a fluid or a solid.”

Examples of sterilization methods are : steam treatment at 121℃, dry heat at 230℃, flushing with a sterilizing
solution such as hydrogen peroxide (H2O2) or ozone (O3), irradiation, and filtration.

Sterility

“The reduction of anticipated levels of contamination in a load to the point where the probability of survival is less
than 10-6.”

Moist Heat Sterilization

Definition

D-value

The time in minutes required for a one-log or 90% reduction of a specific microbial population under specified
lethal conditions. For steam sterilization it is determined at a constant temperature

z-value

The number of degree of temperature change necessary to change the D-value by a factor of 10.

Moist Heat Sterilization

Definition

F value(lethal rate, instantaneous Fo)

The F value is a measurement of sterilization effectiveness. F(T,z) is defined as the equivalent time at temperature
T delivered to a container or unit of product for the purpose of sterilization, calculated using a specific value of z.

Fo value(accumulated Fo)

The term "Fo " is defined as the number of equivalent minutes of steam sterilization at temperature 121.1°C
delivered to a container or unit of product calculated using a z-value of 10°C.

Fo = ♣ 10^((121-T)/z)*′ t

Moist Heat Sterilization

 Methodology

Overkill Sterilization

Provides a minimum 12 log reduction of a resistant BI w/ a known D-value of not less than 1 minute.

Required minimal information on the bioburden

Bioburden/Bioindicator Sterilization

Provides a probability of survival of less than 1 in 106 for the bioburden as demonstrated using a resistant BI w/ a
known D-value.

BI may not be inactivated

Requires information on the numbers and heat resistance of the BI.

Requires ongoing monitoring or control over bioburden.

Moist Heat Sterilization

Methodology

Bioburden Sterilization

Provides a probability of survival of less than 1 in 106 for the most resistance bioburden expected in the load.

Requires information on the numbers and heat resistance of the BI.

Requires ongoing monitoring or control over bioburden.

Moist Heat Sterilization

Sterilizer Cycle

Gravity Displacement

Difference of density

Density of air at 20℃ = 1.2 g/ℓ

Density of steam at 100℃ = 0.6 g/ℓ

Effectiveness of air elimination depends on the rate of steam supply

Air pocket : too rapidly

Diffusion into the steam : too slowly, more difficult to remove

Specially designed steam trap permitting the passage of large volume of air
 Moist Heat Sterilization

Sterilizer Cycle

Prevacuum cycle

A more effective method

By means of a mechanical vacuum pump or a steam eductor

Vacuum as low as 15~20 mmHg, apply for 8~10 min.

Pulsing cycle

A series of alternating steam pulses followed by vacuum excursions

Air-steam mixture

Terminal sterilization of large volume parenterals

Air injection required to compensate the great expansion of air or nitrogen in the head space above the liquid

Well mixed chamber : fan, raining effect by external pump w/ cooling

Moist Heat Sterilization

Cycle Development

Consider factors into account

Nature of the load : porous materials, heat sensitivity of the products

Type of the sterilizer

Employed containers and closures

Heat stable product : overkill approach

Heat liable product : bioburden approach

Bioburden studies : number of microorganisms

D-value studies : only highly resistant spore formers,

BIER(biological indicator evaluator resistometer)

Inoculate the spore into the actual solutions

For solid materials, precut strips

Moist Heat Sterilization

Preparing for Validation

Temperature sensing devices :

 T type thermocouples(copper-constantan) encased in flexible sheaths

 Premium grades of wire having ♣0.1℃ accuracy

Temperature standards

 RTD traceable to the National Bureau of Standards , IPR, HTR

Calibration of thermocouples

At two temperatures : 0 ℃, 130 ℃

Correction factors

Stability : ♣0.03℃

Accuracy : 0.5℃♣

Moist Heat Sterilization

Preparing for Validation

Autoclave

Validation nozzle and adaptor

Data logger : digital output and multi-channel device

BIs or biological challenges

Loads

Moist Heat Sterilization

Validation Protocol

Protocol should include

Objectives of the validation

Responsibilities of validation personnel and operating department personnel

Identification and description of the sterilizer and its process control

Identification of SOPs :equipment

Calibration of instrument : SOPs and/or description

Identification and calibration of the temperature monitoring equipment

Moist Heat Sterilization

Validation Protocol

A description of the following studies

Bioburden determination studies(if applicable)

Empty chamber heat distribution studies

Container mapping studies(if applicable)

Loaded chamber heat penetration studies

Microbiological challenge studies

Evaluation of drug product cooling water(if applicable)

Integrity testing of vent filter

Acceptance criteria

References

Review and approval

Moist Heat Sterilization

Heat Distribution Studies

To demonstrate the temperature uniformity and stability of the sterilizing medium throughout the sterilizer

Conduct on both the empty and loaded chamber with max. and min. load configurations

Acceptance criteria : Less than ±1℃of the mean temperature

Conduct 3 runs to obtain consistent results

Distribution of the thermocouples : geometrical representatives, exhaust drain, adjacent to the control sensor

At least 10 probes, normally 15~20 probes

Moist Heat Sterilization

Heat Distribution Studies

At loaded chamber heat distribution test, the thermocouples should be positioned in the same locations used for
empty chamber heat distribution

Avoid contacting solid surfaces

Do not place within any containers

Data should be obtained at regular intervals

Moist Heat Sterilization

Container Mapping

To determine the coolest point within the liquid filled container

Temperature mapping should be conducted on all the different container types, sizes and fill volume to be
validated

The number of the thermocouples used depends on the container volume

Possible to use a single thermocouple at different positions,

and can be conducted in a smaller autoclave or retort

Penetration thermocouples should be positioned at the cold spot having lowest temperature or Fo

Moist Heat Sterilization

Heat Penetration Studies

To determine the coolest point(s) within the specified load and configuration, and to assure that these points be
consistently exposed to sufficient heat lethality

Prior to conduct heat penetration studies, determine max. and min. load configurations

Probed container at the cold spot should be distributed uniformly throughout the load

Penetration thermocouple are positioned at points within the process equipment suspected to be the most
difficult for steam heat penetration

Moist Heat Sterilization

Heat Penetration Studies

Lethal rate can be determined from the temperature data

by the following formula :

L = log-1(To-Tb)/z = 10^((To-Tb)/z)

A summation over time of the lethal rate at a series of temperature(accumulated lethality)

Fo = ♣ 10^((121-T)/z)*′ t

Regard to product stability

Moist Heat Sterilization

Microbial Challenge Studies

Biological challenges are employed during heat penetration studies in order to demonstrate the degree of process
lethality provided by the sterilization cycle

Microorganism frequently utilized

Overkill : Bacillus stearothermophilus and Clostridium sporogenes

Bioburden : Calibrated BIs from environmental and process

isolates such as E. coli

Type of BI :

Spore strips or spore suspension into the suspending medium

Microbial challenge studies are conducted concurrently with the heat penetration studies

Moist Heat Sterilization

Validation Report

Common elements of all reports :

Identification of the task report by number

Reference to protocol

A brief summary of the range of operational conditions experienced and how they were controlled

A procedure for maintaining control within the approved range

A summary and analysis of the experimental results

A brief description of any deviation

Conclusion

Review and approval

Cycle development reports are not usually a part of the validation report

Moist Heat Sterilization

Maintenance of Validation

A routine calibration program for all instruments critical to the operation of the sterilizer and its support system

A preventative maintenance program including periodic operational rechecks and comparison to OQ record

Routine monitoring of bioburden and periodic BI challenges(optionally)

Operating records and equipment logs

Process and equipment change control procedures including review to establish whether additional validations
are required

On-going validation

Moist Heat Sterilization

Controversial Issues

Incubation of the sterility test : 7 days vs. 14 days

USP provide information concerning critical parameters for Parameteric Release

Reduction extent and frequency of revalidation

Verification of D-value of BIs

Use of alternative to B. stearothermophilus as a BI

Z-value measures the rate of change in the D-value as a function of temperature.

Z=(T2-T1)/Log(D2/D1),
D 값을 1/10 로 단축시키는데 소요되는 온도상승값

Temperature uniformity of the heat distribution studies may be influenced by

Type

Size

Design

Installation of the sterilizer

Container mapping studies can be conducted in a small autoclave or retort.

The temperature profile of the container should remain constant among different sterilizing chamber.