VALIDATION OF STERILIZATION

JM Tech.

Do-Young Ahn

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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.”

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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.

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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

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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.

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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.

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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

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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

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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

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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℃

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Preparing for Validation

Autoclave

Validation nozzle and adaptor

Data logger : digital output and multi-channel device

BIs or biological challenges

Loads

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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),

D값을 1/10로 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.