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.

3 comments:

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Praveen G said...

Autoclave Validation with Fo Value
Very informative blog, Hope this helps many!