Steam Sterilization Cycles and

Microbiological Lethality.Session 20

Steven S. Kuwahara, Ph.D.

GXP BioTechnology

6336 N. Oracle #326-313

Tucson, AZ 85704-5480

E-Mail: s.s.kuwahara@gmail.com

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VALIDATION OF STERILIZATION PROCESSES

Section 211.113, Control of Microbiological

Contamination.

(b) "Appropriate written procedures, designed

to prevent microbiological contamination of

drug products purporting to be sterile, shall be

established and followed. Such procedures shall

include VALIDATION of any sterilization

process."

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INFORMATION FOR TERMINAL MOIST HEAT

STERILIZATION PROCESSES.

The following information should be submitted for each facility to be used in the manufacture of the proposed drug product:

A Description of the Process and Product:

1.The Drug Product and Container-Closure System Descriptions of the drug product and the container-closure system(s) to be sterilized (e.g., size (s), fill volume, or secondary packaging).

2.The Sterilization Process. A description of the sterilization process used to sterilize the drug product in its final container-closure system, as well as a description of any other sterilization process(es) used to sterilize delivery sets, components, packaging, bulk drug substance or bulk product, and related items. Information and data in support of the efficacy of these processes should also be submitted.

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The Autoclave Process and Performance

Specifications

A description of the autoclave process, including

pertinent information such as cycle type (e.g.,

saturated steam, water immersion, and water

spray), cycle parameters and performance

specifications including temperature, pressure,

time, and minimum and maximum F . Identify

the autoclave(s) to be used for production

sterilization, including manufacturer and

model.

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Autoclave Process: Loading Patterns, Methods,

Requalifiaction

A description of representative autoclave loading patterns should be provided.

Methods and controls used to monitor routine production cycles (e.g., thermocouples, pilot bottles, and biological indicators) should be described, including the number and location of each as well as acceptance and rejection specifications.

Requalification of Production Autoclaves: A description of the program for routine and unscheduled requalification of production autoclaves, including frequency, should be provided.

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Autoclave Process: Reprocessing

A description and validation summary of any program

that provides for reprocessing (e.g., additional thermal

processing) of product should be provided. Please note

that the stability program is also affected by additional

thermal processing. For further information concerning

the stability program, reference is made to the Center

for Drug Evaluation and Research "Guideline for

Submitting Documentation for the Stability of Human

Drugs and Biologics"and to the Center for Veterinary

Medicine "Drug Stability Guideline."

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Thermal Qualification of the Cycle

1. Heat Distribution and Penetration Studies: Heat distribution and penetration study protocols and data summaries that demonstrate the uniformity, reproducibility, and conformance to specifications of the production sterilization cycle should be provided. Results from a minimum of three consecutive, successful cycles should be provided to ensure that the results are consistent and meaningful.

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Thermal Qualification of the Cycle

2.Thermal Monitors: The number of thermal monitors used and their location in the chamber should be described. A diagram is helpful.

3.The Effects of Loading on Thermal Input Data should be generated with minimum and maximum load to demonstrate the effects of loading on thermal input to product. Additional studies may be necessary if different fill volumes are used in the same container line. Data summaries are Acceptable for these purposes. A summary should consist of, for example, high and low temperatures (range), average temperature during the dwell period, minimum and maximum F values, dwell time, run date and time, and identification of the autoclave(s) used. These data should have been generated from studies carried out in production autoclave(s) that will be used for sterilization of the product that is the subject of the application.

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Information

Information Included in the Batch Record: The batch

record supplied with the chemistry, manufacturing, and

controls section of the application should identify the

validated processes to be used for sterilization and for

depyrogenation of any container-closure components.

This information can be included in the batch record by

reference to the validation protocol or standard

operating procedure (SOP). Validation information

should be provided as described above.

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Microbiological Efficacy of the Cycle. 1.

Validation studies that demonstrate the efficacy

(lethality) of the production cycle should be provided. A

sterility assurance of 10-6 or better should be

demonstrated for any terminal sterilization process.

This level of sterility assurance should be demonstrated

for all parts of the drug product (including the

container and closure, if applicable), which are claimed

to be sterile. The specific type of study and the methods

used to carry out the study (or studies) are product and

process specific and may vary from manufacturer to

manufacturer.

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Microbiological Efficacy of the Cycle. 2.

In general, the following types of information and data should be provided.

1.Identification and Characterization of Bioburden Organisms: Describe the methods and results from studies used to identify and characterize bioburden organisms. The amount and type of information supplied may be dependent on the validation strategy chosen. For example, more information may be needed for bioburden-based autoclave processes than for overkill processes.

Information concerning the number, type, and resistance of bioburden organisms may be necessary, including those organisms associated with the product solution and the container and closure. It may be necessary to identify the most heat-resistant bioburden organisms.

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Specifications for Bioburden

Specifications (alert and action levels) for

bioburden should be provided. A description

should be included of the program for routinely

monitoring bioburden to ensure that validated

and established limits are not exceeded (e.g.,

frequency of analysis and methods used in

bioburden screening).

The methods provided should be specific.

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Identification, Resistance, and

Stability

3.Identification, Resistance, and Stability of Biological Indicators: Information and data concerning the identification, resistance (D and Z values), and stability of biological indicators used in the biological validation of the cycle should be provided. If biological indicators are purchased from a commercial source, it may be necessary to corroborate the microbial count and resistance, and provide performance specifications.

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Resistance

4.The Resistance of the Biological Indicator Relative to

That of Bioburden Studies. Characterizing the

resistance of the biological Indicator relative to that of

bioburden may be necessary. Resistance in or on the

product (i.e., in the product solution, or on the surface

of container or closure parts or interfaces) should be

determined as necessary. If spore carriers are used (e.g.,

spore strips), the resistance of spores on the carrier

relative to that of directly inoculated product should be

determined, if necessary.

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Microbiological Challenge Studies

Microbiological validation studies should be submitted that demonstrate the efficacy of the minimum cycle to provide a sterility assurance of 10-6 or better to the product under the most difficult to sterilize conditions (e.g., the most difficult to sterilize load with biological indicators at microbiological master sites or in master product or both). Use of a microbiological master product or site should be supported by scientific data. Microbiological master sites or solutions are those sites or solutions in which it is most difficult to kill the biological indicator under sterilization cycles that simulate production conditions.

Steam Sterilization Cycles

There are two main considerations.

1. Sterilization of hard non-permeable substances that

are stable to steam heating.

Sterilization of these materials are fairly straight forward.

2. Sterilization of liquids or substances that may

interact with steam.

With these substances, you must give consideration to the

properties of the liquid as well as the container.

There are intermediate conditions such as a liquid

inside a glass or other non-permeable container-

closure.

Since the liquid will probably be heated above its boiling

point, it is usually necessary to carefully control the cooling

process.ClVal0815S2016

Limitations of Steam Sterilization

The biggest limitation is heat.

Heat sensitive material and material that is reactive

with steam should not be exposed.

Complex culture media can be “cooked” to the point where

nutrients are lost or undesirable degradation products are

produced.

Solid, impermeable material can by corroded by steam.

Steam, or moist heat, usually kills more rapidly than

dry heat, partially because it transfers heat more

efficiently.

Items that contain pockets or limited access regions where

steam cannot easily penetrate will require different cycles

from those where the steam can interact with all parts.

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Problems: Load

The configuration of the load that is being sterilized

can be critical.

Different material will have different heat capacities thus

the rate of heating will be different.

When the steam is first introduced into a cold load,

condensation may take place and pools of water may

form. The kill rate in the “puddles” may be different from

what happens on the surfaces.

Layering of steam and air can occur with a load or a

container. In the case of SIP processes, it is important to

assure an even distribution of the steam.

With hard surfaces and items, the precise configuration of

the load may not be critical once the cycle is validated.ClVal0815S2018

Problems: Steam Quality

The steam used for sanitization or sterilization

normally interacts with product contact

surfaces and must be “clean steam.”

Clean steam is usually defined as steam that, when

condensed, will produce water that meets compendial

criteria for “purified water” or “water for injection.”

Boiling water generates aerosols.

Steam generation systems normally contain

“demisters” to remove the particles from the steam,

but if routine maintenance is not done the demisters

may not work well. Sometimes the steam generation

need “blows” mist through the demister

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Problems: Mist

If the mist is carried with the steam, the particles

can contain entrained material that contaminates

the surfaces being sterilized.

Normally the feed water is “conditioned” to

minimize contaminants.

Corroded piping or low quality feed water can introduce

particles.

In many cases, “briquettes” are added to the distillation

pot. They contain absorbents or anti-scaling material to

protect the pot. These substances can be carried in the

mist.

Heat-resistant spores have been carried over from

contaminated feed water.ClVal0815S2020

The D Value. I.

The D value is the time in minutes to kill 90%

of the organisms present.

D is dependent on the exact conditions used and also

varies with the organism being tested.

Changing the exact conditions or the organism under study

can result in a change in the D value.

Geobacillus stearothermophilus has a D value of about

2 min in steam at 121o C. At 110o C it becomes 20 min

and at 130o C it is around 0.2 – 0.3 min.

G. stearothermophilus is usually used as an indicator

organism (BI), but it is also critical to know the D

values for the bioburden (BB).

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The D Value. II.

The D value for a BI must be known. Otherwise the

fact that you can kill the BI will have no

significance.

This is critical for validation studies.

D values can be calculated for many different

sterilization processes and organisms.

Since the BB represents the organisms that can be

expected in your environment , the D value for the

BB is very important.

The problem develops when the composition of your BB

changes. You need to monitor the composition of your BB.

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The D Value. III.

Survivors

Log10

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6

5

4

3

Exposure Time

Slope = -1/D

The Z Value

The Z value is related to the temperature

dependence of the D value.

Z is the degrees of temperature needed to produce a

10 degree change in the D value. Thus D and z are

related.

It is only useful for sterilization processes where

temperature has an great impact on the D value.

The z value is fairly constant over a range of

temperatures.

G. stearothermophilus has a z value of 8o – 12o C over

the temperature range from 100o - 135o C (Often

assumed to be 10o C)

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The F value. I.

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The F value. II.

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A Calculation of F0 for a Imaginary Steam

Sterilization Process.

Temp T-T0 T-T0/Z 10(T-T0/Z) ∑

101 -20 -2 0.01 0.01

111 -10 -1 0.10 0.11

121 0 0 1.00 1.11

124 3 0.3 2.00 3.11

131 10 1 10.00 13.11

121 0 0 1.00 14.11

111 -10 -1 0.10 14.21

101 101 -2 0.01 14.22

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Physical Process Data

The problem is that the physical data apply

only to the actual conditions that the organisms

experience.

With complex loads there can be locations, (e.g. in the

neck of a vial next to a stopper) where the D value

may be quite different from other locations.

However the physical data do allow one to make

comparisons among data from different parts of a

load or from one sterilizer to another.

Without physical data comparisons become difficult

to make.

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Probability of a Nonsterile Unit (PNSU)

Historically regulators have accepted a target of

no more than 1 nonsterile unit per million.

This is viewed a reasonable goal for patient safety.

A closely related term is the Sterility Assurance

Level or SAL.

The target for the SAL has been given as 1 X 106 or

as 1 X 10-6 leading to confusion.

The PNSU is preferred as being easier to understand.

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Probability of a Nonsterile Unit

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Indicator Organisms

The most common BI for steam sterilization

are:

Geobacillus stearothermophilus, Bacillus subtilis,

Bacillus coagulans, Clostridium sporogenes.

Regardless of the BI chosen, the BB are really the most

interesting as it is important to know and understand

the organisms that would commonly contaminate your

material.

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