Guideline for Sterilization Process Validation

(Date:1995-11-01)

Chapter I. INTRODUCTION
According to the GMP regulations, to avoid microbiological
contamination on aseptic products, a manufacturer must set up
and exactly comply with the documented manufacture process.
This documentation must include the validation of all
sterilization process.
The efficacy of a given sterilization process for a specific drug
product is evaluated on the basis of a series of protocols and
scientific experiments designed to demonstrate that the
sterilization process and associated control procedures can
reproducibly deliver a sterile product.
Regardless of whether the applicant uses terminal sterilization or
aseptic processing to manufacture a drug product that is
purported to be sterile, the information about the validation of
that process should be submitted for all types of sterilization.
Whether a product is sterilized by a terminal sterilization
process or by an aseptic filling process, the efficacy of the
sterilization process may be validated without the manufacture
of three production batches.
Sterilization process validation data, however, should be
generated using procedures and conditions that are fully
representative and descriptive of the procedures and conditions
proposed for manufacture of the product in the application.
This document is intended to provide guidance for the
submission of information and data in support of the efficacy of
sterilization processes in drug applications. This document will
be used as a reference when the manufacturers execute
validation on the aseptic drug products.

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Chapter Ⅱ. TERMINAL MOIST HEAT

STERILIZATION PROCESSES
Section 1. 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

(1)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.
(2) Information and data in support of the efficacy of these
processes should also be submitted. ( See also chapter Ⅱ 2.
and chapter Ⅱ 3. Of this guidance. )

3. The Autoclave Process and Performance Specifications

(1)A description of the autoclave process, including pertinent
information such as cycle type (e.g., saturated steam, water
immersion, and water spray), including the manufacturer and
model, of the autoclave.
(2)A description of performance specifications ( e.g.,
temperature, pressure, time, and minimum and maximum FO) .
Identify the autoclave(s) to be used for production sterilization.

4. Autoclave Loading Patterns

A description of representative autoclave loading patterns
should be provided.
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5. Methods and Controls to Monitor Production Cycles
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.

6. Requalification of Production Autoclaves

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

7. 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.

Section 2. Thermal Qualification of the Cycle

1. Heat Distribution and Penetration Studies
(1)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.
(2) Results from a minimum of three consecutive, successful
cycles should be provided to ensure that the results are
consistent and meaningful.

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

(1)Data should be generated with minimum and maximum load
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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.
(2)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 FO values, and dwell time, run
date and time, and identification of the autoclave(s) used.
(3)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.

Section 3. Microbiological Efficacy of the Cycle

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. In general, the following
types of information and data should be provided.
1. Identification and Characterization of 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
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and closure.
Describe the methods and results from studies used to identify
and characterize bioburden organisms. It may be necessary to
identify the most heat-resistant bioburden organisms.

2. Specifications for Bioburden

(1)Specifications (alert and action levels) for bioburden should
be provided.
(2)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).

3. Identification, Resistance, and Stability of Biological

Indicators
(1) 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.
(2) If biological indicators are purchased from a commercial
source, it may be necessary to corroborate the microbial count
and resistance, and provide performance specifications.

4. The Resistance of the Biological Indicator Relative to That of

Bioburden
(1) A description of characterizing the resistance of the
biological indicator relative to that of bioburden may be
necessary.
(2) 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.
(3) If spore carriers are used the resistance of spores on the
carrier relative to that of directly inoculated product should be
determined, if necessary.

5. Microbiological Challenge Studies

Microbiological validation studies should be submitted that
demonstrate the efficacy of the minimum cycle to provide a
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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.

Section 4. Microbiological Monitoring of the Environment

The establishment of scientifically sound and appropriate
specifications, standards, sampling plans, and test procedures
designed to ensure that components, drug product containers,
closures,in-process materials, and drug products conform to
appropriate quality standards. Therefore, a microbiological
monitoring program for production areas along with a
bioburden-monitoring program for components and process
water should be established. Process water includes
autoclave-cooling water. Applicants should provide information
concerning this program. Frequency, methods used, action levels,
and data summaries should be included. A description of the
actions taken when specifications are exceeded should be
provided.

Section 5. Container-Closure and Package Integrity

1. Simulation of the Stresses from Processing
Experimental designs should simulate the stresses of the
sterilization process, handling, and storage of the drug and their
effects on the container-closure system. Physical, chemical, and
microbiological challenge studies may be necessary.
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2. Demonstrate Integrity Following the Maximum Exposure
(1)Container-closure integrity should be demonstrated on
product units that have been exposed to the maximum
sterilization cycle(s).
(2)If a product is exposed to more than one process, then
exposure to the maximum cycle of all processes should be
incorporated into the study design.

3. Multiple Barriers
Each barrier that separates areas of the drug product claimed to
be sterile should be separately evaluated and validated.

4. The Sensitivity of the Test

The sensitivity of the experimental method used for
container-closure integrity testing should be specified and
provided.

5. Integrity over the Product Shelf Life

Microbial integrity of the container-closure system should be
demonstrated over the shelf life of the product.

Section 6. Bacterial Endotoxins Test and Method

The bacterial endotoxins test used for the product should be
described. The description should include qualification of the
laboratory, inhibition and enhancement tasting and results,
determination of noninhibitory concentration and maximum
valid dilution.

Section 7. Sterility Testing Methods and Release Criteria

1.Sterility test methods should be described and should include
the protocol for the selection of representative units during
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production.
2.Testing performed within barrier systems should be described.
3.Information concerning validation of the barrier system may
be necessary.

Chapter Ⅲ. OTHER TERMINAL STERILIZATION

PROCESSES
Although the information above (Chapter II Section 1 through
Section7 of this guidance) directly addresses moist heat
processes, the same type of information would pertain to other
terminal sterilization processes used singly or in combination to
sterilize a drug product.
The types of information outlined are, in general, also applicable
to ethylene oxide and radiation (gamma and electron beam).
These other processes should be addressed as each applies to the
drug product, sterile packaging and in-process sterilization of
components. Examples of such information might include:
descriptions of loading configurations; qualification and
validation of master load configurations; determination and
validation of the efficacy of the minimum cycle to provide
sterility assurance at the product master sites; requalification of
the cycle; provisions for resterilization; specifications and
monitoring program for product bioburden; and
container-closure integrity. Additional information relating to
the effects of the sterilization process on the chemical and
physical attributes of the drug substance or drug product may be
applicable.

Section 1. Ethylene Oxide

1. Sterilizer
The sterilizer(s) and controlled site(s) for prehumidification and
aeration of the product load should be described (A diagram is
helpful).

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2. Cycle Parameters
(1) The parameters and limits for all phases of the cycle, e.g.,
prehumidification, gas concentration, vacuum and gas pressure
cycles, exposure time and temperature, humidity, degassing,
aeration, and determination of residuals should be specified.
(2)Specific procedures used to monitor and control routine
production cycles to assure that performance is within validated
limits should be provided.

3. Microbiological Methods
The microbiological methods (growth medium, incubation
temperature, and time interval) for cultivating spores from
inoculated samples during validation experiments should be
described as well as the microbiological methods used as part of
routine production cycles.

4. Stability
The program for monitoring the stability of packaging and the
integrity of the container-closure system barrier over the
claimed shelf life should be described.

Section 2. Radiation
1. The Facility and the Process
(1)The radiation facility should be identified (A diagram is
helpful).
(2) The radiation source, method of exposure (i.e., movement
through the irradiator), and the type and location of dosimeters
used to monitor routine production loads should be described. If
the low dose site is not used for routine monitoring, data that
shows the dose relationship between the two sites should be
provided.

2. The Packaging of the Product

The packaging of the drug product within the shipping carton
and within the carrier should be described.
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3. Multiple-Dose Mapping Studies
Multiple-dose mapping studies for identification of low and high
dose sites and demonstration of uniformity and reproducibility
of the process should be described.

4. Microbiological Methods and Controls

The microbiological methods and controls used to establish,
validate, and audit the efficacy of the cycle should be described.

5. Monitoring Stability
The program for monitoring the stability of packaging and the
integrity of the container-closure system barrier over the
claimed shelf life should be described.

Chapter Ⅳ. ASEPTIC FILL MANUFACTURING

PROCESSES
Section 1. Buildings and Facilities
A brief description of the manufacturing building and facilities
should be provided. The following information should be
included:

1. Floor Plan
(1)A floor plan of the areas holding the aseptic filling facilities
including preparation and holding areas, filtering and filling
areas, and gowning rooms should be included.
(2) Class 100, Class 10,000, Class 100,000 should be identified.
(3)Isolators or barrier systems should be identified.

2. Location of Equipment
(1)The placement of all critical equipment, including, but not
limited to, laminar flow hoods, autoclaves, lyophilizers, and
filling heads, should be identified.
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(2) Equipment within barrier or isolation systems should be
noted.

Section 2. Overall Manufacturing Operation

The overall manufacturing operation including, for example,
material flow, filling, capping, and aseptic assembly, should be
described.
The normal flow (movement) of product and components from
formulation to finished dosage form should be identified and
indicated on the floor plan described above.
The following information should be considered when
describing the overall manufacturing operation:

1. Drug Product Solution Filtration

(1)The specific bulk product solution filtration processes,
including tandem filter units, prefilter, and bacterial retentive
filters, should be described.
(2)A summary should be provided containing information and
data concerning the validation of the retention of microbes and
compatibility of the filter used for the specific product. Any
effects of the filter on the product formulation should be
described, e.g., adsorption of preservatives or active drug
substance, or extractable

2. Specifications Concerning Holding Periods

GMP Regulations requires, in part, when appropriate, the
establishment of time limits for completing each phase of
production to ensure the quality of the drug product. Therefore,
specifications concerning any holding periods between the
compounding of the bulk drug product and its filling into final
containers should be provided. These specifications should
include, for example, holding tanks, times, temperatures, and
conditions of storage. Procedures used to protect
microbiological quality of the bulk drug during these holding
periods should be indicated. Maintenance of the microbiological
quality during holding periods may need verification.
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3. Critical Operations
The critical operations that expose product or product contact
surfaces to the environment (such as transfer of sterilized
containers or closures to the aseptic filling areas) should be
described. Any barrier or isolation systems should be described.

Section 3. Sterilization and Depyrogenation of

Containers, Closures, Equipment, and Components

The sterilization and depyrogenation processes used for

containers, closures, equipment, components, and barrier
systems should be described.
A description of the validation of these processes should be
provided including, where applicable, heat distribution and
penetration summaries, biological challenge studies
(microbiological indicators and endotoxin) and routine
monitoring procedures.
Validation information for sterilization processes other than
moist heat should also be included.
Methods and data (including controls) demonstrating
distribution and penetration of the sterilant and microbiological
efficacy of each process should be submitted. (See Chapter II)

1. Bulk Drug Solution Components That are Sterilized

Separately
If the bulk drug solution is aseptically formulated from
components that are sterilized separately, information and data
concerning the validation of each of these separate sterilization
processes should be provided.

2. Sterilization Information in the Batch Records

The completed batch record supplied with the application should
identify the validated processes to be used for sterilization and
depyrogenation of any container-closure components. This
information may be included in the batch record by reference to
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the validation protocol or SOP.

Section 4. Procedures and Specifications for Media Fills

The procedures and specifications used for media fills should be
described.
Summaries of results for validation using the same container-
closure system and filling process that is to be used for the
product should be described.
Any procedural differences between the media fill and the
production process should be indicated.
A summary of recent media fill results, including failures,
should be provided.
These data should be obtained using the same filling line(s) that
are to be used for the drug product.
The following are recommended to be included with the data
summary for each media fill run described
1. The filling room
Identify the aseptic filling area used and relate this to the floor
plan provided in Chapter IV Section 1.1 of this guidance.

2. Container-closure type and size

3. Volume of medium used in each container

4. Type of medium used

5. Number of units filled

6. Number of units incubated

7. Number of units positive

8. Incubation parameters
The incubation time and temperature for each group of units
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incubated and specifications for any group of units subjected to
two (or more) different temperatures should be specified.

9. Date of each media fill

10. Simulations
The procedures used to simulate any steps of a normal
production fill should be described. This might include, for
example, slower line speed, personnel shift changes, equipment
failure and repair, mock lyophilization and substitution of vial
headspace gas.

11. Microbiological monitoring

The microbiological monitoring data obtained during the media
fill runs should be provided (see Chapter 4 section 6 of this
guidance).

12. Process parameters

The parameters used for production filling and for media fills
(e.g., line speed, fill volume, number of containers filled, or
duration of fill) should be compared.

Section 5 Actions Concerning Product When Media Fills Fail

The disposition of product made before and after a failed media
fill should be described. The description should include details
of investigations, reviews, and how decisions are made to reject
or release product.

Section 6 Microbiological Monitoring of the Environment

The microbiological monitoring program used during routine
production and media fills should be described. The frequency
of monitoring, type of monitoring, sites monitored, alert and
action level specifications and precise descriptions of the actions
taken when specifications are exceeded should be included.
1. Microbiological Methods
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The microbiological materials and methods used in the
environmental monitoring program should be described.
Methods may include sample collection, transport, and
neutralization of sanitizers, incubation, and calculation of
results.
The following are sources of microbial contamination and their
monitoring that should be addressed, including specifications:
(1)Airborne microorganisms
(2)Microorganisms on inanimate surfaces
(3) Microorganisms on personnel
(4) Water systems
(5)Product component bioburden

2. Yeasts, Molds, and Anaerobic Microorganisms

A description of periodic or routine monitoring methods used
for yeasts, molds, and anaerobes should be provided.

3. Exceeded Limits
A description of the actions taken when specifications are
exceeded should be provided.

Section 7 Container-Closure and Package Integrity

1.The methods and results demonstrating the integrity of the
microbiological barrier of the container-closure system should
be summarized. This should include testing for initial validation.
The procedures used for the stability protocol also should be
described. For initial validation of microbiological integrity of
container-closure systems, product sterility testing is not
normally considered sufficient.

2. The sensitivity of the experimental method used for

container-closure integrity testing should be specified and
provided.

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Section 8 Bacterial Endotoxins Test and Method
The bacterial endotoxin test used for the product should be
described, if applicable. This description should include
qualification of the laboratory, inhibition and enhancement
testing and results, determination of noninhibitory concentration
and maximum valid dilution.

Section 9 Sterility Testing Methods and Release Criteria

1. Sterility test methods should be described and should include
the protocol for the selection of representative units during
production.
2. For a drug product represented to be a drug recognized in an
official compendium, when test methods differ significantly
from official compendial test methods, a demonstration of the
equivalency to the official compendial method should be
provided.
3. Testing performed within barrier systems should be discussed,
and information concerning validation of the barrier system may
be necessary.

Chapter Ⅴ. MAINTENANCE OF MICROBIOLOGICAL

CONTROL AND QUALITY:

Section 1 Container-Closure Integrity

1.The ability of the container-closure system to maintain the
integrity of its microbial barrier, and, hence, the sterility of a
drug product throughout its shelf life, should be demonstrated.
2.Referencs are made to Chapter II section 5 and Chapter IV
section7 of this guidance. As previously stated, sterility testing
at the initial time point is not considered sufficient to
demonstrate the microbial integrity of a container-closure
system. Documentation of the sensitivity of the
container-closure integrity test should be provided.

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Section 2 Preservative Effectiveness
1.The efficacy of preservative systems inadvertently introduced
during drug product use should be demonstrated at the minimum
concentration specified for drug product release or at the
minimum concentration specified for the end of the expiration
dating period, whichever is less.
2.Since the efficacy of preservative systems is judged by their
effect on microorganisms, microbial challenge assays should be
performed.

3.For purposes of the stability protocol, the stability protocol,

the first three production lots should be tested with a microbial
challenge assay at the beginning and end of the stability period.

4.Chemical assays to monitor the concentration of preservatives

should be performed at all test intervals.

5.For subsequent lots placed on stability, chemical assays may

be adequate to demonstrate the presence of specified
concentrations of preservatives, and such testing should be
carried out according to the approved stability study protocol.

Section 3 Pyrogen or Endotoxin Testing

For drug products purporting to be pyrogen free, it is
recommended that pyrogen or endotoxin tests be carried out at
the beginning and end of the stability period as part of the
approved stability study protocol.

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