Guidance for Industry

for the Submission Documentation for

Sterilization Process Validation in

Applications for Human and Veterinary

Drug Products

Center for Drug Evaluation and Research (CDER)

Center for Veterinary Medicine (CVM)

November 1994

CMC 2
TABLE OF CONTENTS

I. INTRODUCTION ............................................... 1

A. Purpose ................................................. 1

B. Documenting Sterilization Process Validation ................... 2

C. Remarks ................................................ 2

II. INFORMATION FOR TERMINAL MOIST HEAT STERILIZATION

PROCESSES ................................................. 3

A. Description of the Process and Product ........................ 3

1. The Drug Product and Container-Closure System .......... 3

2. The Sterilization Process .............................. 3

3. The Autoclave Process and Performance Specifications ..... 4

4. Autoclave Loading Patterns ............................ 4

5. Methods and Controls to Monitor Production Cycles ......... 4

6. Requalification of Production Autoclaves .................. 4

7. Reprocessing ....................................... 4

B. Thermal Qualification of the Cycle ............................ 4

1. Heat Distribution and Penetration Studies ................. 4

2. Thermal Monitors .................................... 5

3. The Effects of Loading on Thermal Input .................. 5

4. Information Included in the Batch Record ................ . 5

C. Microbiological Efficacy of the Cycle .......................... 5

1. Identification and Characterization of Bioburden Organisms ... 6

2. Specifications for Bioburden ........................... 6
3. Identification, Resistance, and Stability of Biological

Indicators .......................................... 6

4. The Resistance of the Biological Indicator Relative to That

of Bioburden ........................................ 6

5. Microbiological Challenge Studies ....................... 7

D. Microbiological Monitoring of the Environment ................... 7

E. Container-Closure and Package Integrity ........................ 7

1. Simulation of the Stresses From Processing ................ 7

2. Demonstrate Integrity Following the Maximum Exposure ...... 8

3. Multiple Barriers ...................................... 8

4. The Sensitivity of the Test .............................. 8

5. Integrity Over the Product Shelf Life ...................... 8

F. Bacterial Endotoxins Test and Method .......................... 8

G. Sterility Testing Methods and Release Criteria .................... 8

H. Evidence of Formal, Written Procedures ........................ 9

III. OTHER TERMINAL STERILIZATION PROCESSES .................... 9

A. Ethylene Oxide ............................................ 9

1. Description of the Sterilizer ............................. 9

2. Cycle Parameters .................................... 10

3. Microbiological Methods .............................. 10

4. Stability ............................................ 10

B. Radiation ................................................ 10

1. The Facility and the Process ........................... 10
2. The Packaging of the Product .......................... 10

3. Multiple-Dose Mapping Studies ......................... 10

4. Microbiological Methods and Controls ................... 11

5. Monitoring Stability ................................... 11

IV. INFORMATION FOR ASEPTIC FILL MANUFACTURING PROCESSES

WHICH SHOULD BE INCLUDED IN DRUG APPLICATIONS ........... 11

A. Buildings and Facilities ..................................... 11

1. Floor Plan .......................................... 11

2. Location of equipment ................................ 11

B. Overall Manufacturing Operation ............................. 11

1. Drug Product Solution Filtration ......................... 12

2. Specifications Concerning Holding Periods ................ 12

3. Critical Operations ................................... 12

C. Sterilization and Depyrogenation of Containers, Closures,

Equipment, and Components ................................ 12

1. Bulk Drug Solution Components That are Sterilized

Separately ......................................... 13

2. Sterilization Information in the Batch Records .............. 13

D. Procedures and Specifications for Media Fills ................... 13

E. Actions Concerning Product When Media Fills Fail ............... 14

F. Microbiological monitoring of the environment ................... 15

1. Microbiological Methods ............................... 15

2. Yeasts, Molds, and Anaerobic Microorganisms ............. 15

3. Exceeded Limits ..................................... 15
G. Container-Closure and Package Integrity ....................... 15

H. Sterility Testing Methods and Release Criteria ................... 16

I. Bacterial Endotoxins Test and Method ......................... 16

J. Evidence of Formal Written Procedures ........................ 16

V. MAINTENANCE OF MICROBIOLOGICAL CONTROL AND QUALITY: 

STABILITY CONSIDERATIONS .................................. 16

A. Container-Closure Integrity .................................. 16

B. Preservative Effectiveness .................................. 17

C. Pyrogen or Endotoxin Testing ............................... 17

VI. ADDITIONAL INFORMATION .................................... 17
 This guidance has been prepared by the Sterility Technical Committee of the

1

Chemistry Manufacturing Controls Coordinating Committee of the Center for Drug

Evaluation and Research (CDER), and the Center for Veterinary Medicine (CVM), at the

Food and Drug Administration. Although this guidance does not create or confer any

rights for or on any person and does not operate to bind FDA or the industry, it does

represent the agency’s current thinking on sterilization process validation documentation.

For additional copies of this guidance, contact the Division of Communications

Management, HFD-210, CDER, FDA, 5600 Fishers Lane, Rockville, MD 20857 (Phone:

301-594-1012) Send one self-addressed adhesive label to assist the office in processing

your request. An electronic version of this guidance is also available via Internet via World

Wide Web (WWW) (connect to the FDA Home Page at WWW.FDA.GOV/CDER and go

to the “Regulatory Guidance” section).

GUIDANCE FOR INDUSTRY1

FOR THE SUBMISSION OF 

DOCUMENTATION FOR STERILIZATION PROCESS

VALIDATION IN APPLICATIONS FOR HUMAN AND

VETERINARY DRUG PRODUCTS

I. INTRODUCTION

A. Purpose

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 for both human and veterinary drugs. The

recommendations in the guidance apply to applications for sterile drug

products (new drug applications, new animal drug applications,

abbreviated new drug applications, abbreviated antibiotic applications,

and abbreviated new animal drug applications). These recommendations

also apply to previously approved applications when supplements

associated with the sterile processing of approved drugs are submitted. 

Information and data in support of sterility assurance may also be

necessary in investigational new drug and investigational new animal

drug applications.

In the FEDERAL REGISTER of October 11, 1991 (56 FR 51354), the

agency published a proposed rule entitled "Use of Aseptic Processing

and Terminal Sterilization in the Preparation of Sterile Pharmaceuticals
for Human and Veterinary Use." This guidance is not a substitution for or

a supplement to that proposed rule. Regardless of whether the applicant

uses terminal sterilization or aseptic processing to manufacture a drug

product that is purported to be sterile, certain information about the

validation of that process should be submitted for both of those types of

sterilization.

B. Documenting Sterilization Process Validation

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. Data

derived from experiments and control procedures allow conclusions to be

drawn about the probability of nonsterile product units (sterility assurance

level). Based on the scientific validity of the protocols and methods, as

well as on the scientific validity of the results and conclusions, the agency

concludes that the efficacy of the sterilization process is validated. 

Whether a drug 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.

The Center for Drug Evaluation and Research's (CDER's) and the Center

for Veterinary Medicine's (CVM's) review of the validation of the

sterilization process consists of a scientific evaluation of the studies

submitted in the applications. This review is conducted by FDA's review

staff, and is part of a cooperative effort between the review staff,

compliance staff, and field investigators to ensure the overall state of

control of the sterile processing of human and veterinary drug products. 

Information and data in support of sterility assurance may be provided

directly to the application or by specific reference to a drug master file

(DMF), a veterinary master file (VMF), or another application. Letters of

authorization to refer to the referenced files should be included.

C. Remarks

This guidance is intended to provide recommendations for the types of

information applicants should include in human and animal drug

applications. Regulatory requirements for the submission of information

and data in various applications are specified in the sections listed below:
1. Human Drugs:

Investigational new drug applications 21 CFR 312.23(a)(7)

New drug applications 21 CFR 314.50

Abbreviated new drug and abbreviated

antibiotic drug applications 21 CFR 314.94 and 314.50

Supplements to NDA's and ANDA's 21 CFR 314.70

2. Animal Drugs:

Investigational new animal drug applications 21 CFR Part 511

New animal drug applications 21 CFR 514.1

Supplements to NADA's 21 CFR 514.8

II. INFORMATION FOR TERMINAL MOIST HEAT STERILIZATION

PROCESSES

The following types of information should be submitted in support of sterility

assurance for products produced using terminal moist heat sterilization. 

Although the following outline directly addresses moist heat processes, the

same types of information would generally pertain to other terminal sterilization

processes (e.g., ethylene oxide or radiation). (See section III of this guidance.) 

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. (See also

sections II.B. and II.C. of this guidance.) 
3. 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 o

production sterilization, including manufacturer and model.

4. Autoclave Loading Patterns

A description of representative autoclave loading patterns should

be provided.

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. 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."

B. 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.

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 F0 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.

4. 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.

C. Microbiological Efficacy of the Cycle

Validation studies that demonstrate the efficacy (lethality) of the

production cycle should be provided. A sterility assurance of 10 or -6

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

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 heatresistant bioburden organisms. 

2. 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. 

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.

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. 
5. Microbiological Challenge Studies

Microbiological validation studies should be submitted that

demonstrate the efficacy of the minimum cycle to provide a sterility

assurance of 10 or better to the product under the most difficult to -6

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.

D. Microbiological Monitoring of the Environment

Section 211.160 of the Code of Federal Regulations requires, in part, 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 product 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.

E. Container-Closure and Package Integrity

An applicant should provide scientific validation studies (and data) in

support of the microbial integrity of the drug packaging components. The

following types of information should be included:

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.

2. Demonstrate Integrity Following the Maximum Exposure

Container-closure integrity should be demonstrated on product
units that have been exposed to the maximum sterilization

cycle(s). 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 containerclosure 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. (See section V.A.

of this guidance.)

F. 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 testing and results, determination of noninhibitory

concentration and maximum valid dilution. For further information see the

agency guidance entitled "Guideline on Validation of the Limulus

Amebocyte Lysate Test As An End-Product Endotoxin Test for Human

And Animal Parenteral Drugs, Biological Products, and Medical Devices."

G. Sterility Testing Methods and Release Criteria

Sterility test methods should be described and should include the protocol

for the selection of representative units during production. When test

methods differ significantly from compendial test methods, a

demonstration of the equivalency to the compendial method should be

provided. Testing performed within barrier systems should be described,

and information concerning validation of the barrier system may be

necessary.

H. Evidence of Formal, Written Procedures
Section 211.113(b) of the Code of Federal Regulations requires that

written procedures, designed to prevent microbiological contamination of

drug products purporting to be sterile, be established and followed. Such

procedures should include validation of any sterilization process. 

Therefore, evidence should be provided that there are formal, written

procedures describing the elements listed above and that these

procedures are followed. Such evidence may consist of SOP's, listing of

SOP's, and protocols submitted as part of these elements.

III. OTHER TERMINAL STERILIZATION PROCESSES

Although the information above (sections I.A. through I.G. 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 containerclosure integrity. Specific examples are provided below to demonstrate the

application of these concepts to other sterilization processes.

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, and should be supplied to the chemistry, manufacturing, and controls

section of the application.

A. Ethylene Oxide

1. Description of the Sterilizer

The sterilizer(s) and controlled site(s) for prehumidification and

aeration of the product load should be described.
2. Cycle Parameters

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. 

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.

B. Radiation

1. The Facility and the Process

The radiation facility should be identified. 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 show 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.

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.

IV. INFORMATION FOR ASEPTIC FILL MANUFACTURING PROCESSES

WHICH SHOULD BE INCLUDED IN DRUG APPLICATIONS

The following types of information should be submitted in support of sterility

assurance for products manufactured by aseptic processing.

A. Buildings and Facilities

A brief description of the manufacturing building and facilities should be

provided. The following information should be included:

1. Floor Plan

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. The air cleanliness class

of each area should be identified (e.g., Class 100, Class 10,000,

Class 100,000). Isolators or barrier systems should be identified.

2. Location of Equipment

The placement of all critical equipment, including, but not limited to,

laminar flow hoods, autoclaves, lyophilizers, and filling heads,

should be identified. Equipment within barrier or isolation systems

should be noted.

B. 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

The specific bulk drug product solution filtration processes,

including tandem filter units, prefilters, and bacterial retentive

filters, should be described. 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 extractables).

2. Specifications Concerning Holding Periods

Section 211.111 of the Code of Federal 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.

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.

C. 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. The section of this guidance concerning terminal

sterilization contains information that may be of further assistance.

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 chemistry,

manufacturing, and controls section of 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 the

validation protocol or SOP.

D. Procedures and Specifications for Media Fills

The procedures and specifications used for media fills, and 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. The

microbiological testing method(s) used 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 section IV.A.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

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 section IV.F. 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.

E. 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.

F. 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

The microbiological materials and methods used in the

environmental monitoring program should be described. Methods

may include sample collection, transport, neutralization of

sanitizers, incubation, and calculation of results. The following are

sources of microbial contamination and their monitoring that should

be addressed, including specifications:

a. Airborne microorganisms

b. Microorganisms on inanimate surfaces

c. Microorganisms on personnel

d. Water systems

e. 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.

G. Container-Closure and Package Integrity

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. The

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

H. Sterility Testing Methods and Release Criteria

Sterility test methods should be described and should include the protocol

for the selection of representative units during production. 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. Testing performed within barrier systems

should be discussed, and information concerning validation of the barrier

system may be necessary.

I. Bacterial Endotoxins Test and Method

The bacterial endotoxins 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. For further

information see the agency guidance entitled "Guidance on Validation of

the Limulus Amebocyte Lysate Test As An End-Product Endotoxin Test

for Human And Animal Parenteral Drugs, Biological Products, and

Medical Devices."

J. Evidence of Formal Written Procedures

Evidence should be provided that there are formal, written procedures

describing the above elements and that these procedures are followed. 

Such evidence may consist of SOP's or a listing of SOP's or protocols

submitted as part of the elements listed above.

V. MAINTENANCE OF MICROBIOLOGICAL CONTROL AND QUALITY: 

STABILITY CONSIDERATIONS

A. Container-Closure Integrity

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. Reference is made to sections II.E.

and IV.G. 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.
B. Preservative Effectiveness

The efficacy of preservative systems to control bacteria and fungi

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. Since the efficacy of preservative systems is

judged by their effect on microorganisms, microbial challenge assays

should be performed. The United States Pharmacopeia (USP) provides

a microbial challenge assay under the title "Antimicrobial PreservativesEffectiveness." For purposes of 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. Chemical assays to monitor the

concentration of preservatives should be performed at all test intervals. 

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.

C. 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.

VI. ADDITIONAL INFORMATION

Further information concerning content and format of drug applications is

available in the form of guidances and other publications. The following

documents contain information related to the topics discussed in this guidance:

"Guideline for Submitting Documentation for the Stability of Human Drugs and

Biologics" (CDER).

"Guideline on Validation of the Limulus Amebocyte Lysate Test as an EndProduct Endotoxin Test for Human and Animal Parenteral Drugs, Biological

Products, and Medical Devices" (CDER, CVM, CBER, CDRH).

"Guideline on Sterile Drug Products Produced by Aseptic Processing" (CDER).

"Drug Stability Guideline" (CVM).
Submitted by:

_____________________________

Peter H. Cooney, PhD

Center for Drug Evaluation and Research

______________________________

William G. Marnane

Center for Veterinary Medicine

Approved by CMC CC:

_______________________________ ______________________________

Charles Kumkumian, Ph.D. Roger L. Williams, M.D.