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 GUIDANCE FOR INDUSTRY 1
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
     1This guidance has been prepared by the Sterility Technical Committee of th e
Chemistry  Manufacturing  Controls  Coordinating  Committee  of  the  Center  for  Dru g
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 an y
rights for or on any person and does not operate to bind FDA or the industry, it doe s
represent the agencys current thinking  on sterilization process validation documentation.
For  additional  copies  of  this  guidance,  contact  the  Division  of  Communication s
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 v ia Internet via World
Wide Web (WWW) (connect to the FDA Home Page at WWW.FDA.GOV/CDER and go
to the Regulatory Guidance section). 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 F  values, dwell time, run
0
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 -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
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. 
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 10-6 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.
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 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.  (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 container-
closure 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 Preservatives-
Effectiveness."  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 End-
Product 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.