Guidance for Industry 
 
Sterile Drug Products  
Produced by Aseptic Processing   
Current Good Manufacturing Practice 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
Office of Regulatory Affairs (ORA) 
 
September 2004 
Pharmaceutical CGMPs 
 
   
 
Guidance for Industry  
 Sterile Drug Products  
Produced by Aseptic Processing  
Current Good Manufacturing Practice 
 
 
 
 
Additional copies are available from: 
Office of Training and Communication 
Division of Drug Information, HFD-240 
Center for Drug Evaluation and Research 
Food and Drug Administration 
5600 Fishers Lane 
Rockville, MD  20857 
(Tel) 301-827-4573 
 http://www.fda.gov/cder/guidance/index.htm 
 
or 
 
 Office of Communication, Training and  
Manufacturers Assistance, HFM-40  
Center for Biologics Evaluation and Research 
 Food and Drug Administration 
1401 Rockville Pike, Rockville, MD 20852-1448  
 http://www.fda.gov/cber/guidelines.htm.   
(Tel) Voice Information System at 800-835-4709 or 301-827-1800 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
Office of Regulatory affairs (ORA) 
 
September 2004 
Pharmaceutical CGMPs 
 
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
I.  INTRODUCTION.............................................................................................................1 
II.  BACKGROUND...............................................................................................................2 
A.  Regulatory Framework.................................................................................................................2 
B.  Technical Framework....................................................................................................................2 
III.  SCOPE...............................................................................................................................3 
IV.  BUILDINGS AND FACILITIES....................................................................................4 
A.  Critical Area  Class 100 (ISO 5).................................................................................................5 
B.   Supporting Clean Areas................................................................................................................7 
C.   Clean Area Separation..................................................................................................................7 
D.  Air Filtration..................................................................................................................................8 
1. Membrane........................................................................................................................................8 
2. High-Efficiency Particulate Air (HEPA).........................................................................................8 
E.  Design............................................................................................................................................10 
V.  PERSONNEL TRAINING, QUALIFICATION, & MONITORING........................12 
A.  Personnel.......................................................................................................................................13 
B.  Laboratory Personnel..................................................................................................................15 
C.  Monitoring Program....................................................................................................................15 
VI. COMPONENTS AND CONTAINER/CLOSURES..........................................................15 
A.   Components..................................................................................................................................16 
B.   Containers/Closures.....................................................................................................................17 
1. Preparation....................................................................................................................................17 
2.  Inspection of Container Closure System........................................................................................18 
VII. ENDOTOXIN CONTROL.................................................................................................19 
VIII. TIME LIMITATIONS......................................................................................................20 
IX.  VALIDATION OF ASEPTIC PROCESSING AND STERILIZATION..................20 
A.   Process Simulations.....................................................................................................................20 
1.  Study Design.................................................................................................................................21 
2. Frequency and Number of Runs....................................................................................................22 
3. Duration of Runs............................................................................................................................22 
4. Size of Runs....................................................................................................................................23 
5. Line Speed......................................................................................................................................23 
6. Environmental Conditions.............................................................................................................24 
7.  Media............................................................................................................................................24 
8. Incubation and Examination of Media-Filled Units......................................................................24 
9. Interpretation of Test Results.........................................................................................................26 
B.   Filtration Efficacy........................................................................................................................27 
  Contains Nonbinding Recommendations 
C.   Sterilization of Equipment, Containers, and Closures.............................................................28 
1. Qualification and Validation.........................................................................................................29 
2. Equipment Controls and Instrument Calibration..........................................................................30 
X.  LABORATORY CONTROLS......................................................................................31 
A.    Environmental Monitoring.........................................................................................................32 
1.   General Written Program.............................................................................................................32 
2. Establishing Levels and a Trending Program...............................................................................33 
3. Disinfection Efficacy......................................................................................................................34 
4.  Monitoring Methods......................................................................................................................34 
B.   Microbiological Media and Identification.................................................................................35 
C.   Prefiltration Bioburden...............................................................................................................36 
D.   Alternate Microbiological Test Methods...................................................................................36 
E.   Particle Monitoring......................................................................................................................36 
XI.  STERILITY TESTING..................................................................................................37 
A.   Microbiological Laboratory Controls........................................................................................38 
B.   Sampling and Incubation............................................................................................................38 
C.   Investigation of Sterility Positives..............................................................................................39 
XII.  BATCH RECORD REVIEW: PROCESS CONTROL DOCUMENTATION........42 
APPENDIX 1:  ASEPTIC PROCESSING ISOLATORS.......................................................44 
APPENDIX 2:  BLOW-FILL- SEAL TECHNOLOGY..........................................................49 
APPENDIX 3: PROCESSING PRIOR TO FILLING AND SEALING OPERATIONS....52 
REFERENCES............................................................................................................................54 
RELEVANT GUIDANCE DOCUMENTS...............................................................................55 
GLOSSARY.................................................................................................................................56 
   
Contains Nonbinding Recommendations 
 
1
Guidance for Industry  
Sterile Drug Products Produced by  
 
 
Aseptic Processing  Current Good Manufacturing Practice
  
 
 
 
 
This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic.  It 
does not create or confer any rights for or on any person and does not operate to bind FDA or the public.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate 
number listed on the title page of this guidance. 
 
 
 
 
I.  INTRODUCTION 
 
This guidance is intended to help manufacturers meet the requirements in the Agency's current 
good manufacturing practice (CGMP) regulations (2l CFR parts 210 and 211) when 
manufacturing sterile drug and biological products using aseptic processing.  This guidance 
replaces the 1987 Industry Guideline on Sterile Drug Products Produced by Aseptic Processing 
(Aseptic Processing Guideline).  This revision updates and clarifies the 1987 guidance.  
 
For sterile drug products subject to a new or abbreviated drug application (NDA or ANDA) or a 
biologic license application (BLA), this guidance document should be read in conjunction with 
the guidance on the content of sterile drug applications entitled Guideline for the Submission of 
Documentation for Sterilization Process Validation in Applications for Human and Veterinary 
Drug Products (Submission Guidance).  The Submission Guidance describes the types of 
information and data that should be included in drug applications to demonstrate the efficacy of a 
manufacturer's sterilization process. This guidance compliments the Submission Guidance by 
describing procedures and practices that will help enable a sterile drug manufacturing facility to 
meet CGMP requirements relating, for example, to facility design, equipment suitability, process 
validation, and quality control. 
 
FDA's guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the Agency's current thinking on a topic and should 
be viewed only as recommendations, unless specific regulatory or statutory requirements are 
cited.  The use of the word should in Agency guidances means that something is suggested or 
recommended, but not required. 
                                                 
1 This guidance was developed by the Office of Compliance in the Center for Drug Evaluation and Research 
(CDER) in cooperation with the Center for Biologics Evaluation and Research (CBER) and the Office of Regulatory 
Affairs (ORA). 
  1  
Contains Nonbinding Recommendations 
 
 
The text boxes included in this guidance include specific sections of parts 210 and 211 of the 
Code of Federal Regulations (CFR), which address current good manufacturing practice for 
drugs.  The intent of including these quotes in the text boxes is to aid the reader by providing a 
portion of an applicable regulation being addressed in the guidance.  The quotes included in the 
text boxes are not intended to be exhaustive.  Readers of this document should reference the 
complete CFR to ensure that they have complied, in full, with all relevant sections of the 
regulations. 
 
 
II.  BACKGROUND  
 
This section describes briefly both the regulatory and technical reasons why the Agency is 
developing this guidance document.  
 
A.  Regulatory Framework 
 
This guidance pertains to current good manufacturing practice (CGMP) regulations (21 CFR 
parts 210 and 211) when manufacturing sterile drug and biological products using aseptic 
processing.  Although the focus of this guidance is on CGMPs in 21 CFR 210 and 211, 
supplementary requirements for biological products are in 21 CFR 600-680.  For biological 
products regulated under 21 CFR parts 600 through 680,  210.2(a) and 211.1(b) provide that 
where it is impossible to comply with the applicable regulations in both parts 600 through 680 
and parts 210 and 211, the regulation specifically applicable to the drug product in question shall 
supercede the more general regulations. 
 
B.  Technical Framework 
 
There are basic differences between the production of sterile drug products using aseptic 
processing and production using terminal sterilization.   
 
Terminal sterilization usually involves filling and sealing product containers under high-quality 
environmental conditions. Products are filled and sealed in this type of environment to minimize 
the microbial and particulate content of the in-process product and to help ensure that the 
subsequent sterilization process is successful. In most cases, the product, container, and closure 
have low bioburden, but they are not sterile. The product in its final container is then subjected to 
a sterilization process such as heat or irradiation.    
 
In an aseptic process, the drug product, container, and closure are first subjected to sterilization 
methods separately, as appropriate, and then brought together.2  Because there is no process to 
sterilize the product in its final container, it is critical that containers be filled and sealed in an 
extremely high-quality environment.  Aseptic processing involves more variables than terminal 
sterilization. Before aseptic assembly into a final product, the individual parts of the final product 
                                                 
2 Due to their nature, certain products are aseptically processed at an earlier stage in the process, or in their entirety.  
Cellular therapy products are an example. All components and excipients for these products are rendered sterile, and 
release of the final product is contingent on determination of sterility.  See Appendix III. 
 
  2  
Contains Nonbinding Recommendations 
 
are generally subjected to various sterilization processes.  For example, glass containers are 
subjected to dry heat; rubber closures are subjected to moist heat; and liquid dosage forms are 
subjected to filtration.  Each of these manufacturing processes requires validation and control.  
Each process could introduce an error that ultimately could lead to the distribution of a 
contaminated product.  Any manual or mechanical manipulation of the sterilized drug, 
components, containers, or closures prior to or during aseptic assembly poses the risk of 
contamination and thus necessitates careful control.  A terminally sterilized drug product, on the 
other hand, undergoes final sterilization in a sealed container, thus limiting the possibility of 
error.3 
 
Sterile drug manufacturers should have a keen awareness of the public health implications of 
distributing a nonsterile product.  Poor CGMP conditions at a manufacturing facility can 
ultimately pose a life-threatening health risk to a patient.   
 
 
III.  SCOPE 
 
This guidance document discusses selected issues and does not address all aspects of aseptic 
processing.  For example, the guidance addresses primarily finished drug product CGMP issues 
while only limited information is provided regarding upstream bulk processing steps.  This 
guidance updates the 1987 Aseptic Processing Guideline primarily with respect to personnel 
qualification, cleanroom design, process design, quality control, environmental monitoring, and 
review of production records.  The use of isolators for aseptic processing is also discussed. 
 
Although this guidance document discusses CGMP issues relating to the sterilization of 
components, containers, and closures, terminal sterilization of drug products is not addressed.  It 
is a well-accepted principle that sterile drugs should be manufactured using aseptic processing 
only when terminal sterilization is not feasible.  However, some final packaging may afford 
some unique and substantial advantage (e.g., some dual-chamber syringes) that would not be 
possible if terminal sterilization were employed.  In such cases, a manufacturer can explore the 
option of adding adjunct processing steps to increase the level of sterility assurance. 
 
A list of references that may be of value to the reader is included at the conclusion of this 
document. 
                                                 
3 Nearly all drugs recalled due to nonsterility or lack of sterility assurance in the period spanning 1980-2000 were 
produced via aseptic processing. 
  3  
Contains Nonbinding Recommendations 
 
 
IV.  BUILDINGS AND FACILITIES 
 
  
21 CFR 211.42(b) states, in part, that The flow of components, drug product containers, closures, labeling, 
in-process materials, and drug products through the building or buildings shall be designed to prevent 
contamination. 
 
21 CFR 211.42(c) states, in part, that Operations shall be performed within specifically defined areas of 
adequate size.  There shall be separate or defined areas or such other control systems for the firms operations 
as are necessary to prevent contamination or mixups during the course of the following procedures: * * * 
(10)  Aseptic processing, which includes as appropriate:  (i)  Floors, walls, and ceilings of smooth, hard 
surfaces that are easily cleanable; (ii) Temperature and humidity controls;  (iii)  An air supply filtered 
through high-efficiency particulate air filters under positive pressure, regardless of whether flow is laminar or 
nonlaminar; (iv) A system for monitoring environmental conditions;   (v) A system for cleaning and 
disinfecting the room and equipment to produce aseptic conditions; (vi) A system for maintaining any 
equipment used to control the aseptic conditions. 
  
21 CFR 211.46(b) states that Equipment for adequate control over air pressure, micro-organisms, dust, 
humidity, and temperature shall be provided when appropriate for the manufacture, processing, packing, or 
holding of a drug product.   
 
21 CFR 211.46(c) states, in part, that Air filtration systems, including prefilters and particulate matter air 
filters, shall be used when appropriate on air supplies to production areas * * *. 
 
 
21 CFR 211.63 states that Equipment used in the manufacture, processing, packing, or holding of a drug 
product shall be of appropriate design, adequate size, and suitably located to facilitate operations for its 
intended use and for its cleaning and maintenance.   
 
21 CFR 211.65(a) states that Equipment shall be constructed so that surfaces that contact components, in-
process materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, 
identity, strength, quality, or purity of the drug product beyond the official or other established 
requirements. 
 
21 CFR 211.67(a) states that Equipment and utensils shall be cleaned, maintained, and sanitized at 
appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, 
quality, or purity of the drug product beyond the official or other established requirements. 
 
21 CFR 211.113(b) states that Appropriate written procedures, designed to prevent microbiological 
contamination of drug products purporting to be sterile, shall be established and followed.  Such procedures 
shall include validation of any sterilization process. 
 
  
 
 
As provided for in the regulations, separate or defined areas of operation in an aseptic processing 
facility should be appropriately controlled to attain different degrees of air quality depending on 
the nature of the operation.  Design of a given area involves satisfying microbiological and 
particle criteria as defined by the equipment, components, and products exposed, as well as the 
operational activities conducted in the area.  
 
  4  
Contains Nonbinding Recommendations 
 
Clean area control parameters should be supported by microbiological and particle data obtained 
during qualification studies.  Initial cleanroom qualification includes, in part, an assessment of 
air quality under as-built, static conditions.  It is important for area qualification and 
classification to place most emphasis on data generated under dynamic conditions (i.e., with 
personnel present, equipment in place, and operations ongoing).  An adequate aseptic processing 
facility monitoring program also will assess conformance with specified clean area 
classifications under dynamic conditions on a routine basis. 
 
The following table summarizes clean area air classifications and recommended action levels of 
microbiological quality (Ref. 1). 
 
 
TABLE 1- Air Classificationsa  
 
 Clean Area  ISO   > 0.5 m  Microbiological  Microbiological Settling 
Classification  Designationb  particles/m3  Active Air Action  Plates Action Levelsc,d 
(0.5 um particles/ft3)  Levelsc (cfu/m3 )  (diam. 90mm; cfu/4 hours) 
100  5  3,520  1e  1e 
1000  6  35,200  7  3 
10,000  7  352,000  10  5 
100,000  8  3,520,000  100  50 
 
 
a- All classifications based on data measured in the vicinity of exposed materials/articles during periods of activity.  
b- ISO 14644-1 designations provide uniform particle concentration values for cleanrooms in multiple industries.  An ISO 5 particle 
concentration is equal to Class 100 and approximately equals EU Grade A. 
c- Values represent recommended levels of environmental quality.  You may find it appropriate to establish alternate microbiological action 
levels due to the nature of the operation or method of analysis. 
d- The additional use of settling plates is optional. 
e- Samples from Class 100 (ISO 5) environments should normally yield no microbiological contaminants. 
 
Two clean areas are of particular importance to sterile drug product quality: the critical area and 
the supporting clean areas associated with it. 
 
A.  Critical Area  Class 100 (ISO 5) 
 
A critical area is one in which the sterilized drug product, containers, and closures are exposed to 
environmental conditions that must be designed to maintain product sterility ( 211.42(c)(10)).  
Activities conducted in such areas include manipulations (e.g., aseptic connections, sterile 
ingredient additions) of sterile materials prior to and during filling and closing operations.   
 
This area is critical because an exposed product is vulnerable to contamination and will not be 
subsequently sterilized in its immediate container.  To maintain product sterility, it is essential 
that the environment in which aseptic operations (e.g., equipment setup, filling) are conducted be 
controlled and maintained at an appropriate quality.  One aspect of environmental quality is the 
particle content of the air.  Particles are significant because they can enter a product as an 
extraneous contaminant, and can also contaminate it biologically by acting as a vehicle for 
microorganisms (Ref. 2).  Appropriately designed air handling systems minimize particle content 
of a critical area.   
 
Air in the immediate proximity of exposed sterilized containers/closures and filling/closing 
operations would be of appropriate particle quality when it has a per-cubic-meter particle count 
  5  
Contains Nonbinding Recommendations 
 
of no more than 3520 in a size range of 0.5 m and larger when counted at representative 
locations normally not more than 1 foot away from the work site, within the airflow, and during 
filling/closing operations.  This level of air cleanliness is also known as Class 100 (ISO 5).    
 
We recommend that measurements to confirm air cleanliness in critical areas be taken at sites 
where there is most potential risk to the exposed sterilized product, containers, and closures.  The 
particle counting probe should be placed in an orientation demonstrated to obtain a meaningful 
sample.  Regular monitoring should be performed during each production shift.  We recommend 
conducting nonviable particle monitoring with a remote counting system.  These systems are 
capable of collecting more comprehensive data and are generally less invasive than portable 
particle counters.   See Section X.E. for additional guidance on particle monitoring. 
 
Some operations can generate high levels of product (e.g., powder) particles that, by their nature, 
do not pose a risk of product contamination.  It may not, in these cases, be feasible to measure air 
quality within the one-foot distance and still differentiate background levels of particles from air 
contaminants.  In these instances, air can be sampled in a manner that, to the extent possible, 
characterizes the true level of extrinsic particle contamination to which the product is exposed.  
Initial qualification of the area under dynamic conditions without the actual filling function 
provides some baseline information on the non-product particle generation of the operation.  
 
HEPA-filtered4 air should be supplied in critical areas at a velocity sufficient to sweep particles 
away from the filling/closing area and maintain unidirectional airflow during operations.  The 
velocity parameters established for each processing line should be justified and appropriate to 
maintain unidirectional airflow and air quality under dynamic conditions within the critical area 
(Ref. 3).5   
 
Proper design and control prevents turbulence and stagnant air in the critical area.  Once relevant 
parameters are established, it is crucial that airflow patterns be evaluated for turbulence or eddy 
currents that can act as a channel or reservoir for air contaminants (e.g., from an adjoining lower 
classified area).  In situ air pattern analysis should be conducted at the critical area to 
demonstrate unidirectional airflow and sweeping action over and away from the product under 
dynamic conditions.  The studies should be well documented with written conclusions, and 
include evaluation of the impact of aseptic manipulations (e.g., interventions) and equipment 
design.  Videotape or other recording mechanisms have been found to be useful aides in 
assessing airflow initially as well as facilitating evaluation of subsequent equipment 
configuration changes.  It is important to note that even successfully qualified systems can be 
compromised by poor operational, maintenance, or personnel practices. 
 
Air monitoring samples of critical areas should normally yield no microbiological contaminants.  
We recommend affording appropriate investigative attention to contamination occurrences in this 
environment. 
 
                                                 
4High Efficiency Particulate Air filter 
 
5 A velocity of 0.45 meters/second (90 feet per minute) has generally been established, with a range of plus or minus 
20 percent around the setpoint.  Higher velocities may be appropriate in operations generating high levels of 
particulates. 
  6  
Contains Nonbinding Recommendations 
 
B.   Supporting Clean Areas 
 
Supporting clean areas can have various classifications and functions.  Many support areas 
function as zones in which nonsterile components, formulated products, in-process materials, 
equipment, and container/closures are prepared, held, or transferred.  These environments are 
soundly designed when they minimize the level of particle contaminants in the final product and 
control the microbiological content (bioburden) of articles and components that are subsequently 
sterilized.   
 
The nature of the activities conducted in a supporting clean area determines its classification.  
FDA recommends that the area immediately adjacent to the aseptic processing line meet, at a 
minimum, Class 10,000 (ISO 7) standards (see Table 1) under dynamic conditions.  
Manufacturers can also classify this area as Class 1,000 (ISO 6) or maintain the entire aseptic 
filling room at Class 100 (ISO 5).  An area classified at a Class 100,000 (ISO 8) air cleanliness 
level is appropriate for less critical activities (e.g., equipment cleaning). 
 
C.   Clean Area Separation  
 
An essential part of contamination prevention is the adequate separation of areas of operation.  
To maintain air quality, it is important to achieve a proper airflow from areas of higher 
cleanliness to adjacent less clean areas.  It is vital for rooms of higher air cleanliness to have a 
substantial positive pressure differential relative to adjacent rooms of lower air cleanliness.  For 
example, a positive pressure differential of at least 10-15 Pascals (Pa)6 should be maintained 
between adjacent rooms of differing classification (with doors closed).  When doors are open, 
outward airflow should be sufficient to minimize ingress of contamination, and it is critical that 
the time a door can remain ajar be strictly controlled (Ref. 4).   
 
In some cases, the aseptic processing room and adjacent cleanrooms have the same 
classification.  Maintaining a pressure differential (with doors closed) between the aseptic 
processing room and these adjacent rooms can provide beneficial separation.  In any facility 
designed with an unclassified room adjacent to the aseptic processing room, a substantial 
overpressure (e.g., at least 12.5 Pa) from the aseptic processing room should be maintained at all 
times to prevent contamination.  If this pressure differential drops below the minimum limit, it is 
important that the environmental quality of the aseptic processing room be restored and 
confirmed. 
 
The Agency recommends that pressure differentials between cleanrooms be monitored 
continuously throughout each shift and frequently recorded.  All alarms should be documented 
and deviations from established limits should be investigated. 
 
Air change rate is another important cleanroom design parameter.  For Class 100,000 (ISO 8) 
supporting rooms, airflow sufficient to achieve at least 20 air changes per hour is typically 
acceptable.  Significantly higher air change rates are normally needed for Class 10,000 and Class 
100 areas. 
 
                                                 
6 Equal to 0.04-0.06 inches of water gauge.   
  7  
Contains Nonbinding Recommendations 
 
A suitable facility monitoring system will rapidly detect atypical changes that can compromise 
the facilitys environment.  An effective system facilitates restoration of operating conditions to 
established, qualified levels before reaching action levels.  For example, pressure differential 
specifications should enable prompt detection (i.e., alarms) of an emerging low pressure problem 
to preclude ingress of unclassified air into a classified room. 
 
D.  Air Filtration  
 
1.  Membrane  
 
A compressed gas should be of appropriate purity (e.g., free from oil) and its microbiological and 
particle quality after filtration should be equal to or better than that of the air in the environment 
into which the gas is introduced.  Compressed gases such as air, nitrogen, and carbon dioxide are 
often used in cleanrooms and are frequently employed in purging or overlaying. 
 
Membrane filters can be used to filter a compressed gas to meet an appropriate high-quality 
standard.  These filters are often used to produce a sterile compressed gas to conduct operations 
involving sterile materials, such as components and equipment.  For example, we recommend 
that sterile membrane filters be used for autoclave air lines, lyophilizer vacuum breaks, and tanks 
containing sterilized materials.  Sterilized holding tanks and any contained liquids should be held 
under positive pressure or appropriately sealed to prevent microbial contamination.  Safeguards 
should be in place to prevent a pressure change that can result in contamination due to back flow 
of nonsterile air or liquid. 
 
Gas filters (including vent filters) should be dry.  Condensate on a gas filter can cause blockage 
during use or allow for the growth of microorganisms.  Use of hydrophobic filters, as well as 
application of heat to these filters where appropriate, prevents problematic moisture residues.  
We recommend that filters that serve as sterile boundaries or supply sterile gases that can affect 
product be integrity tested upon installation and periodically thereafter (e.g., end of use).  
Integrity tests are also recommended after activities that may damage the filter.  Integrity test 
failures should be investigated, and filters should be replaced at appropriate, defined intervals. 
 
2.  High-Efficiency Particulate Air (HEPA)7 
 
HEPA filter integrity should be maintained to ensure aseptic conditions.  Leak testing should be 
performed at installation to detect integrity breaches around the sealing gaskets, through the 
frames, or through various points on the filter media.  Thereafter, leak tests should be performed 
at suitable time intervals for HEPA filters in the aseptic processing facility.  For example, such 
testing should be performed twice a year for the aseptic processing room.  Additional testing may 
be appropriate when air quality is found to be unacceptable, facility renovations might be the 
cause of disturbances to ceiling or wall structures, or as part of an investigation into a media fill 
or drug product sterility failure.  Among the filters that should be leak tested are those installed 
in dry heat depyrogenation tunnels and ovens commonly used to depyrogenate glass vials.  
Where justified, alternate methods can be used to test HEPA filters in the hot zones of these 
tunnels and ovens. 
                                                 
7 The same broad principles can be applied to ULPA filters. 
 
  8  
Contains Nonbinding Recommendations 
 
 
Any aerosol used for challenging a HEPA filter should meet specifications for critical 
physicochemical attributes such as viscosity.  Dioctylphthalate (DOP) and poly-alpha-olefin 
(PAO) are examples of appropriate leak testing aerosols.  Some aerosols are problematic because 
they pose the risk of microbial contamination of the environment being tested.  Accordingly, the 
evaluation of any alternative aerosol involves ensuring it does not promote microbial growth. 
 
There is a major difference between filter leak testing and efficiency testing. An efficiency test is 
a general test used to determine the rating of the filter.8  An intact HEPA filter should be capable 
of retaining at least 99.97 percent of particulates greater than 0.3 m in diameter.   
 
The purpose of performing regularly scheduled leak tests, on the other hand, is to detect leaks 
from the filter media, filter frame, or seal.  The challenge involves use of a polydispersed aerosol 
usually composed of particles with a light-scattering mean droplet diameter in the submicron size 
range,9 including a sufficient number of particles at approximately 0.3 m.  Performing a leak 
test without introducing a sufficient upstream challenge of particles of known size upstream of 
the filter is ineffective for detecting leaks.  It is important to introduce an aerosol upstream of the 
filter in a concentration that is appropriate for the accuracy of the aerosol photometer.  The leak 
test should be done in place, and the filter face scanned on the downstream side with an 
appropriate photometer probe, at a sampling rate of at least one cubic foot per minute.  The 
downstream leakage measured by the probe should then be calculated as a percent of the 
upstream challenge.  An appropriate scan should be conducted on the entire filter face and frame, 
at a position about one to two inches from the face of the filter.  This comprehensive scanning of 
HEPA filters should be fully documented.   
 
A single probe reading equivalent to 0.01 percent of the upstream challenge would be considered 
as indicative of a significant leak and calls for replacement of the HEPA filter or, when 
appropriate, repair in a limited area.  A subsequent confirmatory retest should be performed in 
the area of any repair.  
 
HEPA filter leak testing alone is insufficient to monitor filter performance.  It is important to 
conduct periodic monitoring of filter attributes such as uniformity of velocity across the filter 
(and relative to adjacent filters).  Variations in velocity can cause turbulence that increases the 
possibility of contamination.  Velocities of unidirectional air should be measured 6 inches from 
the filter face and at a defined distance proximal to the work surface for HEPA filters in the 
critical area.  Velocity monitoring at suitable intervals can provide useful data on the critical area 
in which aseptic processing is performed.  The measurements should correlate to the velocity 
range established at the time of in situ air pattern analysis studies.  HEPA filters should be 
replaced when nonuniformity of air velocity across an area of the filter is detected or airflow 
patterns may be adversely affected.  
 
                                                 
8 The efficiency test uses a monodispersed aerosol of 0.3 micron sized particles and assesses filter media.  
Downstream readings represent an average over the entire filter surface.  Efficiency tests are not intended to test for 
filter leaks. 
 
9 Although the mean is normally less than one micron, it is greater than 0.3m. 
  9  
Contains Nonbinding Recommendations 
 
Although contractors often provide these services, drug manufacturers are responsible for 
ensuring that equipment specifications, test methods, and acceptance criteria are defined, and 
that these essential certification activities are conducted satisfactorily. 
 
E.  Design 
 
Note: The design concepts discussed within this section are not intended to be exhaustive.  Other 
appropriate technologies that achieve increased sterility assurance are also encouraged. 
 
Aseptic processes are designed to minimize exposure of sterile articles to the potential 
contamination hazards of the manufacturing operation.  Limiting the duration of exposure of sterile 
product elements, providing the highest possible environmental control, optimizing process flow, 
and designing equipment to prevent entrainment of lower quality air into the Class 100 (ISO 5) 
clean area are essential to achieving high assurance of sterility (Ref. 4). 
 
Both personnel and material flow should be optimized to prevent unnecessary activities that 
could increase the potential for introducing contaminants to exposed product, container-closures, 
or the surrounding environment.  The layout of equipment should provide for ergonomics that 
optimize comfort and movement of operators.  The number of personnel in an aseptic processing 
room should be minimized.  The flow of personnel should be designed to limit the frequency 
with which entries and exits are made to and from an aseptic processing room and, most 
significant, its critical area.  Regarding the latter, the number of transfers into the critical area of 
a traditional cleanroom, or an isolator, should be minimized.  To prevent changes in air currents 
that introduce lower quality air, movement adjacent to the critical area should be appropriately 
restricted.  
 
Any intervention or stoppage during an aseptic process can increase the risk of contamination.  
The design of equipment used in aseptic processing should limit the number and complexity of 
aseptic interventions by personnel.  For example, personnel intervention can be reduced by 
integrating an on-line weight check device, thus eliminating a repeated manual activity within 
the critical area.  Rather than performing an aseptic connection, sterilizing the preassembled 
connection using sterilize-in-place (SIP) technology also can eliminate a significant aseptic 
manipulation.  Automation of other process steps, including the use of technologies such as 
robotics, can further reduce risk to the product. 
 
Products should be transferred under appropriate cleanroom conditions.  For example, 
lyophilization processes include transfer of aseptically filled product in partially sealed 
containers.  To prevent contamination, a partially closed sterile product should be transferred 
only in critical areas.10  Facility design should ensure that the area between a filling line and the 
lyophilizer provide for Class 100 (ISO 5) protection. Transport and loading procedures should 
afford the same protection.  
 
The sterile drug product and its container-closures should be protected by equipment of suitable 
design.  Carefully designed curtains and rigid plastic shields are among the barriers that can be 
                                                 
10 Appropriately designed transfer equipment provides these conditions and can be qualified for this purpose. 
 
  10  
Contains Nonbinding Recommendations 
 
used in appropriate locations to achieve segregation of the aseptic processing line.  Use of an 
isolator system further enhances product protection (see Appendix 1). 
 
Due to the interdependence of the various rooms that make up an aseptic processing facility, it is 
essential to carefully define and control the dynamic interactions permitted between cleanrooms.  
Use of a double-door or integrated sterilizer helps ensure direct product flow, often from a lower 
to a higher classified area.  Airlocks and interlocking doors will facilitate better control of air 
balance throughout the aseptic processing facility.  Airlocks should be installed between the 
aseptic manufacturing area entrance and the adjoining unclassified area.  Other interfaces such as 
personnel transitions or material staging areas are appropriate locations for air locks.  It is critical 
to adequately control material (e.g., in-process supplies, equipment, utensils) as it transfers from 
lesser to higher classified clean areas to prevent the influx of contaminants.  For example, written 
procedures should address how materials are to be introduced into the aseptic processing room to 
ensure that room conditions remain uncompromised.  In this regard, materials should be 
disinfected according to appropriate procedures or, when used in critical areas, rendered sterile 
by a suitable method. 
 
If stoppered vials exit an aseptic processing zone or room prior to capping, appropriate 
assurances should be in place to safeguard the product, such as local protection until completion 
of the crimping step.  Use of devices for on-line detection of improperly seated stoppers can 
provide additional assurance. 
 
Cleanrooms are normally designed as functional units with specific purposes.  The materials of 
construction of cleanrooms ensure ease of cleaning and sanitizing.  Examples of adequate design 
features include seamless and rounded floor to wall junctions as well as readily accessible 
corners.  Floors, walls, and ceilings should be constructed of smooth, hard surfaces that can be 
easily cleaned.  Ceilings and associated HEPA filter banks should be designed to protect sterile 
materials from contamination.  Cleanrooms also should not contain unnecessary equipment, 
fixtures, or materials.   
 
Processing equipment and systems should be equipped with sanitary fittings and valves. With 
rare exceptions, drains are considered inappropriate for classified areas of the aseptic processing 
facility other than Class 100,000 (ISO 8) areas.  It is essential that any drain installed in an 
aseptic processing facility be of suitable design.   
 
Equipment should be appropriately designed ( 211.63) to facilitate ease of sterilization.  It is 
also important to ensure ease of installation to facilitate aseptic setup.  The effect of equipment 
design on the cleanroom environment should be addressed.  Horizontal surfaces or ledges that 
accumulate particles should be avoided.  Equipment should not obstruct airflow and, in critical 
areas, its design should not disturb unidirectional airflow. 
 
Deviation or change control systems should address atypical conditions posed by shutdown of air 
handling systems or other utilities, and the impact of construction activities on facility control.  
Written procedures should address returning a facility to operating conditions following a 
shutdown. 
 
  11  
Contains Nonbinding Recommendations 
 
 
V.  PERSONNEL TRAINING, QUALIFICATION, & MONITORING 
 
21 CFR 211.22(a) states that There shall be a quality control unit that shall have the responsibility and authority to 
approve or reject all components, drug product containers, closures, in-process materials, packaging material, 
labeling, and drug products, and the authority to review production records to assure that no errors have occurred or, 
if errors have occurred, that they have been fully investigated.  The quality control unit shall be responsible for 
approving or rejecting drug products manufactured, processed, packed, or held under contract by another company.
 
21 CFR 211.22(c) states that The quality control unit shall have the responsibility for approving or rejecting all 
procedures or specifications impacting on the identity, strength, quality, and purity of the drug product. 
 
21 CFR 211.25(a) states that Each person engaged in the manufacture, processing, packing, or holding of a drug 
product shall have education, training, and experience, or any combination thereof, to enable that person to perform 
the assigned functions.  Training shall be in the particular operations that the employee performs and in current 
good manufacturing practice (including the current good manufacturing practice regulations in this chapter and 
written procedures required by these regulations) as they relate to the employee's functions. Training in current 
good manufacturing practice shall be conducted by qualified individuals on a continuing basis and with sufficient 
frequency to assure that employees remain familiar with CGMP requirements applicable to them.   
 
21 CFR 211.25(b) states that Each person responsible for supervising the manufacture, processing, packing, or 
holding of a drug product shall have the education, training, and experience, or any combination thereof, to perform 
assigned functions in such a manner as to provide assurance that the drug product has the safety, identity, strength, 
quality, and purity that it purports or is represented to possess.   
 
21 CFR 211.25(c) states that There shall be an adequate number of qualified personnel to perform and supervise 
the manufacture, processing, packing, or holding of each drug product. 
 
21 CFR 211.28(a) states that Personnel engaged in the manufacture, processing, packing, or holding of a drug 
product shall wear clean clothing appropriate for the duties they perform.  Protective apparel, such as head, face, 
hand, and arm coverings, shall be worn as necessary to protect drug products from contamination.   
 
21 CFR 211.28(b) states that Personnel shall practice good sanitation and health habits.   
 
21 CFR 211.28(c) states that Only personnel authorized by supervisory personnel shall enter those areas of the 
buildings and facilities designated as limited-access areas.   
 
21 CFR 211.28(d) states that Any person shown at any time (either by medical examination or supervisory 
observation) to have an apparent illness or open lesions that may adversely affect the safety or quality of drug 
products shall be excluded from direct contact with components, drug product containers, closures, in-process 
materials, and drug products until the condition is corrected or determined by competent medical personnel not to 
jeopardize the safety or quality of drug products.  All personnel shall be instructed to report to supervisory 
personnel any health conditions that may have an adverse effect on drug products. 
 
21 CFR 211.42(c) states, in part, that Operations shall be performed within specifically defined areas of adequate 
size.  There shall be separate or defined areas or such other control systems for the firms operations as are 
necessary to prevent contamination or mixups during the course of the following procedures: * * * (10)  Aseptic 
processing, which includes as appropriate: * * * (iv) A system for monitoring environmental conditions * * *.   
 
21 CFR 211.113(b) states that Appropriate written procedures, designed to prevent microbiological contamination 
of drug products purporting to be sterile, shall be established and followed.  Such procedures shall include 
validation of any sterilization process. 
  12  
Contains Nonbinding Recommendations 
 
 
A.  Personnel 
 
A well-designed, maintained, and operated aseptic process minimizes personnel intervention.  As 
operator activities increase in an aseptic processing operation, the risk to finished product 
sterility also increases. To ensure maintenance of product sterility, it is critical for operators 
involved in aseptic activities to use aseptic technique at all times.  
 
Appropriate training should be conducted before an individual is permitted to enter the aseptic 
manufacturing area.  Fundamental training topics should include aseptic technique, cleanroom 
behavior, microbiology, hygiene, gowning, patient safety hazards posed by a nonsterile drug 
product, and the specific written procedures covering aseptic manufacturing area operations.  
After initial training, personnel should participate regularly in an ongoing training program.  
Supervisory personnel should routinely evaluate each operators conformance to written 
procedures during actual operations.  Similarly, the quality control unit should provide regular 
oversight of adherence to established, written procedures and aseptic technique during 
manufacturing operations.   
 
Some of the techniques aimed at maintaining sterility of sterile items and surfaces include:  
 
  Contact sterile materials only with sterile instruments 
 
Sterile instruments should always be used in the handling of sterilized materials.  
Between uses, sterile instruments should be held under Class 100 (ISO 5) conditions and 
maintained in a manner that prevents contamination (e.g., placed in sterilized containers).  
Instruments should be replaced as necessary throughout an operation.  
 
After initial gowning, sterile gloves should be regularly sanitized or changed, as 
appropriate, to minimize the risk of contamination.  Personnel should not directly contact 
sterile products, containers, closures, or critical surfaces with any part of their gown or 
gloves.  
 
  Move slowly and deliberately   
 
Rapid movements can create unacceptable turbulence in a critical area.  Such movements 
disrupt the unidirectional airflow, presenting a challenge beyond intended cleanroom 
design and control parameters.  The principle of slow, careful movement should be 
followed throughout the cleanroom. 
 
  Keep the entire body out of the path of unidirectional airflow  
 
Unidirectional airflow design is used to protect sterile equipment surfaces, container-
closures, and product.  Disruption of the path of unidirectional flow air in the critical area 
can pose a risk to product sterility.   
 
  Approach a necessary manipulation in a manner that does not compromise sterility of 
the product  
  13  
Contains Nonbinding Recommendations 
 
 
To maintain sterility of nearby sterile materials, a proper aseptic manipulation should be 
approached from the side and not above the product (in vertical unidirectional flow 
operations).  Also, operators should refrain from speaking when in direct proximity to the 
critical area.  
 
  Maintain Proper Gown Control 
 
Prior to and throughout aseptic operations, an operator should not engage in any activity 
that poses an unreasonable contamination risk to the gown. 
 
Only personnel who are qualified and appropriately gowned should be permitted access to the 
aseptic manufacturing area.  The gown should provide a barrier between the body and exposed 
sterilized materials and prevent contamination from particles generated by, and microorganisms 
shed from, the body.  The Agency recommends gowns that are sterilized and nonshedding, and 
cover the skin and hair (face-masks, hoods, beard/moustache covers, protective goggles, and 
elastic gloves are examples of common elements of gowns).  Written procedures should detail 
the methods used to don each gown component in an aseptic manner.  An adequate barrier 
should be created by the overlapping of gown components (e.g., gloves overlapping sleeves).  If 
an element of a gown is found to be torn or defective, it should be changed immediately.  Gloves 
should be sanitized frequently. 
 
There should be an established program to regularly assess or audit conformance of personnel to 
relevant aseptic manufacturing requirements.  An aseptic gowning qualification program should 
assess the ability of a cleanroom operator to maintain the quality of the gown after performance 
of gowning procedures.  We recommend that this assessment include microbiological surface 
sampling of several locations on a gown (e.g., glove fingers, facemask, forearm, chest).  
Sampling sites should be justified.  Following an initial assessment of gowning, periodic 
requalification will provide for the monitoring of various gowning locations over a suitable 
period to ensure consistent acceptability of aseptic gowning techniques.  Annual requalification 
is normally sufficient for those automated operations where personnel involvement is minimized 
and monitoring data indicate environmental control.  For any aseptic processing operation, if 
adverse conditions occur, additional or more frequent requalification could be indicated. 
 
To protect exposed sterilized product, personnel should to maintain gown quality and strictly 
adhere to appropriate aseptic techniques.  Written procedures should adequately address 
circumstances under which personnel should be retrained, requalified, or reassigned to other 
areas. 
  14  
Contains Nonbinding Recommendations 
 
 
B.  Laboratory Personnel 
 
The basic principles of training, aseptic technique, and personnel qualification in aseptic 
manufacturing also are applicable to those performing aseptic sampling and microbiological 
laboratory analyses.  Processes and systems cannot be considered to be in control and 
reproducible if the validity of data produced by the laboratory is in question.   
 
C.  Monitoring Program  
 
Personnel can significantly affect the quality of the environment in which the sterile product is 
processed.  A vigilant and responsive personnel monitoring program should be established.  
Monitoring should be accomplished by obtaining surface samples of each operator's gloves on a 
daily basis, or in association with each lot.  This sampling should be accompanied by an 
appropriate sampling frequency for other strategically selected locations of the gown (Ref. 5).  
The quality control unit should establish a more comprehensive monitoring program for 
operators involved in operations which are especially labor intensive (i.e., those requiring 
repeated or complex aseptic manipulations).   
 
Asepsis is fundamental to an aseptic processing operation.  An ongoing goal for manufacturing 
personnel in the aseptic processing room is to maintain contamination-free gloves and gowns 
throughout operations.  Sanitizing gloves just prior to sampling is inappropriate because it can 
prevent recovery of microorganisms that were present during an aseptic manipulation.  When 
operators exceed established levels or show an adverse trend, an investigation should be 
conducted promptly.  Follow-up actions can include increased sampling, increased observation, 
retraining, gowning requalification, and in certain instances, reassignment of the individual to 
operations outside of the aseptic manufacturing area.  Microbiological trending systems, and 
assessment of the impact of atypical trends, are discussed in more detail under Section X. 
Laboratory Controls. 
 
 
VI. COMPONENTS AND CONTAINER/CLOSURES 
 
 
 
21 CFR 210.3(b)(3) states that Component means any ingredient intended for use in the manufacture of a drug product, 
including those that may not appear in such drug product. 
 
21 CFR 211.80(a) states that There shall be written procedures describing in sufficient detail the receipt, identification, 
storage, handling, sampling, testing, and approval or rejection of components and drug product containers and closures; 
such written procedures shall be followed.  
 
21 CFR 211.80(b) states that Components and drug product containers and closures shall at all times be handled and stored 
in a manner to prevent contamination. 
 
21 CFR 211.84(d) states, in part, that Samples shall be examined and tested as follows: * * *  (6) Each lot of a component, 
drug product container, or closure that is liable to microbiological contamination that is objectionable in view of its 
intended use shall be subjected to microbiological tests before use. 
 
  15  
Contains Nonbinding Recommendations 
 
21 CFR 211.94(c) states that Drug product containers and closures shall be clean and, where indicated by the nature of the 
drug, sterilized and processed to remove pyrogenic properties to assure that they are suitable for their intended use.  
 
21 CFR 211.94(d) states that Standards or specifications, methods of testing, and, where indicated, methods of cleaning, 
sterilizing, and processing to remove pyrogenic properties shall be written and followed for drug product containers and 
closures. 
  
21 CFR 211.113(b) states that Appropriate written procedures, designed to prevent microbiological contamination of drug 
products purporting to be sterile, shall be established and followed.  Such procedures shall include validation of any 
sterilization process. 
 
A.   Components 
 
A drug product produced by aseptic processing can become contaminated through the use of one 
or more components that are contaminated with microorganisms or endotoxins.  Examples of 
components include active ingredients, Water for Injection (WFI), and other excipients.  It is 
important to characterize the microbial content (e.g., bioburden, endotoxin) of each component 
that could be contaminated and establish appropriate acceptance limits.   
 
Endotoxin load data are significant because parenteral products are intended to be nonpyrogenic.  
There should be written procedures and appropriate specifications for acceptance or rejection of 
each lot of components that might contain endotoxins.  Any components failing to meet defined 
endotoxin limits should be rejected.   
 
In aseptic processing, each component is individually sterilized or several components are 
combined, with the resulting mixture sterilized.11  Knowledge of bioburden is important in 
assessing whether a sterilization process is adequate.  Several methods can be suitable for 
sterilizing components (see relevant discussion in Section IX).  A widely used method is 
filtration of a solution formed by dissolving the component(s) in a solvent such as Water For 
Injection, USP.  The solution is passed through a sterilizing membrane or cartridge filter.  Filter 
sterilization is used where the component is soluble and is likely to be adversely affected by heat.  
A variation of this method includes subjecting the filtered solution to aseptic crystallization and 
precipitation (or lyophilization) of the component as a sterile powder.  However, this method 
involves more handling and manipulation and therefore has a higher potential for contamination 
during processing.    
 
Dry heat sterilization is a suitable method for components that are heat stable and insoluble.  
However, conducting carefully designed heat penetration and distribution studies is of particular 
significance for powder sterilization because of the insulating effects of the powder. 
 
Irradiation can be used to sterilize some components.  Studies should be conducted to 
demonstrate that the process is appropriate for the component.   
                                                 
11 See Appendix III for discussion of certain biologic components that are aseptically handled from the start of the 
process. 
  16  
Contains Nonbinding Recommendations 
 
 
B.   Containers/Closures 
 
1.  Preparation  
 
Containers and closures should be rendered sterile and, for parenteral drug products, 
nonpyrogenic.  The process used will depend primarily on the nature of the container and/or 
closure materials.  The validation study for such a process should be adequate to demonstrate its 
ability to render materials sterile and non-pyrogenic.  Written procedures should specify the 
frequency of revalidation of these processes as well as time limits for holding sterile, 
depyrogenated containers and closures. 
 
Pre-sterilization preparation of glass containers usually involves a series of wash and rinse 
cycles. These cycles serve an important role in removing foreign matter.  We recommend use of 
rinse water of high purity so as not to contaminate containers.  For parenteral products, final 
rinse water should meet the specifications of WFI, USP.   
 
The adequacy of the depyrogenation process can be assessed by spiking containers and closures 
with known quantities of endotoxin, followed by measuring endotoxin content after 
depyrogenation.  The challenge studies can generally be performed by directly applying a 
reconstituted endotoxin solution onto the surfaces being tested.  The endotoxin solution should 
then be allowed to air dry.  Positive controls should be used to measure the percentage of 
endotoxin recovery by the test method.  Validation study data should demonstrate that the 
process reduces the endotoxin content by at least 99.9 percent (3 logs) (see Section VII).12 
 
Subjecting glass containers to dry heat generally accomplishes both sterilization and 
depyrogenation.  Validation of dry heat sterilization and depyrogenation should include 
appropriate heat distribution and penetration studies as well as the use of worst-case process 
cycles, container characteristics (e.g., mass), and specific loading configurations to represent 
actual production runs.  See Section IX.C.  Plastic containers used for parenteral products also 
should be non-pyrogenic.  Where applicable, multiple WFI rinses can be effective in removing 
pyrogens from these containers. 
 
Plastic containers can be sterilized with an appropriate gas, irradiation, or other suitable means.   
For gases such as Ethylene Oxide (EtO), certain issues should receive attention.  For example, 
the parameters and limits of the EtO sterilization cycle (e.g., temperature, pressure, humidity, gas 
concentration, exposure time, degassing, aeration, and determination of residuals) should be 
specified and monitored closely.  EtO is an effective surface sterilant and is also used to 
penetrate certain packages with porous overwrapping.  Biological indicators are of special 
importance in demonstrating the effectiveness of EtO and other gas sterilization processes.  We 
recommend that these methods be carefully controlled and validated to evaluate whether 
consistent penetration of the sterilant can be achieved and to minimize residuals.  Residuals from 
EtO processes typically include ethylene oxide as well as its byproducts, and should be within 
specified limits.  
 
                                                 
12 When this level of depyrogenation by dry heat has been successfully validated using endotoxin challenge, a 
sterilization validation using a biological indicator challenge would not be indicated. 
  17  
Contains Nonbinding Recommendations 
 
Rubber closures (e.g., stoppers and syringe plungers) can be cleaned by multiple cycles of 
washing and rinsing prior to final steam or irradiation sterilization.  At minimum, the initial 
rinses for the washing process should employ at least Purified Water, USP, of minimal endotoxin 
content, followed by final rinse(s) with WFI for parenteral products.  Normally, depyrogenation 
can be achieved by multiple rinses of hot WFI.  The time between washing, drying (where 
appropriate), and sterilizing should be minimized because residual moisture on the stoppers can 
support microbial growth and the generation of endotoxins.  Because rubber is a poor conductor 
of heat, extra attention is indicated in the validation of processes that use heat with respect to its 
penetration into the rubber stopper load (See Section IX.C).  Validation data from the washing 
procedure should demonstrate successful endotoxin removal from rubber materials. 
 
A potential source of contamination is the siliconization of rubber stoppers.  Silicone used in the 
preparation of rubber stoppers should meet appropriate quality control criteria and not have an 
adverse effect on the safety, quality, or purity of the drug product.  
 
Contract facilities that perform sterilization and/or depyrogenation of containers and closures are 
subject to the same CGMP requirements as those established for in-house processing.  The 
finished dosage form manufacturer should review and assess the contractor's validation protocol 
and final validation report.  In accord with 211.84(d)(3), a manufacturer who establishes the 
reliability of the suppliers test results at appropriate intervals may accept containers or closures 
based on visual identification and Certificate of Analysis review. 
 
2.   Inspection of Container Closure System 
 
A container closure system that permits penetration of microorganisms is unsuitable for a sterile 
product.  Any damaged or defective units should be detected, and removed, during inspection of 
the final sealed product.  Safeguards should be implemented to strictly preclude shipment of 
product that may lack container closure integrity and lead to nonsterility.  Equipment suitability 
problems or incoming container or closure deficiencies can cause loss of container closure 
system integrity.  For example, failure to detect vials fractured by faulty machinery as well as by 
mishandling of bulk finished stock has led to drug recalls.  If damage that is not readily detected 
leads to loss of container closure integrity, improved procedures should be rapidly implemented 
to prevent and detect such defects. 
 
Functional defects in delivery devices (e.g., syringe device defects, delivery volume) can also 
result in product quality problems and should be monitored by appropriate in-process testing.   
 
Any defects or results outside the specifications established for in-process and final inspection 
are to be investigated in accord with  211.192.   
 
 
  18  
Contains Nonbinding Recommendations 
 
VII. ENDOTOXIN CONTROL 
 
 
21 CFR 211.63 states that Equipment used in the manufacture, processing, packing, or holding of a drug product shall be 
of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning 
and maintenance.   
 
21 CFR 211.65(a) states that Equipment shall be constructed so that surfaces that contact components, in-process 
materials, or drug products shall not be reactive, additive, or absorptive so as to alter the safety, identity, strength, quality, 
or purity of the drug product beyond the official or other established requirements. 
 
21 CFR 211.67(a) states that Equipment and utensils shall be cleaned, maintained, and sanitized at appropriate intervals 
to prevent malfunctions or contamination that would alter the safety, identify, strength, quality, or purity of the drug 
product beyond the official or other established requirements. 
  
21 CFR 211.94(c) states that Drug product containers and closures shall be clean and, where indicated by the nature of 
the drug, sterilized and processed to remove pyrogenic properties to assure that they are suitable for their intended use.   
 
21 CFR 211.167(a) states that For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be 
appropriate laboratory testing to determine conformance to such requirements.  The test procedures shall be in writing and 
shall be followed. 
 
 
Endotoxin contamination of an injectable product can occur as a result of poor CGMP controls. 
Certain patient populations (e.g., neonates), those receiving other injections concomitantly, or 
those administered a parenteral in atypically large volumes or doses can be at greater risk for 
pyrogenic reaction than anticipated by the established limits based on body weight of a normal 
healthy adult (Ref. 6, 7).  Such clinical concerns reinforce the importance of exercising 
appropriate CGMP controls to prevent generation of endotoxins.  Drug product components, 
containers, closures, storage time limitations, and manufacturing equipment are among the areas 
to address in establishing endotoxin control.  
 
Adequate cleaning, drying, and storage of equipment will control bioburden and prevent 
contribution of endotoxin load.  Equipment should be designed to be easily assembled and 
disassembled, cleaned, sanitized, and/or sterilized.  If adequate procedures are not employed, 
endotoxins can be contributed by both upstream and downstream processing equipment.   
 
Sterilizing-grade filters and moist heat sterilization have not been shown to be effective in 
removing endotoxin.  Endotoxin on equipment surfaces can be inactivated by high-temperature 
dry heat, or removed from equipment surfaces by cleaning procedures.  Some clean-in-place 
procedures employ initial rinses with appropriate high purity water and/or a cleaning agent (e.g., 
acid, base, surfactant), followed by final rinses with heated WFI.  Equipment should be dried 
following cleaning, unless the equipment proceeds immediately to the sterilization step.   
 
 
  19  
Contains Nonbinding Recommendations 
 
VIII. TIME LIMITATIONS 
 
 
21 CFR 211.111 states that When appropriate, time limits for the completion of each phase of production shall be 
established to assure the quality of the drug product.  Deviation from established time limits may be acceptable if such 
deviation does not compromise the quality of the drug product.  Such deviation shall be justified and documented. 
 
 
When appropriate, time limits must be established for each phase of aseptic processing 
( 211.111).  Time limits should include, for example, the period between the start of bulk 
product compounding and its sterilization, filtration processes, product exposure while on the 
processing line, and storage of sterilized equipment, containers and closures.  The time limits 
established for the various production phases should be supported by data.  Bioburden and 
endotoxin load should be assessed when establishing time limits for stages such as the 
formulation processing stage. 
 
The total time for product filtration should be limited to an established maximum to prevent 
microorganisms from penetrating the filter.  Such a time limit should also prevent a significant 
increase in upstream bioburden and endotoxin load.  Because they can provide a substrate for 
microbial attachment, maximum use times for those filters used upstream for solution 
clarification or particle removal should also be established and justified. 
 
 
IX.  VALIDATION OF ASEPTIC PROCESSING AND STERILIZATION  
 
 
21 CFR 211.63, 211.65, and 211.67 address, respectively, Equipment design, size, and location, Equipment construction, 
and Equipment cleaning and maintenance.   
 
21 CFR 211.84(c) states, in part, that Samples shall be collected in accordance with the following procedures: * * * (3) 
Sterile equipment and aseptic sampling techniques shall be used when necessary. 
 
21 CFR 211.100(a) states, in part, that There shall be written procedures for production and process control designed to 
assure that the drug products have the identity, strength, quality, and purity they purport or are represented to possess.  Such 
procedures shall include all requirements in this subpart * * *. 
 
21 CFR 211.113(b) states that Appropriate written procedures, designed to prevent microbiological contamination of drug 
products purporting to be sterile, shall be established and followed.  Such procedures shall include validation of any 
sterilization process. 
 
This section primarily discusses routine qualification and validation study recommendations.  
Change control procedures are addressed only briefly, but are an important part of the quality 
systems established by a firm.  A change in facility, equipment, process, or test method should be 
evaluated through the written change control program, triggering an evaluation of the need for 
revalidation or requalification.   
 
A.   Process Simulations 
 
  20  
Contains Nonbinding Recommendations 
 
To ensure the sterility of products purporting to be sterile, sterilization, aseptic filling and closing 
operations must be adequately validated ( 211.113).  The goal of even the most effective 
sterilization processes can be defeated if the sterilized elements of a product (the drug 
formulation, the container, and the closure) are brought together under conditions that 
contaminate any of those elements.   
 
An aseptic processing operation should be validated using a microbiological growth medium in 
place of the product.  This process simulation, also known as a media fill, normally includes 
exposing the microbiological growth medium to product contact surfaces of equipment, 
container closure systems, critical environments, and process manipulations to closely simulate 
the same exposure that the product itself will undergo.  The sealed containers filled with the 
medium are then incubated to detect microbial contamination.  Results are then interpreted to 
assess the potential for a unit of drug product to become contaminated during actual operations 
(e.g., start-up, sterile ingredient additions, aseptic connections, filling, closing).  Environmental 
monitoring data from the process simulation can also provide useful information for the 
processing line evaluation.   
 
1.   Study Design  
 
A media fill program should incorporate the contamination risk factors that occur on a 
production line, and accurately assesses the state of process control.  Media fill studies should 
closely simulate aseptic manufacturing operations incorporating, as appropriate, worst-case 
activities and conditions that provide a challenge to aseptic operations.  FDA recommends that 
the media fill program address applicable issues such as: 
 
  Factors associated with the longest permitted run on the processing line that can pose 
contamination risk (e.g., operator fatigue) 
  Representative number, type, and complexity of normal interventions that occur with 
each run, as well as nonroutine interventions and events (e.g., maintenance, 
stoppages, equipment adjustments) 
  Lyophilization, when applicable  
  Aseptic assembly of equipment (e.g., at start-up, during processing) 
  Number of personnel and their activities  
  Representative number of aseptic additions (e.g., charging containers and closures as 
well as sterile ingredients) or transfers 
  Shift changes, breaks, and gown changes (when applicable)  
  Type of aseptic equipment disconnections/connections 
  Aseptic sample collections 
  Line speed and configuration 
  Weight checks  
  Container closure systems (e.g., sizes, type, compatibility with equipment) 
  21  
Contains Nonbinding Recommendations 
 
  Specific provisions in written procedures relating to aseptic processing (e.g., 
conditions permitted before line clearance is mandated)   
 
A written batch record, documenting production conditions and simulated activities, should be 
prepared for each media fill run.  The same vigilance should be observed in both media fill and 
routine production runs.  The firms rationale for the conditions and activities simulated during 
the media fill should be clearly defined.  Media fills should not be used to justify practices that 
pose unnecessary contamination risks. 
 
2.  Frequency and Number of Runs   
 
When a processing line is initially qualified, individual media fills should be repeated enough 
times to ensure that results are consistent and meaningful.  This approach is important because a 
single run can be inconclusive, while multiple runs with divergent results signal a process that is 
not in control.  We recommend that at least three consecutive separate successful runs be 
performed during initial line qualification.  Subsequently, routine semi-annual qualification 
conducted for each processing line will evaluate the state of control of the aseptic process.  
Activities and interventions representative of each shift, and shift changeover, should be 
incorporated into the design of the semi-annual qualification program.  For example, the 
evaluation of a production shift should address its unique time-related and operational features.13  
All personnel who are authorized to enter the aseptic processing room during manufacturing, 
including technicians and maintenance personnel, should participate in a media fill at least once 
a year.  Participation should be consistent with the nature of each operators duties during routine 
production.   
 
Each change to a product or line change should be evaluated using a written change control 
system.  Any changes or events that have the potential to affect the ability of the aseptic process 
to exclude contamination from the sterilized product should be assessed through additional 
media fills.  For example, facility and equipment modifications, line configuration changes, 
significant changes in personnel, anomalies in environmental testing results, container closure 
system changes, extended shutdowns, or end product sterility testing showing contaminated 
products may be cause for revalidation of the system.   
 
When data from a media fill indicate the process may not be in control, an investigation should 
be conducted to determine the origin of the contamination and the scope of the problem.  Once 
corrections are instituted, process simulation run(s) should be performed to confirm that 
deficiencies have been corrected and the process has returned to a state of control.   When an 
investigation fails to reach well-supported, substantive conclusions as to the cause of the media 
fill failure, three consecutive successful runs in tandem with increased scrutiny of the production 
process may be warranted.    
 
3.  Duration of Runs   
 
                                                 
13 One example might be the movement of personnel into and out of the aseptic processing and gowning change 
rooms during a shift change. 
  22  
Contains Nonbinding Recommendations 
 
The duration of aseptic processing operations is a major consideration in media fill design.  
Although the most accurate simulation model would be the full batch size and duration because 
it most closely simulates the actual production operations, other appropriate models can be 
justified.  The duration of the media fill run should be determined by the time it takes to 
incorporate manipulations and interventions, as well as appropriate consideration of the duration 
of the actual aseptic processing operation.  Interventions that commonly occur should be 
routinely simulated, while those occurring rarely can be simulated periodically. 
 
While conventional manufacturing lines are usually automated, operated at relatively high 
speeds, and designed to limit operator intervention, some processes still include considerable 
operator involvement.  When aseptic processing employs manual filling or closing, or extensive 
manual manipulations, the duration of the process simulation should generally be no less than the 
length of the actual manufacturing process to best simulate contamination risks posed by 
operators. 
 
For lyophilization operations, FDA recommends that unsealed containers be exposed to partial 
evacuation of the chamber in a manner that simulates the process.  Vials should not be frozen, 
and precautions should be taken that ensure that the medium remains in an aerobic state to avoid 
potentially inhibiting the growth of microorganisms. 
 
4.  Size of Runs  
 
The simulation run sizes should be adequate to mimic commercial production conditions and 
accurately assess the potential for commercial batch contamination.  The number of units filled 
during the process simulation should be based on contamination risk for a given process and 
sufficient to accurately simulate activities that are representative of the manufacturing process.  
A generally acceptable starting point for run size is in the range of 5,000 to 10,000 units.  For 
operations with production sizes under 5,000, the number of media filled units should at least 
equal the maximum batch size made on the processing line (Ref. 8). 
 
When the possibility of contamination is higher based on the process design (e.g., manually 
intensive filling lines), a larger number of units, generally at or approaching the full production 
batch size, should be used.  In contrast, a process conducted in an isolator (see Appendix 1) can 
have a low risk of contamination because of the lack of direct human intervention and can be 
simulated with a lower number of units as a proportion of the overall operation.  
 
Media fill size is an especially important consideration because some batches are produced over 
multiple shifts or yield an unusually large number of units.  These factors should be carefully 
evaluated when designing the simulation to adequately encompass conditions and any potential 
risks associated with the larger operation.   
 
5.  Line Speed  
 
The media fill program should adequately address the range of line speeds employed during 
production.  Each media fill run should evaluate a single line speed, and the speed chosen should 
be justified.  For example, use of high line speed is often most appropriate in the evaluation of 
manufacturing processes characterized by frequent interventions or a significant degree of 
  23  
Contains Nonbinding Recommendations 
 
manual manipulation.  Use of slow line speed is generally appropriate for evaluating 
manufacturing processes with prolonged exposure of the sterile drug product and 
containers/closures in the aseptic area. 
 
6.  Environmental Conditions  
 
Media fills should be adequately representative of the conditions under which actual 
manufacturing operations are conducted.  An inaccurate assessment (making the process appear 
cleaner than it actually is) can result from conducting a media fill under extraordinary air 
particulate and microbial quality, or under production controls and precautions taken in 
preparation for the media fill.  To the extent standard operating procedures permit stressful 
conditions (e.g., maximum number of personnel present and elevated activity level), it is 
important that media fills include analogous challenges to support the validity of these studies.  
Stressful conditions do not include artificially created environmental extremes, such as 
reconfiguration of HVAC systems to operate at worst-case limits. 
 
7.   Media  
 
In general, a microbiological growth medium, such as soybean casein digest medium, should be 
used.  Use of anaerobic growth media (e.g., fluid thioglycollate medium) should be considered in 
special circumstances.  The media selected should be demonstrated to promote growth of gram-
positive and gram-negative bacteria, and yeast and mold (e.g., USP indicator organisms).  The 
QC laboratory should determine if USP indicator organisms sufficiently represent production-
related isolates.  Environmental monitoring and sterility test isolates can be substituted (as 
appropriate) or added to the growth promotion challenge.  Growth promotion units should be 
inoculated with a <100 CFU challenge.  If the growth promotion testing fails, the origin of any 
contamination found during the simulation should nonetheless be investigated and the media fill 
promptly repeated.14  
 
The production process should be accurately simulated using media and conditions that optimize 
detection of any microbiological contamination.  Each unit should be filled with an appropriate 
quantity and type of microbial growth medium to contact the inner container closure surfaces 
(when the unit is inverted or thoroughly swirled) and permit visual detection of microbial 
growth.   
 
Some drug manufacturers have expressed concern over the possible contamination of the facility 
and equipment with nutrient media during media fill runs.  However, if the medium is handled 
properly and is promptly followed by the cleaning, sanitizing, and, where necessary, sterilization 
of equipment, subsequently processed products are not likely to be compromised.   
 
8.  Incubation and Examination of Media-Filled Units  
 
Media units should be incubated under conditions adequate to detect microorganisms that might 
otherwise be difficult to culture.  Incubation conditions should be established in accord with the 
following general guidelines:   
 
                                                 
14 The cause of the growth promotion failure should also be investigated. 
  24  
Contains Nonbinding Recommendations 
 
  Incubation temperature should be suitable for recovery of bioburden and environmental 
isolates and should at no time be outside the range of 20-35oC.  Incubation temperature 
should be maintained within +2.5oC of the target temperature.   
 
  Incubation time should not be less than 14 days.  If two temperatures are used for the 
incubation of the media filled units, the units should be incubated for at least 7 days at 
each temperature (starting with the lower temperature). 
 
Each media-filled unit should be examined for contamination by personnel with appropriate 
education, training, and experience in inspecting media fill units for microbiological 
contamination.  If QC personnel do not perform the inspection, there should be QC unit 
oversight throughout any such examination.  All suspect units identified during the examination 
should be brought to the immediate attention of the QC microbiologist.  To allow for visual 
detection of microbial growth, we recommend substituting clear containers (with otherwise 
identical physical properties) for amber or other opaque containers.  If appropriate, other 
methods can also be considered to ensure visual detection.   
 
When a firm performs a final product inspection of units immediately following the media fill 
run, all integral units should proceed to incubation.  Units found to have defects not related to 
integrity (e.g., cosmetic defect) should be incubated; units that lack integrity should be rejected.  
Erroneously rejected units should be returned promptly for incubation with the media fill lot. 
 
After incubation is underway, any unit found to be damaged should be included in the data for 
the media fill run, because the units can be representative of drug product released to the market.  
Any decision to exclude such incubated units (i.e., non-integral) from the final run tally should 
be fully justified and the deviation explained in the media fill report.  If a correlation emerges 
between difficult to detect damage and microbial contamination, a thorough investigation should 
be conducted to determine its cause (see Section VI.B).  
 
Written procedures regarding aseptic interventions should be clear and specific (e.g., intervention 
type; quantity of units removed), providing for consistent production practices and assessment of 
these practices during media fills.  If written procedures and batch documentation are adequate to 
describe an associated clearance, the intervention units removed during media fills do not need to 
be incubated.15  Where procedures lack specificity, there would be insufficient justification for 
exclusion of units removed during an intervention from incubation.  For example, if a production 
procedure requires removal of 10 units after an intervention at the stoppering station infeed, 
batch records (i.e., for production and media fills) should clearly document conformance with 
this procedure.  In no case should more units be removed during a media fill intervention than 
would be cleared during a production run. 
 
The ability of a media fill run to detect potential contamination from a given simulated activity 
should not be compromised by a large-scale line clearance.  We recommend incorporating 
                                                 
15
 To assess contamination risks during initial aseptic setup (before fill), valuable information can be obtained by 
incubating all such units that may be normally removed.  These units are typically incubated separately, and would 
not necessarily be included in the acceptance criteria for the media fill. 
 
  25  
Contains Nonbinding Recommendations 
 
appropriate study provisions to avoid and address a large line clearance that results in the 
removal of a unit possibly contaminated during an unrelated event or intervention. 
 
Appropriate criteria should be established for yield16 and accountability (reconciliation of filled 
units).  Media fill record reconciliation documentation should include a full accounting and 
description of units rejected from a batch. 
 
9.  Interpretation of Test Results   
 
The process simulation run should be observed by the QC Unit, and contaminated units should 
be reconcilable with the approximate time and the activity being simulated during the media fill.  
Video recording of a media fill may serve as a useful aide in identifying personnel practices that 
could negatively affect the aseptic process. 
 
Any contaminated unit should be considered objectionable and investigated.  The 
microorganisms should be identified to species level.  The investigation should survey the 
possible causes of contamination.  In addition, any failure investigation should assess the impact 
on commercial drugs produced on the line since the last media fill.  
 
Whenever contamination exists in a media fill run, it should be considered indicative of a 
potential sterility assurance problem, regardless of run size.  The number of contaminated units 
should not be expected to increase in a directly proportional manner with the number of vials in 
the media fill run.  Test results should reliably and reproducibly show that the units produced by 
an aseptic processing operation are sterile.  Modern aseptic processing operations in suitably 
designed facilities have demonstrated a capability of meeting contamination levels approaching 
zero (Ref. 8, 9) and should normally yield no media fill contamination.  Recommended criteria 
for assessing state of aseptic line control are as follows: 
 
  When filling fewer than 5000 units, no contaminated units should be detected. 
-- One (1) contaminated unit is considered cause for revalidation, following an 
investigation. 
 
  When filling from 5,000 to 10,000 units: 
-- One (1) contaminated unit should result in an investigation, including consideration of 
a repeat media fill. 
-- Two (2) contaminated units are considered cause for revalidation, following 
investigation. 
 
  When filling more than 10,000 units:  
-- One (1) contaminated unit should result in an investigation. 
-- Two (2) contaminated units are considered cause for revalidation, following 
investigation.  
 
For any run size, intermittent incidents of microbial contamination in media filled runs can be 
indicative of a persistent low-level contamination problem that should be investigated.  
                                                 
16Total units incubated/total number of units filled. 
  26  
Contains Nonbinding Recommendations 
 
Accordingly, recurring incidents of contaminated units in media fills for an individual line, 
regardless of acceptance criteria, would be a signal of an adverse trend on the aseptic processing 
line that should lead to problem identification, correction, and revalidation.   
 
A firm's use of media fill acceptance criteria allowing infrequent contamination does not mean 
that a distributed lot of drug product purporting to be sterile may contain a nonsterile unit.  The 
purpose of an aseptic process is to prevent any contamination.  A manufacturer is fully liable for 
the shipment of any nonsterile unit, an act that is prohibited under the FD&C Act (Section 301(a) 
21 U.S.C. 331(a)).  FDA also recognizes that there might be some scientific and technical 
limitations on how precisely and accurately process simulations can characterize a system of 
controls intended to exclude contamination. 
 
As with any process validation run, it is important to note that invalidation of a media fill run 
should be a rare occurrence.  A media fill run should be aborted only under circumstances in 
which written procedures require commercial lots to be equally handled.  Supporting 
documentation and justification should be provided in such cases.  
 
B.   Filtration Efficacy  
 
Filtration is a common method of sterilizing drug product solutions.  A sterilizing grade filter 
should be validated to reproducibly remove viable microorganisms from the process stream, 
producing a sterile effluent.17  Currently, such filters usually have a rated pore size of 0.2 m or 
smaller.18  Use of redundant sterilizing filters should be considered in many cases.  Whatever 
filter or combination of filters is used, validation should include microbiological challenges to 
simulate worst-case production conditions for the material to be filtered and integrity test results 
of the filters used for the study.  Product bioburden should be evaluated when selecting a suitable 
challenge microorganism to assess which microorganism represents the worst-case challenge to 
the filter.  The microorganism Brevundimonas diminuta (ATCC 19146) when properly grown, 
harvested and used, is a common challenge microorganism for 0.2 m rated filters because of its 
small size (0.3 m mean diameter).  The manufacturing process controls should be designed to 
minimize the bioburden of the unfiltered product.  Bioburden of unsterilized bulk solutions 
should be determined to trend the characteristics of potentially contaminating organisms.   
 
In certain cases, when justified as equivalent or better than use of B. diminuta, it may be 
appropriate to conduct bacterial retention studies with a bioburden isolate.  The number of 
microorganisms in the challenge is important because a filter can contain a number of pores 
larger than the nominal rating, which has the potential to allow passage of microorganisms.  The 
probability of such passage is considered to increase as the number of organisms (bioburden) in 
the material to be filtered increases.  A challenge concentration of at least 107 organisms per cm2 
of effective filtration area should generally be used, resulting in no passage of the challenge 
microorganism.  The challenge concentration used for validation is intended to provide a margin 
of safety well beyond what would be expected in production. 
 
                                                 
17 This document does not address virus removal. 
 
18 0.22 and 0.2 are considered interchangeable nominal pore size ratings. 
  27  
Contains Nonbinding Recommendations 
 
Direct inoculation into the drug formulation is the preferred method because it provides an 
assessment of the effect of drug product on the filter matrix and on the challenge organism.  
However, directly inoculating B. diminuta into products with inherent bactericidal activity 
against this microbe, or into oil-based formulations, can lead to erroneous conclusions.  When 
sufficiently justified, the effects of the product formulation on the membrane's integrity can be 
assessed using an appropriate alternate method.  For example, a drug product could be filtered in 
a manner in which the worst-case combination of process specifications and conditions are 
simulated.  This step could be followed by filtration of the challenge organism for a significant 
period of time, under the same conditions, using an appropriately modified product (e.g., lacking 
an antimicrobial preservative or other antimicrobial component) as the vehicle.  Any divergence 
from a simulation using the actual product and conditions of processing should be justified.   
 
Factors that can affect filter performance generally include (1) viscosity and surface tension of 
the material to be filtered, (2) pH, (3) compatibility of the material or formulation components 
with the filter itself, (4) pressures, (5) flow rates, (6) maximum use time, (7) temperature, (8) 
osmolality, (9) and the effects of hydraulic shock.  When designing the validation protocol, it is 
important to address the effect of the extremes of processing factors on the filter capability to 
produce sterile effluent.  Filter validation should be conducted using the worst-case conditions, 
such as maximum filter use time and pressure (Ref. 12).  Filter validation experiments, including 
microbial challenges, need not be conducted in the actual manufacturing areas.  However, it is 
essential that laboratory experiments simulate actual production conditions.  The specific type of 
filter membrane used in commercial production should be evaluated in filter validation studies.  
There are advantages to using production filters in these bacterial retention validation studies.  
When the more complex filter validation tests go beyond the capabilities of the filter user, tests 
are often conducted by outside laboratories or by filter manufacturers.  However, it is the 
responsibility of the filter user to review the validation data on the efficacy of the filter in 
producing a sterile effluent.  The data should be applicable to the user's products and conditions 
of use because filter performance may differ significantly for various conditions and products. 
 
After a filtration process is properly validated for a given product, process, and filter, it is 
important to ensure that identical filters (e.g., of identical polymer construction and pore size 
rating) are used in production runs.  Sterilizing filters should be routinely discarded after 
processing of a single lot.  However, in those instances when repeated use can be justified, the 
sterile filter validation should incorporate the maximum number of lots to be processed.  
Integrity testing of the filter(s) can be performed prior to processing, and should be routinely 
performed post-use.   It is important that integrity testing be conducted after filtration to detect 
any filter leaks or perforations that might have occurred during the filtration.  Forward flow and 
bubble point tests, when appropriately employed, are two integrity tests that can be used.  A 
production filters integrity test specification should be consistent with data generated during 
bacterial retention validation studies. 
 
C.   Sterilization of Equipment, Containers, and Closures 
 
Equipment surfaces that contact sterilized drug product or its sterilized containers or closures 
must be sterile so as not to alter purity of the drug (211.67 and 211.113).  Where reasonable 
contamination potential exists, surfaces that are in the vicinity of the sterile product should also 
be rendered free of viable organisms.  It is as important in aseptic processing to validate the 
  28  
Contains Nonbinding Recommendations 
 
processes used to sterilize such critical equipment as it is to validate processes used to sterilize 
the drug product and its container and closure.  Moist heat and dry heat sterilization, the most 
widely used, are the primary processes discussed in this document.  However, many of the heat 
sterilization principles discussed in this guidance are also applicable to other sterilization 
methods.   
 
Sterility of aseptic processing equipment should normally be maintained by sterilization between 
each batch.19  Following sterilization, transportation and assembly of equipment, containers, and 
closures should be performed with strict adherence to aseptic methods in a manner that protects 
and sustains the product's sterile state. 
 
1.  Qualification and Validation 
 
Validation studies should be conducted to demonstrate the efficacy of the sterilization cycle.  
Requalification studies should also be performed on a periodic basis.  The specific load 
configurations, as well as biological indicator and temperature sensor locations, should be 
documented in validation records.  Batch production records should subsequently document 
adherence to the validated load patterns. 
 
It is important to remove air from the autoclave chamber as part of a steam sterilization cycle.  
The insulating properties of air interfere with the ability of steam to transfer its energy to the 
load, achieving lower lethality than associated with saturated steam.  It also should be noted that 
the resistance of microorganisms can vary widely depending on the material to be sterilized.  For 
this reason, careful consideration should be given during sterilization validation to the nature or 
type of material chosen as the carrier of the biological indicator to ensure an appropriately 
representative study. 
 
Potentially difficult to reach locations within the sterilizer load or equipment train (for SIP 
applications) should be evaluated.  For example, filter installations in piping can cause a 
substantial pressure differential across the filter, resulting in a significant temperature drop on the 
downstream side.  We recommend placing biological indicators at appropriate downstream 
locations of the filter.  
 
Empty chamber studies evaluate numerous locations throughout a sterilizing unit (e.g., steam 
autoclave, dry heat oven) or equipment train (e.g., large tanks, immobile piping) to confirm 
uniformity of conditions (e.g., temperature, pressure).  These uniformity or mapping studies 
should be conducted with calibrated measurement devices. 
 
Heat penetration studies should be performed using the established sterilizer loads.  Validation of 
the sterilization process with a loaded chamber demonstrates the effects of loading on thermal 
input to the items being sterilized and may identify difficult to heat or penetrate items where 
there could be insufficient lethality to attain sterility.  The placement of biological indicators at 
numerous positions in the load, including the most difficult to sterilize places, is a direct means 
of confirming the efficacy of any sterilization procedure.  In general, the biological indicator 
should be placed adjacent to the temperature sensor so as to assess the correlation between 
microbial lethality and predicted lethality based on thermal input.  When determining which 
                                                 
19 If appropriate, alternate intervals can be defined, justified, and supported by validation studies. 
  29  
Contains Nonbinding Recommendations 
 
articles are difficult to sterilize, special attention should be given to the sterilization of filters, 
filling manifolds, and pumps.  Some other examples include certain locations of tightly wrapped 
or densely packed supplies, securely fastened load articles, lengthy tubing, the sterile filter 
apparatus, hydrophobic filters, and stopper load. 
 
Ultimately, cycle specifications for such sterilization methods should be based on the delivery of 
adequate lethality to the slowest to heat locations.  A sterility assurance level of 10-6 or better 
should be demonstrated for a sterilization process.  For more information, please also refer to the 
FDA guidance entitled Guideline for the Submission of Documentation for Sterilization Process 
Validation in Applications for Human and Veterinary Drug Products.  
 
The sterilizer validation program should continue to focus on the load areas identified as most 
difficult to penetrate or heat.  The suitability of the sterilizer should be established by 
qualification, maintenance, change control, and periodic verification of the cycle, including 
biological challenges.  Change control procedures should adequately address issues such as a 
load configuration change or a modification of a sterilizer. 
 
2.  Equipment Controls and Instrument Calibration  
 
For both validation and routine process control, the reliability of the data generated by 
sterilization cycle monitoring devices should be considered to be of the utmost importance.  
Devices that measure cycle parameters should be routinely calibrated.  Written procedures 
should be established to ensure that these devices are maintained in a calibrated state.  For 
example, we recommend that procedures address the following:  
 
  Temperature and pressure monitoring devices for heat sterilization should be 
calibrated at suitable intervals.  The sensing devices used for validation studies should 
be calibrated before and after validation runs. 
  Devices used to monitor dwell time in the sterilizer should be periodically calibrated. 
  The microbial count of a biological indicator should be confirmed. Biological 
indicators should be stored under appropriate conditions.   
  If the reliability of a vendors Certificate of Analysis is established through an 
appropriate qualification program, the D-value of a biological indicator (e.g., spore 
strips, glass ampuls) can be accepted in lieu of confirmatory testing of each lot.  
However, a determination of resistance (D-value) should be performed for any 
biological indicator inoculated onto a substrate, or used in a way that is other than 
described by the vendor.  D-value determinations can be conducted by an 
independent laboratory.   
  Where applicable, instruments used to determine the purity of steam should be 
calibrated. 
  For dry heat depyrogenation tunnels, devices (e.g. sensors and transmitters) used to 
measure belt speed should be routinely calibrated.  Bacterial endotoxin challenges 
should be appropriately prepared and measured by the laboratory. 
 
To ensure robust process control, equipment should be properly designed with attention to 
features such as accessibility to sterilant, piping slope, and proper condensate removal (as 
applicable).  Equipment control should be ensured through placement of measuring devices at 
  30  
Contains Nonbinding Recommendations 
 
those control points that are most likely to rapidly detect unexpected process variability.  Where 
manual manipulations of valves are required for sterilizer or SIP operations, these steps should 
be documented in manufacturing procedures and batch records.  Sterilizing equipment should be 
properly maintained to allow for consistent, satisfactory function.  Routine evaluation of 
sterilizer performance-indicating attributes, such as equilibrium (come up) time is important in 
assuring that the unit continues to operate as per the validated conditions. 
 
 
X.  LABORATORY CONTROLS 
 
 
 
21 CFR 211.22(b) states that Adequate laboratory facilities for the testing and approval (or rejection) of components, 
drug product containers, closures, packaging materials, in-process materials, and drug products shall be available to 
the quality control unit. 
 
21 CFR 211.22(c) states that The quality control unit shall have the responsibility for approving or rejecting all 
procedures or specifications impacting on the identity, strength, quality, and purity of the drug product. 
 
21 CFR 211.42(c) states, in part, that Operations shall be performed within specifically defined areas of adequate 
size.  There shall be separate or defined areas or such other control systems for the firms operations as are necessary 
to prevent contamination or mixups during the course of the following procedures: * * * (10) Aseptic processing, 
which includes as appropriate: * * * (iv) A system for monitoring environmental conditions; * * *. 
 
21 CFR 211.56(b) states that There shall be written procedures assigning responsibility for sanitation and describing 
in sufficient detail the cleaning schedules, methods, equipment, and materials to be used in cleaning the buildings and 
facilities; such written procedures shall be followed.  
 
21 CFR 211.56(c) states, in part, that There shall be written procedures for use of suitable rodenticides, insecticides, 
fungicides, fumigating agents, and cleaning and sanitizing agents.  Such written procedures shall be designed to 
prevent the contamination of equipment, components, drug product containers, closures, packaging, labeling 
materials, or drug products and shall be followed * * *. 
 
21 CFR 211.110(a) states, in part, that To assure batch uniformity and integrity of drug products, written procedures 
shall be established and followed that describe the in-process controls, and tests, or examinations to be conducted on 
appropriate samples of in-process materials of each batch.  Such control procedures shall be established to monitor the 
output and to validate the performance of those manufacturing processes that may be responsible for causing 
variability in the characteristics of in-process material and the drug product * * *. 
 
 
21 CFR 211.113(b) states that Appropriate written procedures, designed to prevent microbiological contamination of 
drug products purporting to be sterile, shall be established and followed.  Such procedures shall include validation of 
any sterilization process. 
 
21 CFR 211.160(b) states that Laboratory controls shall include the establishment of scientifically sound and 
appropriate specifications, standards, sampling plans, and test procedures designed to assure that components, drug 
product containers, closures, in-process materials, labeling, and drug products conform to appropriate standards of 
identity, strength, quality, and purity.  Laboratory controls shall include: (1) Determination of conformance to 
appropriate written specifications for the acceptance of each lot within each shipment of components, drug product 
containers, closures, and labeling used in the manufacture, processing, packing, or holding of drug products.  The 
specifications shall include a description of the sampling and testing procedures used.  Samples shall be representative 
and adequately identified.  Such procedures shall also require appropriate retesting of any component, drug product 
container, or closure that is subject to deterioration.  (2) Determination of conformance to written specifications and a 
description of sampling and testing procedures for in-process materials.  Such samples shall be representative and 
  31  
Contains Nonbinding Recommendations 
 
properly identified. (3) Determination of conformance to written descriptions of sampling procedures and appropriate 
specifications for drug products. Such samples shall be representative and properly identified.  (4) The calibration of 
instruments, apparatus, gauges, and recording devices at suitable intervals in accordance with an established written 
program containing specific directions, schedules, limits for accuracy and precision, and provisions for remedial 
action in the event accuracy and/or precision limits are not met.  Instruments, apparatus, gauges, and recording devices 
not meeting established specifications shall not be used.  
 
21 CFR 211.165(e) states that The accuracy, sensitivity, specificity, and reproducibility of test methods employed by 
the firm shall be established and documented.  Such validation and documentation may be accomplished in 
accordance with  211.194(a)(2). 
 
21 CFR 211.192 states, in part, that All drug product production and control records, including those for packaging 
and labeling, shall be reviewed and approved by the quality control unit to determine compliance with all established, 
approved written procedures before a batch is released or distributed * * *. 
  
 
A.    Environmental Monitoring    
 
1.    General Written Program 
 
In aseptic processing, one of the most important laboratory controls is the environmental 
monitoring program.  This program provides meaningful information on the quality of the 
aseptic processing environment (e.g., when a given batch is being manufactured) as well as 
environmental trends of ancillary clean areas.  Environmental monitoring should promptly 
identify potential routes of contamination, allowing for implementation of corrections before 
product contamination occurs (211.42 and 211.113). 
 
Evaluating the quality of air and surfaces in the cleanroom environment should start with a well-
defined written program and scientifically sound methods.  The monitoring program should 
cover all production shifts and include air, floors, walls, and equipment surfaces, including the 
critical surfaces that come in contact with the product, container, and closures.  Written 
procedures should include a list of locations to be sampled.  Sample timing, frequency, and 
location should be carefully selected based upon their relationship to the operation performed. 
Samples should be taken throughout the classified areas of the aseptic processing facility (e.g., 
aseptic corridors, gowning rooms) using scientifically sound sampling procedures.  Sample sizes 
should be sufficient to optimize detection of environmental contaminants at levels that might be 
expected in a given clean area. 
 
It is important that locations posing the most microbiological risk to the product be a key part of 
the program. It is especially important to monitor the microbiological quality of the critical area 
to determine whether or not aseptic conditions are maintained during filling and closing 
activities.  Air and surface samples should be taken at the locations where significant activity or 
product exposure occurs during production.  Critical surfaces that come in contact with the 
sterile product should remain sterile throughout an operation.  When identifying critical sites to 
be sampled, consideration should be given to the points of contamination risk in a process, 
including factors such as difficulty of setup, length of processing time, and impact of 
interventions.  Critical surface sampling should be performed at the conclusion of the aseptic 
processing operation to avoid direct contact with sterile surfaces during processing.  Detection of 
microbial contamination on a critical site would not necessarily result in batch rejection.  The 
  32  
Contains Nonbinding Recommendations 
 
contaminated critical site sample should prompt an investigation of operational information and 
data that includes an awareness of the potential for a low incidence of false positives. 
 
Environmental monitoring methods do not always recover microorganisms present in the 
sampled area.  In particular, low-level contamination can be particularly difficult to detect.  
Because false negatives can occur, consecutive growth results are only one type of adverse trend.  
Increased incidence of contamination over a given period is an equal or more significant trend to 
be tracked.  In the absence of any adverse trend, a single result above an action level should 
trigger an evaluation and a determination about whether remedial measures may be appropriate.  
In all room classes, remedial measures should be taken in response to unfavorable trends. 
 
All environmental monitoring locations should be described in SOPs with sufficient detail to 
allow for reproducible sampling of a given location surveyed.  Written SOPs should also address 
elements such as (1) frequency of sampling, (2) when the samples are taken (i.e., during or at the 
conclusion of operations), (3) duration of sampling, (4) sample size (e.g., surface area, air 
volume), (5) specific sampling equipment and techniques, (6) alert and action levels, and (7) 
appropriate response to deviations from alert or action levels.   
 
2.  Establishing Levels and a Trending Program 
 
Microbiological monitoring levels should be established based on the relationship of the sampled 
location to the operation.  The levels should be based on the need to maintain adequate 
microbiological control throughout the entire sterile manufacturing facility.  One should also 
consider environmental monitoring data from historical databases, media fills, cleanroom 
qualification, and sanitization studies, in developing monitoring levels.  Data from similar 
operations can also be helpful in setting action and alert levels, especially for a new operation.   
 
Environmental monitoring data will provide information on the quality of the manufacturing 
environment.  Each individual sample result should be evaluated for its significance by 
comparison to the alert or action levels.  Averaging of results can mask unacceptable localized 
conditions.  A result at the alert level urges attention to the approaching action conditions.  A 
result at the action level should prompt a more thorough investigation.  Written procedures 
should be established, detailing data review frequency and actions to be taken.  The quality 
control unit should provide routine oversight of near-term (e.g., daily, weekly, monthly, 
quarterly) and long-term trends in environmental and personnel monitoring data.  
 
Trend reports should include data generated by location, shift, room, operator, or other 
parameters.  The quality control unit should be responsible for producing specialized data reports 
(e.g., a search on a particular isolate over a year period) with the goal of investigating results 
beyond established levels and identifying any appropriate follow-up actions.  Significant changes 
in microbial flora should be considered in the review of the ongoing environmental monitoring 
data. 
 
Written procedures should define the system whereby the most responsible managers are 
regularly informed and updated on trends and investigations. 
 
  33  
Contains Nonbinding Recommendations 
 
3.  Disinfection Efficacy 
 
The suitability, efficacy, and limitations of disinfecting agents and procedures should be 
assessed.  The effectiveness of these disinfectants and procedures should be measured by their 
ability to ensure that potential contaminants are adequately removed from surfaces.   
 
To prevent introduction of contamination, disinfectants should be sterile, appropriately handled 
in suitable (e.g., sterile) containers and used for no longer than the predefined period specified by 
written procedures.  Routinely used disinfectants should be effective against the normal 
microbial vegetative flora recovered from the facility.  Many common disinfectants are 
ineffective against spores.  For example, 70 percent isopropyl alcohol is ineffective against 
Bacillus spp. spores.  Therefore, a sound disinfectant program also includes a sporicidal agent, 
used according to a written schedule and when environmental data suggest the presence of 
sporeforming organisms.   
 
Disinfection procedures should be described in sufficient detail (e.g., preparation, work 
sequence, contact time) to enable reproducibility.  Once the procedures are established, their 
adequacy should be evaluated using a routine environmental monitoring program.  If indicated, 
microorganisms associated with adverse trends can be investigated as to their sensitivity to the 
disinfectants employed in the cleanroom in which the organisms were isolated.   
 
4.   Monitoring Methods 
 
Acceptable methods for monitoring the microbiological quality of the environment include:    
 
a.  Surface Monitoring 
 
Environmental monitoring involves sampling various surfaces for microbiological 
quality.  For example, product contact surfaces, floors, walls, and equipment should be 
tested on a regular basis.  Touch plates, swabs, and contact plates can be used for such 
tests.   
 
b.  Active Air Monitoring 
 
Assessing microbial quality of air should involve the use of active devices including but 
not limited to impaction, centrifugal, and membrane (or gelatin) samplers.  Each device 
has certain advantages and disadvantages, although all allow testing of the number of 
organisms per volume of air sampled.  We recommend that such devices be used during 
each production shift to evaluate aseptic processing areas at carefully chosen locations.  
Manufacturers should be aware of a device's air monitoring capabilities, and the air 
sampler should be evaluated for its suitability for use in an aseptic environment based on 
collection efficiency, cleanability, ability to be sterilized, and disruption of unidirectional 
airflow.20  Because devices vary, the user should assess the overall suitability of a 
monitoring device before it is placed into service.  Manufacturers should ensure that such 
devices are calibrated and used according to appropriate procedures.   
                                                 
20 For example, the volume of air sampled should be sufficient to yield meaningful measurements of air quality in a 
given environment. 
  34  
Contains Nonbinding Recommendations 
 
 
c.  Passive Air Monitoring (Settling Plates) 
 
Another method is the use of passive air samplers, such as settling plates (petri dishes 
containing nutrient growth medium exposed to the environment).  Because only 
microorganisms that settle onto the agar surface are detected, settling plates can be used 
as qualitative, or semi-quantitative, air monitors.  Their value in critical areas will be 
enhanced by ensuring that plates are positioned in locations posing the greatest risk of 
product contamination.  As part of methods validation, the quality control laboratory 
should evaluate what media exposure conditions optimize recovery of low levels of 
environmental isolates.  Exposure conditions should preclude desiccation (e.g., caused by 
lengthy sampling periods and/or high airflows), which inhibits recovery of 
microorganisms.  The data generated by passive air sampling can be useful when 
considered in combination with results from other types of air samples. 
 
B.   Microbiological Media and Identification  
 
Characterization of recovered microorganisms provides vital information for the environmental 
monitoring program.  Environmental isolates often correlate with the contaminants found in a 
media fill or product sterility testing failure, and the overall environmental picture provides 
valuable information for an investigation.  Monitoring critical and immediately surrounding 
clean areas as well as personnel should include routine identification of microorganisms to the 
species (or, where appropriate, genus) level.  In some cases, environmental trending data have 
revealed migration of microorganisms into the aseptic processing room from either uncontrolled 
or lesser controlled areas.  Establishing an adequate program for differentiating microorganisms 
in the lesser-controlled environments, such as Class 100,000 (ISO 8), can often be instrumental 
in detecting such trends.  At minimum, the program should require species (or, where 
appropriate, genus) identification of microorganisms in these ancillary environments at frequent 
intervals to establish a valid, current database of contaminants present in the facility during 
processing (and to demonstrate that cleaning and sanitization procedures continue to be 
effective).   
 
Genotypic methods have been shown to be more accurate and precise than traditional 
biochemical and phenotypic techniques.  These methods are especially valuable for 
investigations into failures (e.g., sterility test; media fill contamination).  However, appropriate 
biochemical and phenotypic methods can be used for the routine identification of isolates. 
 
The goal of microbiological monitoring is to reproducibly detect microorganisms for purposes of 
monitoring the state of environmental control.  Consistent methods will yield a database that 
allows for sound data comparisons and interpretations.  The microbiological culture media used 
in environmental monitoring should be validated as capable of detecting fungi (i.e., yeasts and 
molds) as well as bacteria and incubated at appropriate conditions of time and temperature.  
Total aerobic bacterial count can be obtained by incubating at 30 to 35oC for 48 to 72 hours.  
Total combined yeast and mold count can generally be obtained by incubating at 20 to 25oC for 5 
to 7 days.  
 
  35  
Contains Nonbinding Recommendations 
 
Incoming lots of environmental monitoring media should be tested for their ability to reliably 
recover microorganisms.  Growth promotion testing should be performed on all lots of prepared 
media.  Where appropriate, inactivating agents should be used to prevent inhibition of growth by 
cleanroom disinfectants or product residuals (e.g., antibiotics). 
 
C.   Prefiltration Bioburden 
 
Manufacturing process controls should be designed to minimize the bioburden in the unfiltered 
product.  In addition to increasing the challenge to the sterilizing filter, bioburden can contribute 
impurities (e.g., endotoxin) to, and lead to degradation of, the drug product.  A prefiltration 
bioburden limit should be established.  
 
D.   Alternate Microbiological Test Methods    
 
Other suitable microbiological test methods (e.g., rapid test methods) can be considered for 
environmental monitoring, in-process control testing, and finished product release testing after it 
is demonstrated that the methods are equivalent or better than traditional methods (e.g.,USP).   
 
E.   Particle Monitoring 
 
Routine particle monitoring is useful in rapidly detecting significant deviations in air cleanliness 
from qualified processing norms (e.g., clean area classification).  A result outside the established 
classification level at a given location should be investigated as to its cause.  The extent of 
investigation should be consistent with the severity of the excursion and include an evaluation of 
trending data.  Appropriate corrective action should be implemented, as necessary, to prevent 
future deviations. 
 
See Section IV.A for additional guidance on particle monitoring. 
  36  
Contains Nonbinding Recommendations 
 
 
XI.  STERILITY TESTING 
 
 
21 CFR 210.3(b)(21) states that Representative sample means a sample that consists of a number of units that are drawn 
based on rational criteria such as random sampling and intended to assure that the sample accurately portrays the material 
being sampled.   
 
21 CFR 211.110(a) states, in part, that To assure batch uniformity and integrity of drug products, written procedures shall 
be established and followed that describe the in-process controls, and tests, or examinations to be conducted on appropriate 
samples of in-process materials of each batch.  Such control procedures shall be established to monitor the output and to 
validate the performance of those manufacturing processes that may be responsible for causing variability in the 
characteristics of in-process material and the drug product. 
 
21 CFR 211.160(b) states that Laboratory controls shall include the establishment of scientifically sound and appropriate 
specifications, standards, sampling plans, and test procedures designed to assure that components, drug product containers, 
closures, in-process materials, labeling, and drug products conform to appropriate standards of identity, strength, quality, 
and purity.  Laboratory controls shall include: (1) Determination of conformance to appropriate written specifications for 
the acceptance of each lot within each shipment of components, drug product containers, closures, and labeling used in the 
manufacture, processing, packing, or holding of drug products.  The specifications shall include a description of the 
sampling and testing procedures used.  Samples shall be representative and adequately identified.  Such procedures shall 
also require appropriate retesting of any component, drug product container, or closure that is subject to deterioration.  (2) 
Determination of conformance to written specifications and a description of sampling and testing procedures for in-process 
materials.  Such samples shall be representative and properly identified. (3) Determination of conformance to written 
descriptions of sampling procedures and appropriate specifications for drug products. Such samples shall be representative 
and properly identified.  (4) The calibration of instruments, apparatus, gauges, and recording devices at suitable intervals 
in accordance with an established written program containing specific directions, schedules, limits for accuracy and 
precision, and provisions for remedial action in the event accuracy and/or precision limits are not met.  Instruments, 
apparatus, gauges, and recording devices not meeting established specifications shall not be used.  
 
21 CFR 211.165(a) states, in part, that For each batch of drug product, there shall be appropriate laboratory determination 
of satisfactory conformance to final specifications for the drug product, including the identity and strength of each active 
ingredient, prior to release * * *.  
 
21 CFR 211.165(e) states that The accuracy, sensitivity, specificity, and reproducibility of test methods employed by the 
firm shall be established and documented.  Such validation and documentation may be accomplished in accordance with   
211.194(a)(2). 
 
21 CFR 211.167(a) states that For each batch of drug product purporting to be sterile and/or pyrogen-free, there shall be 
appropriate laboratory testing to determine conformance to such requirements.  The test procedures shall be in writing and 
shall be followed. 
 
21 CFR 211.180(e) states, in part, that Written records required by this part shall be maintained so that data therein can be 
used for evaluating, at least annually, the quality standards of each drug product to determine the need for changes in drug 
product specifications or manufacturing or control procedures * * *. 
 
21 CFR 211.192 states that All drug product production and control records, including those for packaging and labeling, 
shall be reviewed and approved by the quality control unit to determine compliance with all established, approved written 
procedures before a batch is released or distributed.  Any unexplained discrepancy (including a percentage of theoretical 
yield exceeding the maximum or minimum percentages established in master production and control records) or the failure 
of a batch or any of its components to meet any of its specifications shall be thoroughly investigated, whether or not the 
batch has already been distributed.  The investigation shall extend to other batches of the same drug product and other drug 
products that may have been associated with the specific failure or discrepancy.  A written record of the investigation shall 
be made and shall include the conclusions and followup. 
 
  37  
Contains Nonbinding Recommendations 
 
 
Certain aspects of sterility testing are of particular importance, including control of the testing 
environment, understanding the test limitations, and investigating manufacturing systems 
following a positive test. 
 
The testing laboratory environment should employ facilities and controls comparable to those 
used for aseptic filling operations.  Poor or deficient sterility test facilities or controls can result 
in test failure.  If production facilities and controls are significantly better than those for sterility 
testing, the danger exists of mistakenly attributing a positive sterility test result to a faulty 
laboratory even when the product tested could have, in fact, been nonsterile.  Therefore, a 
manufacturing deficiency may go undetected.  The use of isolators for sterility testing minimizes 
the chance of a false positive test result. 
 
A.   Microbiological Laboratory Controls  
 
Sterility testing methods are required to be accurate and reproducible, in accordance with 
211.194 and 211.165.  USP <71> Sterility Tests is the principal source used for sterility 
testing methods, including information on test procedures and media.21   
 
As a part of methods validation, appropriate microbiological challenge testing will demonstrate 
reproducibility of the method to reliably recover representative microorganisms.  If growth is 
inhibited, modifications (e.g., increased dilution, additional membrane filter washes, addition of 
inactivating agents) to the test method should be implemented to optimize recovery.  Ultimately, 
methods validation studies should demonstrate that the method does not provide an opportunity 
for false negatives.   
 
It is essential that the media used to perform sterility testing be rendered sterile and demonstrated 
as growth promoting.  Personnel performing sterility testing should be qualified and trained for 
the task.  A written program should be in place to maintain updated training of personnel and 
confirm acceptable sterility testing practices.  
 
B.   Sampling and Incubation 
 
Sterility tests are limited in their ability to detect contamination because of the small sample size 
typically used.  For example, as described by USP, statistical evaluations indicate that the 
sterility test sampling plan "only enables the detection of contamination in a lot in which 10% of 
the units are contaminated about nine times out of ten in making the test" (Ref. 13).  To further 
illustrate, if a 10,000-unit lot with a 0.1 percent contamination level was sterility tested using 20 
units, there is a 98 percent chance that the batch would pass the test.   
 
It is important that the samples represent the entire batch and processing conditions.  Samples 
should be taken: 
 
  at the beginning, middle, and end of the aseptic processing operation 
  in conjunction with processing interventions or excursions 
                                                 
. 
  38  
Contains Nonbinding Recommendations 
 
 
Because of the limited sensitivity of the test, any positive result is considered a serious CGMP 
issue that should be thoroughly investigated.  
 
C.   Investigation of Sterility Positives 
 
Care should be taken in the performance of the sterility test to preclude any activity that allows 
for possible sample contamination.  When microbial growth is observed, the lot should be 
considered nonsterile and an investigation conducted.  An initial positive test would be invalid 
only in an instance in which microbial growth can be unequivocally ascribed to laboratory error.  
 
Only if conclusive and documented evidence clearly shows that the contamination occurred as 
part of testing should a new test be performed.  When available evidence is inconclusive, batches 
should be rejected as not conforming to sterility requirements. 
 
After considering all relevant factors concerning the manufacture of the product and testing of 
the samples, the comprehensive written investigation should include specific conclusions and 
identify corrective actions.  The investigation's persuasive evidence of the origin of the 
contamination should be based on at least the following: 
 
1.  Identification (speciation) of the organism in the sterility test   
 
Sterility test isolates should be identified to the species level.  Microbiological monitoring data 
should be reviewed to determine if the organism is also found in laboratory and production 
environments, personnel, or product bioburden.  Advanced identification methods (e.g., nucleic-
acid based) are valuable for investigational purposes.  When comparing results from 
environmental monitoring and sterility positives, both identifications should be performed using 
the same methodology. 
 
2.  Record of laboratory tests and deviations 
 
Review of laboratory deviation and investigation findings can help to eliminate or implicate the 
laboratory as the source of contamination.  For example, if the organism is seldom found in the 
laboratory environment, product contamination is more likely than laboratory error.  If the 
organism is found in laboratory and production environments, it can still indicate product 
contamination. 
 
The proper handling of deviations is an essential aspect of laboratory control.  When a deviation 
occurs during sterility testing, it should be documented, investigated, and remedied.  If any 
deviation is considered to have compromised the integrity of the sterility test, the test should be 
invalidated immediately without incubation.   
 
A sterility positive result can be viewed as indicative of production or laboratory problems, and 
the entire manufacturing process should be comprehensively investigated since such problems 
often can extend beyond a single batch.  To more accurately monitor potential contamination 
sources, we recommend keeping separate trends by appropriate categories such as product, 
container type, filling line, sampling, and testing personnel.  Where the degree of sterility test 
  39  
Contains Nonbinding Recommendations 
 
sample manipulation is similar for a terminally sterilized product and an aseptically processed 
product, a higher rate of initial sterility failures for the latter should be taken as indicative of 
aseptic processing production problems.  
 
Microbial monitoring of the aseptic area of the laboratory and personnel can also reveal trends 
that are informative.  Upward trends in the microbial load in the aseptic area of the laboratory 
should be promptly investigated as to cause, and corrected.  In some instances, such trends can 
appear to be more indicative of laboratory error as a possible source of a sterility test failure.   
 
Where a laboratory has a good track record with respect to errors, this history can lower 
suspicion of the lab as a source of contamination since chances are higher that the contamination 
arose from production.  However, the converse is not true.  Specifically, where a laboratory has a 
poor track record, firms should not assume that the contamination is automatically more 
attributable to the laboratory and consequently overlook a genuine production problem.  
Accordingly, it is essential that all sterility positives be thoroughly investigated. 
 
3.  Monitoring of production area environment   
 
Trend analysis of microorganisms in the critical and immediately adjacent areas is especially 
helpful in determining the source of contamination in a sterility failure investigation. 
Consideration of environmental microbial data should not be limited to results of monitoring the 
production environment for the lot, day, or shift associated with the suspect lot.  For example, 
results showing little or no recovery of microorganisms can be misleading, especially when 
preceded or followed by a finding of an adverse trend or atypically high microbial counts.  It is 
therefore important to look at both short- and long-term environmental trend analyses. 
 
4.  Monitoring Personnel   
 
The review of data and associated trends from daily monitoring of personnel can provide 
important information indicating a route of contamination.  The adequacy of personnel practices 
and training also merit significant review and consideration. 
 
5.  Product Presterilization Bioburden  
 
We recommend review of trends in product bioburden and consideration of whether adverse 
bioburden trends have occurred.  
 
6.  Production record review   
 
Complete batch and production control records should be reviewed to detect any signs of failures 
or anomalies that could have a bearing on product sterility.  For example, the investigation 
should include elements such as: 
 
  Events that could have impacted on the critical zone 
  Batch and trending data that indicate whether utility and/or support systems are 
functioning properly.  For instance, records of air quality monitoring for filling lines 
  40  
Contains Nonbinding Recommendations 
 
could show a time at which there was improper air balance or an unusually high particle 
count.  
  Whether construction or maintenance activities could have had an adverse impact 
 
7.  Manufacturing history  
 
The manufacturing history of a product or similar products should be reviewed as part of the 
investigation.  Past deviations, problems, or changes (e.g., process, components, equipment) are 
among the factors that can provide an indication of the origin of the problem.   
  41  
Contains Nonbinding Recommendations 
 
 
XII.  BATCH RECORD REVIEW: PROCESS CONTROL DOCUMENTATION 
 
 
 
21 CFR 211.100(a) states that There shall be written procedures for production and process control designed to assure that 
the drug products have the identity, strength, quality, and purity they purport or are represented to possess.  Such procedures 
shall include all requirements in this subpart.  These written procedures, including any changes, shall be drafted, reviewed, 
and approved by the appropriate organizational units and reviewed and approved by the quality control unit.  
 
21 CFR 211.100(b) states that Written production and process control procedures shall be followed in the execution of the 
various production and process control functions and shall be documented at the time of performance.  Any deviation from 
the written procedures shall be recorded and justified. 
 
21 CFR 211.186 and 211.188 address, respectively, "Master production and control records" and "Batch production and 
control records."  
 
21 CFR 211.192 states that All drug product production and control records, including those for packaging and labeling, 
shall be reviewed and approved by the quality control unit to determine compliance with all established, approved written 
procedures before a batch is released or distributed.  Any unexplained discrepancy (including a percentage of theoretical 
yield exceeding the maximum or minimum percentages established in master production and control records) or the failure 
of a batch or any of its components to meet any of its specifications shall be thoroughly investigated, whether or not the 
batch has already been distributed.  The investigation shall extend to other batches of the same drug product and other drug 
products that may have been associated with the specific failure or discrepancy.  A written record of the investigation shall 
be made and shall include the conclusions and followup. 
 
 
Manufacturers should build process and environmental control activities into their aseptic 
processing operation.  It is critical that these activities be maintained and strictly implemented on 
a daily basis.  The requirement for review of all batch records and data for conformance with 
written procedures, operating parameters, and product specifications prior to arriving at the final 
release decision for an aseptically processed product calls for an overall review of process and 
system performance for that given cycle of manufacture.  All in-process and laboratory control 
results must be included with the batch record documentation in accordance with section 
211.188.  Review of environmental and personnel monitoring data, as well as other data relating 
to acceptability of output from support systems (e.g., HEPA / HVAC, WFI, steam generator) and 
proper functioning of equipment (e.g., batch alarms report; integrity of various filters) are 
considered essential elements of the batch release decision. 
 
While interventions and/or stoppages are normally recorded in the batch record, the manner of 
documenting these occurrences varies.  In particular, line stoppages and any unplanned 
interventions should be sufficiently documented in batch records with the associated time and 
duration of the event.  In addition to lengthened dwell time of sterile product elements in the 
critical area, an extensive intervention can increase contamination risk.  Sterility failures have 
often been attributed to atypical or extensive interventions that have occurred as a response to an 
undesirable event during the aseptic process.  Written procedures describing the need for line 
clearances in the event of certain interventions, such as machine adjustments and any repairs, 
should be established.  Such interventions should be documented with more detail than minor 
events.  Interventions that result in substantial activity near exposed product or container closures 
  42  
Contains Nonbinding Recommendations 
 
or that last beyond a reasonable exposure time should, where appropriate, result in a local or full 
line clearance. 
 
Any disruption in power supply, however momentary, that could affect product quality is a 
manufacturing deviation and must be included in batch records (211.100, 211.192). 
  43  
Contains Nonbinding Recommendations 
 
 
APPENDIX 1:  ASEPTIC PROCESSING ISOLATORS 
 
Aseptic processing using isolation systems separates the external cleanroom environment from 
the aseptic processing line and minimizes its exposure to personnel.  A well-designed positive 
pressure isolator, supported by adequate procedures for its maintenance, monitoring, and control, 
offers tangible advantages over traditional aseptic processing, including fewer opportunities for 
microbial contamination during processing.  However, users should remain vigilant to potential 
sources of operational risk. Manufacturers should also be aware of the need to establish new 
procedures addressing issues unique to isolators. 
 
A.   Maintenance 
 
1.  General 
 
Maintenance of isolator systems differs in some significant respects from the traditional, non-
isolated aseptic processing operations.  Although no isolator forms an absolute seal, very high 
integrity can be achieved in a well-designed unit.  However, a leak in certain components of the 
system can constitute a significant breach of integrity.  The integrity of gloves, half-suits, and 
seams should receive daily attention and be addressed by a comprehensive preventative 
maintenance program.  Replacement frequencies should be established in written procedures that 
ensure parts will be changed before they breakdown or degrade.  Transfer systems, gaskets, and 
seals are among the other parts that should be covered by the maintenance program.   
 
2.  Glove Integrity 
 
A faulty glove or sleeve (gauntlet) assembly represents a route of contamination and a critical 
breach of isolator integrity.  A preventative maintenance program should be established.  The 
choice of durable glove materials, coupled with a well-justified replacement frequency, are key 
aspects of good manufacturing practice to be addressed.  With every use, gloves should be 
visually evaluated for any macroscopic physical defect.  Physical integrity tests should also be 
performed routinely.  A breach in glove integrity can be of serious consequence.  The monitoring 
and maintenance program should identify and eliminate any glove lacking integrity and 
minimize the possibility of placing a sterile product at risk. 
 
Due to the potential for microbial migration through microscopic holes in gloves and the lack of 
a highly sensitive glove integrity test, we recommend affording attention to the sanitary quality 
of the inner surface of the installed glove and to integrating the use of a second pair of thin 
gloves. 
 
B.  Design  
  
1.  Airflow 
 
There are two types of aseptic processing isolators: open and closed.  Closed isolators employ 
connections with auxiliary equipment for material transfer.  Open isolators have openings to the 
  44  
Contains Nonbinding Recommendations 
 
surrounding environment that are carefully engineered to segregate the inner isolator 
environment from the surrounding room via overpressure.   
 
Turbulent flow can be acceptable within closed isolators, which are normally compact in size 
and do not house processing lines.  Other aseptic processing isolators employ unidirectional 
airflow that sweeps over and away from exposed sterile materials, avoiding any turbulence or 
stagnant airflow in the area of exposed sterilized materials, product, and container closures.  In 
most sound designs, air showers over the critical area once and then is systematically exhausted 
from the enclosure.  The air handling system should be capable of maintaining the requisite 
environmental conditions within the isolator. 
 
2.  Materials of Construction 
 
As in any aseptic processing design, suitable materials should be chosen based on durability, as 
well as ease of cleaning and decontamination.  For example, rigid wall construction 
incorporating stainless steel and glass materials is widely used. 
 
3.  Pressure Differential  
 
Isolators that include an open portal should be designed to ensure complete physical separation 
from the external environment.  A positive air pressure differential adequate to achieve this 
separation should be employed and supported by qualification studies.  Positive air pressure 
differentials from the isolator to the surrounding environment have largely ranged from 
approximately 17.5 to 50 Pascals.22  The appropriate minimum pressure differential  established 
by a firm will depend on the systems design and, when applicable, its exit port.  Air balance 
between the isolator and other direct interfaces (e.g., dry heat tunnel) should also be qualified. 
 
The positive pressure differential should be coupled with an appropriately designed opening to 
the external environment to prevent potential ingress of surrounding room air by induction.  
Induction can result from local turbulent flow causing air swirls or pressure waves that might 
push extraneous particles into the isolator.  Local Class 100 (ISO 5) protection at an opening is 
an example of a design provision that can provide a further barrier to the external environment.  
 
4.  Clean Area Classifications 
 
The interior of the isolator should meet Class 100 (ISO 5) standards.  The classification of the 
environment surrounding the isolator should be based on the design of its interfaces (e.g., 
transfer ports), as well as the number of transfers into and out of the isolator.  A Class 100,000 
(ISO 8) background is commonly used based on consideration of isolator design and 
manufacturing situations.  An aseptic processing isolator should not be located in an unclassified 
room. 
 
C.  Transfer of Materials/Supplies 
 
                                                 
22 0.07 to 0.20 water gauge 
  45  
Contains Nonbinding Recommendations 
 
The ability to maintain integrity of a decontaminated isolator can be affected impacted by the 
design of transfer ports.  Various adaptations, of differing capabilities, allow for the transfer of 
supplies into and out of the isolator.   
 
Multiple material transfers are generally made during the processing of a batch.  Frequently, 
transfers are performed via direct interface with manufacturing equipment.  Properly maintained 
and operated rapid transfer ports (RTPs) are an effective transfer mechanism for aseptic transfer 
of materials into and out of isolators.  Some transfer ports might have significant limitations, 
including marginal decontaminating capability (e.g., ultraviolet) or a design that has the potential 
to compromise isolation by allowing ingress of air from the surrounding room.  In the latter case, 
localized HEPA-filtered unidirectional airflow cover in the area of such a port should be 
implemented.  Isolators often include a mousehole or other exit port through which product is 
discharged, opening the isolator to the outside environment.  Sufficient overpressure should be 
supplied and monitored on a continuous basis at this location to ensure that isolation is 
maintained.   
 
D.  Decontamination 
 
1.  Surface Exposure  
 
Decontamination procedures should ensure full exposure of all isolator surfaces to the chemical 
agent.  The capability of a decontaminant to penetrate obstructed or covered surfaces is limited.  
For example, to facilitate contact with the decontaminant, the glove apparatus should be fully 
extended with glove fingers separated during the decontamination cycle.  It is also important to 
clean the interior of the isolator per appropriate procedures to allow for a robust decontamination 
process.   
 
2.  Efficacy 
 
The decontamination method should render the inner surfaces of the isolator free of viable 
microorganisms.  Multiple available vaporized agents are suitable for achieving decontamination.  
Process development and validation studies should include a thorough determination of cycle capability.  
The characteristics of these agents generally preclude the reliable use of statistical methods (e.g., 
fraction negative) to determine process lethality (Ref.  13).  An appropriate, quantified Biological 
Indicator (BI) challenge should be placed on various materials23 and in many locations throughout the 
isolator, including difficult to reach areas.  Cycles should be developed with an appropriate margin of 
extra kill to provide confidence in robustness of the decontamination processes.  Normally, a four- to 
six-log reduction can be justified depending on the application.  The specific BI spore titer used and the 
selection of BI placement sites should be justified.  For example, demonstration of a four-log reduction 
should be sufficient for controlled, very low bioburden materials introduced into a transfer isolator, 
including wrapped sterile supplies that are briefly exposed to the surrounding cleanroom environment. 
 
                                                 
23 If the various isolator materials are thoroughly evaluated during cycle development, a firm might consider placing 
more focus on material texture and porosity during validation of the decontamination process.   
 
  46  
Contains Nonbinding Recommendations 
 
The uniform distribution of a defined concentration of decontaminating agent should also be 
evaluated as part of these studies (Ref. 14).  Chemical indicators may also be useful as a 
qualitative tool to show that the decontaminating agent reached a given location. 
 
3.  Frequency 
 
The design of the interior and content of an isolator should provide for its frequent 
decontamination.  When an isolator is used for multiple days between decontamination cycles, 
the frequency adopted should be justified.  This frequency, established during validation studies, 
should be reevaluated and increased if production data indicate deterioration of the 
microbiological quality of the isolator environment.   
 
A breach of isolator integrity should normally lead to a decontamination cycle.  Integrity can be 
affected by power failures, valve failure, inadequate overpressure, holes in gloves and seams, or 
other leaks.  Breaches of integrity should be investigated.  If it is determined that the 
environment may have been compromised, any product potentially impacted by the breach 
should be rejected.  
 
E.  Filling Line Sterilization 
 
To ensure sterility of product contact surfaces from the start of each operation, the entire path of 
the sterile processing stream should be sterilized.  In addition, aseptic processing equipment or 
ancillary supplies to be used within the isolator should be chosen based on their ability to 
withstand steam sterilization (or equivalent method).  It is expected that materials that permit 
heat sterilization (e.g., SIP) will be rendered sterile by such methods.  Where decontamination 
methods are used to render certain product contact surfaces free of viable organisms, a minimum 
of a six-log reduction should be demonstrated using a suitable biological indicator. 
 
F.   Environmental Monitoring 
 
An environmental monitoring program should be established that routinely ensures acceptable 
microbiological quality of air, surfaces, and gloves (or half-suits) as well as particle levels, 
within the isolator.  Nutrient media should be cleaned off of surfaces following a contact plate 
sample.  Air quality should be monitored periodically during each shift.  For example, we 
recommend monitoring the exit port for particles to detect any unusual results.  Media used for 
environmental monitoring should not be exposed to decontamination cycle residues, as recovery 
of microorganisms would be inhibited. 
 
G.  Personnel 
 
Although cleanroom apparel considerations are generally reduced in an isolator operation, the 
contamination risk contributed by manual factors can not be overlooked.  Isolation processes 
generally include periodic or even frequent use of one or more gloves for aseptic manipulations 
and handling of material transfers into and out of the isolator.  One should be aware that 
locations on gloves, sleeves, or half suits can be among the more difficult to reach places during 
decontamination, and glove integrity defects might not be promptly detected.  Traditional aseptic 
processing vigilance remains critical, with an understanding that contaminated isolator gloves 
  47  
Contains Nonbinding Recommendations 
 
can lead to product nonsterility.  Accordingly, meticulous aseptic technique standards must be 
observed (211.113), including appropriate use of sterile tools for manipulations. 
  48  
Contains Nonbinding Recommendations 
 
 
APPENDIX 2:  BLOW-FILL- SEAL TECHNOLOGY 
 
Blow-fill-seal (BFS) technology is an automated process by which containers are formed, filled, 
and sealed in a continuous operation.  This manufacturing technology includes economies in 
container closure processing and reduced human intervention and is often used for filling and 
packaging ophthalmics, respiratory care products, and, less frequently, injectables.  This 
appendix discusses some of the critical control points of this technology.  Except where 
otherwise noted below, the aseptic processing standards discussed elsewhere in this document 
should apply to blow-fill-seal technology.   
 
A.   Equipment Design and Air Quality 
 
Most BFS machines operate using the following steps.  
 
  Heat a plastic polymer resin 
  Extrude it to form a parison (a tubular form of the hot resin) 
  Cut the parison with a high-temperature knife 
  Move the parison under the blow-fill needle (mandrel) 
  Inflate it to the shape of the mold walls 
  Fill the formed container with the liquid product 
  Remove the mandrel 
  Seal 
 
Throughout this operation, sterile-air is used, for example, to form the parison and inflate it prior 
to filling.  In most operations, the three steps with the greatest potential for exposure to particle 
contamination and/or surrounding air are those in which (1) the parison is cut, (2) the parison is 
moved under the blow-fill mandrel, and (3) the mandrel is removed (just prior to sealing). 
 
BFS machinery and its surrounding barriers should be designed to prevent the potential for 
extraneous contamination.  As with any aseptic processing operation, it is critical that product 
contact surfaces be sterile.  A validated steam-in-place cycle, or equivalent process, should be 
used to sterilize the equipment path through which the product is conveyed.  In addition, any 
other surface that represents a potential contamination risk to the sterile product should be sterile. 
 
The classified environment surrounding BFS machinery should generally meet Class 100,000 
(ISO 8), or better, standards, depending on the design of the BFS machinery and the surrounding 
room.  HEPA-filtered or sterile air provided by membrane filters should be used during the steps 
when sterile products or materials are exposed (e.g., parison formation, container molding or 
filling steps).  Air in the critical area should meet Class 100 (ISO 5) microbiological standards 
during operations.  A well-designed BFS system should also normally achieve Class 100 (ISO 5) 
airborne particle levels.  Only personnel who have been qualified and appropriately gowned 
should enter the classified environment surrounding the BFS machinery.  Refer to Section V of 
this document for guidance on personnel training, qualification, and monitoring. 
 
BFS equipment design typically calls for use of specialized measures to reduce particle levels 
that can contaminate the exposed product.  In contrast to nonpharmaceutical applications using 
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BFS machinery, control of air quality (i.e., particles) is critical for sterile drug product 
manufacture.  Particles generated during the plastic extrusion, cutting, and sealing processes 
should be controlled.  Provisions for carefully controlled airflow can protect the product by 
forcing generated particles outward while preventing any ingress from the adjacent environment.  
Furthermore, equipment designs that separate the filling zone from the surrounding environment 
provide additional product protection.  Barriers, pressure vacuums, microenvironments, and 
appropriately directed high velocities of sterile air have been found useful in preventing 
contamination (Ref. 15).  Smoke studies and multi-location particle data can provide valuable 
information when performing qualification studies to assess whether proper particle control 
dynamics have been achieved throughout the critical area.   
 
In addition to suitable design, it is important to establish an adequate preventative maintenance 
program.  For example, because of its potential to contaminate the sterile drug product, the 
integrity of the cooling, heating and other utility systems associated with the BFS machine 
should be maintained and routinely monitored. 
 
B.  Validation/Qualification 
 
Advantages of BFS processing are known to include rapid container closure processing and 
minimized aseptic interventions.  However, only a properly functioning process can realize these 
advantages.  We recommend affording special attention to setup, troubleshooting of equipment, 
and related aseptic personnel procedures.  Equipment sterilization, media fills, polymer 
extrusion/sterilization, product-plastic compatibility, forming and sealing integrity, and unit 
weight variation are among the key issues to address in validation and qualification studies.   
 
Data gathered during such studies should ensure that BFS containers are sterile and, if used for 
parenteral drugs, nonpyrogenic.  This can generally be achieved by validating that time 
temperature conditions of the extrusion process are effective against endotoxin or spore 
challenges in the polymeric material. 
 
The choice of appropriate polymer material for a BFS operation includes assessing if a material 
is pharmaceutical grade, safe, pure, and passes appropriate criteria (Ref. 17) for plastics.  
Polymer suppliers should be qualified and monitored for raw material quality. 
 
C.   Batch Monitoring and Control 
 
Various in-process control parameters (e.g., container weight variation, fill weight, leakers, air 
pressure) provide information to monitor and facilitate ongoing process control.   It is essential to 
monitor the microbial air quality.  Samples should be taken according to a comprehensive 
sampling plan that provides data representative of the entire filling operation.  Continuous 
monitoring of particles can provide valuable data relative to the control of a blow-fill-seal 
operation.  
 
Container closure defects can be a major problem in control of a BFS operation.  It is critical that 
the operation be designed and set-up to uniformly manufacture integral units.  As a final 
measure, the inspection of each unit of a batch should include a reliable, sensitive, final product 
examination that is capable of identifying defective units (e.g., leakers).  Significant defects due 
  50  
Contains Nonbinding Recommendations 
 
to heat or mechanical problems, such as wall thickness, container or closure interface 
deficiencies, poorly formed closures, or other deviations should be investigated in accordance 
with  211.100 and 211.192.  
 
  51  
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APPENDIX 3: PROCESSING PRIOR TO FILLING AND SEALING OPERATIONS 
 
The purpose of this appendix is to supplement the guidance provided in this document with 
information on products regulated by CBER or CDER that are subject to aseptic processing at 
points early in the manufacturing process, or that require aseptic processing through the entire 
manufacturing process because it is impossible to sterile filter the final drug product.  The scope 
of this appendix includes aseptic processing activities that take place prior to the filling and 
sealing of the finished drug product.  Special considerations include those for: 
 
A.  Aseptic processing from early manufacturing steps 
 
Some products undergo aseptic processing at some or all manufacturing steps preceding the final 
product closing step. With other products, there is a point in the process after which they can no 
longer be rendered sterile by filtration.  In such cases, the product would be handled aseptically 
at all steps subsequent to sterile filtration.  In other instances, the final drug product cannot be 
sterile-filtered and, therefore, each component in the formulation would be rendered sterile and 
mixed aseptically.  For example, products containing aluminum adjuvant are formulated 
aseptically because once they are alum adsorbed, they cannot be sterile-filtered. 
 
When a product is processed aseptically from the early stages, the product and all components or 
other additions are rendered sterile prior to entering the manufacturing process.  It is critical that 
all transfers, transports, and storage stages be carefully controlled at each step of the process to 
maintain sterility of the product.  In some cases, bulk drug substances or products should be 
tested for sterility.24 
 
Procedures (e.g., aseptic connection) that expose a product or product contact surfaces should be 
performed under unidirectional airflow in a Class 100 (ISO 5) environment.  The environment of 
the room surrounding the Class 100 (ISO 5) environment should be Class 10,000 (ISO 7) or 
better.  Microbiological and airborne particle monitoring should be performed during operations.  
Microbial surface monitoring should be performed at the end of operations, but prior to cleaning.  
Personnel monitoring should be performed in association with operations. 
 
Process simulation studies covering the steps preceding filling and sealing should be designed to 
incorporate all conditions, product manipulations, and interventions that could impact on the 
sterility of the product.  The process simulation, from the early process steps, should demonstrate 
that process controls are adequate to protect the product during manufacturing. These studies 
should incorporate all product manipulations, additions, and procedures involving exposure of 
product contact surfaces to the environment.  The studies should include worst-case conditions 
such as maximum duration of open operations and maximum number of participating operators.  
However, the process simulations do not need to mimic total manufacturing time if the 
manipulations that occur during manufacturing are adequately represented.  
 
It is also important that process simulations incorporate storage of sterile bulk drug substances or 
product and transport to other manufacturing areas.  For instance, there should be assurance of 
bulk vessel integrity for specified holding times.  The transport of sterile bulk tanks or other 
containers should be simulated as part of the media fill.  Please refer to Section IX.A for more 
                                                 
24 See 21 CFR 610.12 for general biological product standards for sterility. 
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guidance on media simulation studies.  Process simulation studies for the formulation stage 
should be performed at least twice per year. 
 
B.  Aseptic processing of cellular therapy products and cell-derived products   
 
Cellular therapy and some cell-derived products (e.g., lysates, semi-purified extracts) represent a 
subset of the products that cannot be filter-sterilized and therefore undergo aseptic manipulations 
throughout the manufacturing process.  Where possible, closed systems should be used during 
manufacturing.  Cellular therapy products often have short processing times at each 
manufacturing stage, particularly between the harvest, formulation of the final product, and 
product release.  These products are frequently released from the manufacturing facility and 
administered to patients before final product sterility testing results are available.  In situations 
where results of final sterility testing are not available before the product is administered, 
additional controls and testing should be considered.  For example, additional sterility tests can 
be performed at intermediate stages of manufacture, such as after the last manipulation of the 
product prior to harvest.  Other tests that may indicate microbial contamination, such as 
microscopic examination, Gram stain (or other bacterial and fungal stain), and endotoxin testing 
should be performed and meet acceptance criteria prior to product release. 
 
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REFERENCES 
 
 
1.  ISO 14644-1: Cleanrooms and Associated Controlled Environments, Classification of Air 
Cleanliness.  
2.  NASA Standard for Cleanroom and Work Stations for Microbially Controlled Environment, 
Publication NHB 5340.2 (August l967). 
3.  Technical Order 00-25-203, Contamination Control of Aerospace Facilities, U.S. Air Force, 
December l, l972. 
4.  Ljungqvist, B., and Reinmuller, B., Cleanroom Design: Minimizing Contamination Through 
Proper Design; Interpharm Press, 1997. 
5.  Lord, A. and J. W. Levchuk, "Personnel Issues in Aseptic Processing," Biopharm, 1989. 
6.  Morbidity and Mortality Weekly Report, "Clinical Sepsis and Death in a Newborn Nursery 
Associated with Contaminated Medications"  Brazil, 1996, Centers for Disease Control and 
Prevention, July, 1998; 47(29);610-2. 
7.  Grandics, Peter, Pyrogens in Parenteral Pharmaceuticals, Pharmaceutical Technology, 
April 2000.  
8.  Recommendations of PQRI Aseptic Processing Working Group, Product Quality Research 
Institute; March, 2003. 
9.  Technical Report No. 36, Current Practices in the Validation of Aseptic Processing," 
Parenteral Drug Association, Inc., 2002. 
10. Leahy, T. J. and M. J. Sullivan, Validation of Bacterial - Retention Capabilities of 
Membrane Filters," Pharmaceutical Technology, Nov., l978. 
11. Pall, D. B. and E. A. Kirnbauer, et al., "Particulate Retention by Bacteria Retentive 
Membrane Filters," Pall Corporation Colloids and Surfaces, l (l980) 235-256, Elsevier 
Scientific Publishing Company, Amsterdam. 
12. Technical Report No. 26, "Sterilizing Filtration of Liquids," Parenteral Drug Association, 
Inc., 1998. 
 
13. Sigwarth, V. and A. Stark, Effect of Carrier Materials on the Resistance of Spores of 
Bacillus stearothermophilus to Gaseous Hydrogen Peroxide, PDA Journal of 
Pharmaceutical Science and Technology, Vol. 57, No. 1, January/February 2003.   
14. Isolators used for Aseptic Processing and Sterility Testing, Pharmaceutical Inspection 
Convention Cooperation Scheme (PIC/S); June, 2002. 
15. Price, J., Blow-Fill-Seal Technology: Part I, A Design for Particulate Control, 
Pharmaceutical Technology, February, 1998. 
16. United States Pharmacopoeia 
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Contains Nonbinding Recommendations 
 
RELEVANT GUIDANCE DOCUMENTS 
 
Some relevant FDA guidance documents include: 
 
(cid:131)  Guidance for the Submission of Documentation for Sterilization Process Validation in 
Applications for Human and Veterinary Drug Products 
(cid:131)  Guideline for Validation of Limulus Amebocyte Lysate Test as an End Product Endotoxin 
Test for Human and Animal Parenteral Drugs, Biological Products, and Medical Devices 
(cid:131)  Guide to Inspections of Lyophilization of Parenterals 
(cid:131)  Guide to Inspections of High Purity Water Systems 
(cid:131)  Guide To Inspections of Microbiological Pharmaceutical Quality Control Laboratories 
(cid:131)  Guide To Inspections of Sterile Drug Substance Manufacturers 
(cid:131)  Pyrogens: Still a Danger; (Inspection Technical Guide) 
(cid:131)  Bacterial Endotoxins/Pyrogens; (Inspection Technical Guide) 
(cid:131)  Heat Exchangers to Avoid Contamination;  (Inspection Technical Guide) 
(cid:131)  Compliance Program Guidance Manual 7356.002 A, Sterile Drug Process Inspections 
(cid:131)  ICH Q5A, Guidance on Viral Safety Evaluation of Biotechnology Products Derived from       
Cell Lines of Human or Animal Origin 
(cid:131)  See also the draft guidance Container and Closure Integrity Testing in Lieu of Sterility 
Testing as a Component of the Stability Protocol for Sterile Products, which was issued in 
1998.  Once final, it will represent the Agency's thinking on this topic.  
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GLOSSARY 
 
Air lock- A small room with interlocked doors, constructed to maintain air pressure control 
between adjoining rooms (generally with different air cleanliness standards).  The intent of an 
aseptic processing airlock is to preclude ingress of particulate matter and microorganism 
contamination from a lesser controlled area.  
 
Alert Level- An established microbial or airborne particle level giving early warning of potential 
drift from normal operating conditions and triggers appropriate scrutiny and follow-up to address 
the potential problem.  Alert levels are always lower than action levels. 
 
Action Level- An established microbial or airborne particle level that, when exceeded, should 
trigger appropriate investigation and corrective action based on the investigation. 
 
Aseptic Manufacturing Area- The classified part of a facility that includes the aseptic processing 
room and ancillary cleanrooms.  For purposes of this document, this term is synonymous with 
aseptic processing facility as used in the segregated segment context. 
 
Aseptic Processing Facility- A building, or segregated segment of it, containing cleanrooms in 
which air supply, materials, and equipment are regulated to control microbial and particle 
contamination.   
 
Aseptic Processing Room- A room in which one or more aseptic activities or processes is 
performed. 
 
Asepsis- A state of control attained by using an aseptic work area and performing activities in a 
manner that precludes microbiological contamination of the exposed sterile product. 
 
Bioburden- The total number of microorganisms associated with a specific item prior to 
sterilization. 
 
Barrier- A physical partition that affords aseptic processing area (ISO 5) protection by partially 
separating it from the surrounding area.  
 
Biological Indicator (BI)- A population of microorganisms inoculated onto a suitable medium 
(e.g., solution, container or closure) and placed within appropriate sterilizer load locations to 
determine the sterilization cycle efficacy of a physical or chemical process.   The challenge 
microorganism is selected based upon its resistance to the given process.  Incoming lot D-value 
and microbiological count define the quality of the BI. 
 
Clean Area- An area with defined particle and microbiological cleanliness standards. 
 
Cleanroom- A room designed, maintained, and controlled to prevent particle and microbiological 
contamination of drug products.  Such a room is assigned and reproducibly meets an appropriate 
air cleanliness classification. 
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Component- Any ingredient intended for use in the manufacture of a drug product, including 
those that may not appear in the final drug product. 
 
Colony Forming Unit (CFU)- A microbiological term that describes the formation of a single 
macroscopic colony after the introduction of one or more microorganisms to microbiological 
growth media.  One colony forming unit is expressed as 1 CFU. 
 
Critical Area - An area designed to maintain sterility of sterile materials.  Sterilized product, 
containers, closures, and equipment may be exposed in critical areas.  
 
Clean Zone- See Clean Area. 
 
Critical surfaces- Surfaces that may come into contact with or directly affect a sterilized product 
or its containers or closures. Critical surfaces are rendered sterile prior to the start of the 
manufacturing operation, and sterility is maintained throughout processing. 
 
Decontamination- A process that eliminates viable bioburden via use of sporicidal chemical 
agents.   
 
Disinfection- Process by which surface bioburden is reduced to a safe level or eliminated.  Some 
disinfection agents are effective only against vegetative microbes, while others possess 
additional capability to effectively kill bacterial and fungal spores. 
 
Depyrogenation- A process used to destroy or remove pyrogens (e.g., endotoxin).                 
 
D value- The time (in minutes) of exposure at a given temperature that causes a one-log or 90 
percent reduction in the population of a specific microorganism.    
 
Dynamic- Conditions relating to clean area classification under conditions of normal production.  
 
Endotoxin- A pyrogenic product (e.g., lipopolysaccharide) present in the bacterial cell wall.  
Endotoxin can lead to reactions in patients receiving injections ranging from fever to death. 
 
Gowning Qualification- A program that establishes, both initially and on a periodic basis, the 
capability of an individual to don the complete sterile gown in an aseptic manner.    
 
HEPA filter- High efficiency particulate air filter with minimum 0.3 m particle retaining 
efficiency of 99.97 percent. 
 
HVAC- Heating, ventilation, and air conditioning. 
 
Intervention- An aseptic manipulation or activity that occurs at the critical area. 
 
Isolator- A decontaminated unit, supplied with Class 100 (ISO 5) or higher air quality, that 
provides uncompromised, continuous isolation of its interior from the external environment (e.g., 
surrounding cleanroom air and personnel).  There are two major types of isolators: 
 
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Closed isolator systems exclude external contamination from the isolators interior by 
accomplishing material transfer via aseptic connection to auxiliary equipment, rather than 
use of openings to the surrounding environment.  Closed systems remain sealed 
throughout operations.   
 
Open isolator systems are designed to allow for the continuous or semi-continuous 
ingress and/or egress of materials during operations through one or more openings.  
Openings are engineered (e.g., using continuous overpressure) to exclude the entry of 
external contamination into the isolator. 
 
Laminar flow- An airflow moving in a single direction and in parallel layers at constant velocity 
from the beginning to the end of a straight line vector.   
 
Operator- Any individual participating in the aseptic processing operation, including line set-up, 
filler, maintenance, or other personnel associated with aseptic line activities. 
 
Overkill sterilization process- A process that is sufficient to provide at least a 12 log reduction of 
microorganisms having a minimum D value of 1 minute. 
 
Pyrogen- A substance that induces a febrile reaction in a patient. 
 
Sterile Product- For purposes of this guidance, sterile product refers to one or more of the 
elements exposed to aseptic conditions and ultimately making up the sterile finished drug 
product.  These elements include the containers, closures, and components of the finished drug 
product. 
 
Sterilizing grade filter- A filter that, when appropriately validated, will remove all 
microorganisms from a fluid stream, producing a sterile effluent. 
 
Quality Control Unit- An organizational element with authority and responsibility as defined by 
211.22.   
 
Unidirectional flow- An airflow moving in a single direction, in a robust and uniform manner, 
and at sufficient speed to reproducibly sweep particles away from the critical processing or 
testing area.   
 
Terminal sterilization- The application of a lethal agent to sealed, finished drug products for the 
purpose of achieving a predetermined sterility assurance level (SAL) of usually less than 10-6 
(i.e., a probability of a nonsterile unit of greater than one in a million).  
 
ULPA filter- Ultra-low penetration air filter with minimum 0.3 m particle retaining efficiency 
of 99.999 percent. 
 
Validation- Establishing documented evidence that provides a high degree of assurance that a 
specific process will consistently produce a product meeting its predetermined specifications and 
quality attributes. 
 
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Worst case- A set of conditions encompassing upper and lower processing limits and 
circumstances, including those within standard operating procedures, that pose the greatest 
chance of process or product failure (when compared to ideal conditions).  Such conditions do 
not necessarily induce product or process failure. 
  59