**Sterile Drug Products Produced by**

**Aseptic Processing**

[]{#__RefHeading___Toc20279591 .anchor}**Draft**

#########  {#section .TOC-9}

**TABLE OF CONTENTS**

#  {#section-1 .TOC-1 .unnumbered}

# Draft [1](#__RefHeading___Toc20279591)I. Introduction [1](#i.-introduction) {#draft-1i.-introduction-1 .TOC-1}

# II. Background [1](#ii.-background) {#ii.-background-1 .TOC-1}

# III. Scope [2](#iii.-scope) {#iii.-scope-2 .TOC-1}

# IV. BUILDINGS AND FACILITIES [2](#iv.-buildings-and-facilities) {#iv.-buildings-and-facilities-2 .TOC-1}

## **A.** **Critical Area (Class 100)** [3](#a.-critical-area-class-100) {#a.-critical-area-class-100-3 .TOC-2}

## **B.** **Supporting Clean Areas** [4](#b.-supporting-clean-areas) {#b.-supporting-clean-areas-4 .TOC-2}

## **C.** **Clean Area Separation** [5](#c.-clean-area-separation) {#c.-clean-area-separation-5 .TOC-2}

## **D.** **Air Filtration** [5](#d.-air-filtration) {#d.-air-filtration-5 .TOC-2}

### 1. Membrane (Compressed Gases) [5](#membrane-compressed-gases) {#membrane-compressed-gases-5 .TOC-3}

### 2. High Efficiency Particulate Air (HEPA) [6](#high-efficiency-particulate-air-hepa) {#high-efficiency-particulate-air-hepa-6 .TOC-3}

## **E.** **Design** [7](#e.-design) {#e.-design-7 .TOC-2}

# V. PERSONNEL TRAINING, QUALIFICATION, & MONITORING [9](#v.-personnel-training-qualification-monitoring) {#v.-personnel-training-qualification-monitoring-9 .TOC-1}

## **A.** **Manufacturing Personnel** [10](#a.-manufacturing-personnel) {#a.-manufacturing-personnel-10 .TOC-2}

## **B.** **Laboratory Personnel** [12](#b.-laboratory-personnel) {#b.-laboratory-personnel-12 .TOC-2}

## **C.** **Monitoring Program** [12](#c.-monitoring-program) {#c.-monitoring-program-12 .TOC-2}

# VI. COMPONENTS AND CONTAINER/CLOSURES [12](#vi.-components-and-containerclosures) {#vi.-components-and-containerclosures-12 .TOC-1}

## **A.** **Components** [12](#a.-components) {#a.-components-12 .TOC-2}

## **B.** **Containers/Closures** [13](#b.-containersclosures) {#b.-containersclosures-13 .TOC-2}

### 1. Preparation [14](#preparation) {#preparation-14 .TOC-3}

### 2. Inspection of Container-Closure System [15](#inspection-of-container-closure-system) {#inspection-of-container-closure-system-15 .TOC-3}

# VII. ENDOTOXIN CONTROL [15](#vii.-endotoxin-control) {#vii.-endotoxin-control-15 .TOC-1}

# VIII. TIME LIMITATIONS [16](#viii.-time-limitations) {#viii.-time-limitations-16 .TOC-1}

# IX. PROCESS VALIDATION AND EQUIPMENT QUALIFICATION [17](#ix.-process-validation-and-equipment-qualification) {#ix.-process-validation-and-equipment-qualification-17 .TOC-1}

## **A.** **Process Simulations** [17](#a.-process-simulations) {#a.-process-simulations-17 .TOC-2}

### 1. Study Design [18](#study-design) {#study-design-18 .TOC-3}

### 2. Frequency and number of runs [18](#frequency-and-number-of-runs) {#frequency-and-number-of-runs-18 .TOC-3}

### 3. Size and Duration of runs [19](#size-and-duration-of-runs) {#size-and-duration-of-runs-19 .TOC-3}

### 4. Line Speed [20](#line-speed) {#line-speed-20 .TOC-3}

### 5. Environmental Conditions [20](#environmental-conditions) {#environmental-conditions-20 .TOC-3}

### 6. Media [20](#media) {#media-20 .TOC-3}

### 7. Incubation and Examination of Media Filled Units [21](#incubation-and-examination-of-media-filled-units) {#incubation-and-examination-of-media-filled-units-21 .TOC-3}

### 8. Interpretation of Test Results [22](#interpretation-of-test-results) {#interpretation-of-test-results-22 .TOC-3}

## **B.** **Filtration Efficacy** [23](#b.-filtration-efficacy) {#b.-filtration-efficacy-23 .TOC-2}

## **C.** **Sterilization of Equipment and Container/Closures** [24](#c.-sterilization-of-equipment-and-containerclosures) {#c.-sterilization-of-equipment-and-containerclosures-24 .TOC-2}

### 1. Sterilizer Qualification and Validation [25](#sterilizer-qualification-and-validation) {#sterilizer-qualification-and-validation-25 .TOC-3}

### 2. Equipment Controls and Instrument Calibration [26](#equipment-controls-and-instrument-calibration) {#equipment-controls-and-instrument-calibration-26 .TOC-3}

# X. LABORATORY CONTROLS [27](#x.-laboratory-controls) {#x.-laboratory-controls-27 .TOC-1}

## **A.** **Environmental Monitoring** [27](#a.-environmental-monitoring) {#a.-environmental-monitoring-27 .TOC-2}

### 1. General Written Program [27](#general-written-program) {#general-written-program-27 .TOC-3}

### 2. Establishing Limits and a Trending Program [28](#establishing-limits-and-a-trending-program) {#establishing-limits-and-a-trending-program-28 .TOC-3}

### 3. Sanitization Efficacy [29](#sanitization-efficacy) {#sanitization-efficacy-29 .TOC-3}

### 4. Monitoring Methods [29](#monitoring-methods) {#monitoring-methods-29 .TOC-3}

## **B.** **Microbiological Media and Identification** [30](#b.-microbiological-media-and-identification) {#b.-microbiological-media-and-identification-30 .TOC-2}

## **C.** **Pre-filtration Bioburden** [31](#c.-pre-filtration-bioburden) {#c.-pre-filtration-bioburden-31 .TOC-2}

## **D.** **Particulate Monitoring** [31](#d.-particulate-monitoring) {#d.-particulate-monitoring-31 .TOC-2}

# XI. STERILITY TESTING [31](#xi.-sterility-testing) {#xi.-sterility-testing-31 .TOC-1}

## **A.** **Choice of Methods** [32](#a.-choice-of-methods) {#a.-choice-of-methods-32 .TOC-2}

## **B.** **Media** [32](#b.-media) {#b.-media-32 .TOC-2}

## **C.** **Personnel** [33](#c.-personnel) {#c.-personnel-33 .TOC-2}

## **D.** **Sampling and Incubation** [33](#d.-sampling-and-incubation) {#d.-sampling-and-incubation-33 .TOC-2}

## **E.** **Investigation of Sterility Positives** [33](#e.-investigation-of-sterility-positives) {#e.-investigation-of-sterility-positives-33 .TOC-2}

# XII. BATCH RECORD REVIEW: PROCESS CONTROL DOCUMENTATION [36](#xii.-batch-record-review-process-control-documentation) {#xii.-batch-record-review-process-control-documentation-36 .TOC-1}

# [APPENDIX 1:]{.underline} ASEPTIC PROCESSING ISOLATORS [37](#appendix-1-aseptic-processing-isolators) {#appendix-1-aseptic-processing-isolators-37 .TOC-1}

## **A) Maintenance** [37](#a-maintenance) {#a-maintenance-37 .TOC-2}

### 1. General [37](#general) {#general-37 .TOC-3}

### 2. Glove Integrity [37](#glove-integrity) {#glove-integrity-37 .TOC-3}

## **B) Design** [37](#b-design) {#b-design-37 .TOC-2}

### 1. Airflow [37](#airflow) {#airflow-37 .TOC-3}

### 2. Materials of Construction [38](#materials-of-construction) {#materials-of-construction-38 .TOC-3}

### 3. Pressure Differential [38](#pressure-differential) {#pressure-differential-38 .TOC-3}

### 4. Clean Area Classifications [38](#clean-area-classifications) {#clean-area-classifications-38 .TOC-3}

## **C) Transfer of Materials/Supplies** [38](#c-transfer-of-materialssupplies) {#c-transfer-of-materialssupplies-38 .TOC-2}

### 1. Introduction: [39](#introduction) {#introduction-39 .TOC-3}

### 2. Discharge: [39](#discharge) {#discharge-39 .TOC-3}

## **D) Decontamination** [39](#d-decontamination) {#d-decontamination-39 .TOC-2}

### 1. Surface Exposure [39](#surface-exposure) {#surface-exposure-39 .TOC-3}

### 2. Efficacy [39](#efficacy) {#efficacy-39 .TOC-3}

### 3. Frequency [40](#frequency) {#frequency-40 .TOC-3}

## **E) Filling Line Sterilization** [40](#e-filling-line-sterilization) {#e-filling-line-sterilization-40 .TOC-2}

## **F) Environmental Monitoring** [40](#f-environmental-monitoring) {#f-environmental-monitoring-40 .TOC-2}

## **G) Personnel** [40](#g-personnel) {#g-personnel-40 .TOC-2}

# [APPENDIX 2:]{.underline} BLOW-FILL- SEAL TECHNOLOGY [41](#appendix-2-blow-fill--seal-technology) {#appendix-2-blow-fill--seal-technology-41 .TOC-1}

## **A) Equipment Design and Air Quality** [41](#a-equipment-design-and-air-quality) {#a-equipment-design-and-air-quality-41 .TOC-2}

## **B) Validation/Qualification** [42](#b-validationqualification) {#b-validationqualification-42 .TOC-2}

## **C) Batch Monitoring and Control** [42](#c-batch-monitoring-and-control) {#c-batch-monitoring-and-control-42 .TOC-2}

# [APPENDIX 3:]{.underline} PROCEsSING PRIOR TO FILLING/SEALING OPERATIONS [43](#appendix-3-processing-prior-to-fillingsealing-operations) {#appendix-3-processing-prior-to-fillingsealing-operations-43 .TOC-1}

## **A) Aseptic processing from early manufacturing steps** [43](#a-aseptic-processing-from-early-manufacturing-steps) {#a-aseptic-processing-from-early-manufacturing-steps-43 .TOC-2}

## **B) Aseptic processing of cell-based therapy products (or of products intended for use as cell based therapies)** [44](#b-aseptic-processing-of-cell-based-therapy-products-or-of-products-intended-for-use-as-cell-based-therapies) {#b-aseptic-processing-of-cell-based-therapy-products-or-of-products-intended-for-use-as-cell-based-therapies-44 .TOC-2}

# REFERENCES [45](#references) {#references-45 .TOC-1}

# RELEVANT GUIDANCE DOCUMENTS [46](#relevant-guidance-documents) {#relevant-guidance-documents-46 .TOC-1}

# DRAFT GLOSSARY [47](#draft-glossary) {#draft-glossary-47 .TOC-1}

#####  {#section-2 .TOC-5 .unnumbered}

Sterile Drug Products Produced by Aseptic Processing

Draft

#  

# I. Introduction

# 

# 

# II. Background 

There are basic differences between the production of sterile drug
products by aseptic processing and by terminal sterilization.

Terminal sterilization usually involves filling and sealing product
containers under conditions of a high quality environment; the product,
container, and closure in most cases have low bioburden but are not
sterile. The environment in which filling and sealing is performed is of
high quality in order to minimize the microbial content of the
in-process product, and to help ensure that the subsequent sterilization
process is successful. The product in its final container is then
subjected to a sterilization process such as heat or radiation.

In aseptic processing, the drug product, container, and closure are
subjected to sterilization processes separately, as appropriate, and
then brought together.[^1] Because there is no further processing to
sterilize the product after it is in its final container, it is critical
that containers be filled and sealed in an environment of extremely high
quality. Manufacturers should be aware that there are more variables
associated with aseptic processing than terminal sterilization. Before
aseptic assembly, different parts of the final product are generally
subjected to different sterilization processes, such as dry heat for
glass containers, moist heat sterilization for rubber closures, and
sterile filtration for a liquid dosage form. Each of the processes of
the aseptic manufacturing operation requires thorough validation and
control. Each also introduces the possibility of error that might
ultimately lead to the distribution of contaminated product. Any manual
or mechanical manipulation of the sterilized drug, components,
containers, and closures prior to or during aseptic assembly poses a
risk of contamination and thus necessitates careful control. The
terminally sterilized drug product, on the other hand, undergoes a
single sterilization process in a sealed container, thus limiting the
possibilities for error.[^2]

Manufacturers should have a keen awareness of the public health
implication of distributing a non-sterile drug purporting to be sterile.
Poor CGMP conditions at a manufacturing facility can ultimately pose a
life threatening health risk to a patient.

# 

# III. Scope

This document discusses only selected issues and thus does not address
all aspects of aseptic processing. Finished drug product CGMP issues are
primarily addressed, with only limited guidance regarding upstream bulk
processing steps. Updates relative to the 1987 document include guidance
on: personnel qualification, clean room classifications under dynamic
conditions, room design, quality control, environmental monitoring, and
review of production records. The aseptic processing isolator is also
discussed.

Although this document discusses CGMP issues relating to the
sterilization of components, containers, and closures, terminal
sterilization of the drug product is not addressed. It is a
well-accepted principle that sterile drugs should be manufactured by
aseptic processing only when terminal sterilization is not feasible.
However, unacceptable degradation of the product can occur as a result
of terminal sterilization, or the market presentation can afford some
unique and substantial clinical advantage not possible if terminal
sterilization were employed. In such cases, adjunct processing steps
(e.g., heat exposure conditions which provide some F~0~) to increase the
level of sterility confidence should be considered.

A list of references, which may be of value to the reader, is included
at the conclusion of this document.

# 

# IV. BUILDINGS AND FACILITIES 

+-----------------------------------------------------------------------+
| Section 211.42 (design and construction features) requires, in part,  |
| that aseptic processing operations be "performed within specifically  |
| defined areas of adequate size. There shall be separate or defined    |
| areas for the firm's operations to prevent contamination or mixups."  |
| Aseptic processing operations must also "include, as appropriate, an  |
| air supply filtered through high efficiency particulate air (HEPA)    |
| filters under positive pressure," as well as systems for "monitoring  |
| environmental conditions..."and "maintaining any equipment used to    |
| control aseptic conditions."                                          |
|                                                                       |
| Section 211.46 (ventilation, air filtration, air heating and cooling) |
| states, in part, that "equipment for adequate control over air        |
| pressure, microorganisms, dust, humidity, and temperature shall be    |
| provided when appropriate for the manufacture, processing, packing or |
| holding of a drug product." This regulation also states that "air     |
| filtration systems, including pre-filters and particulate matter air  |
| filters, shall be used when appropriate on air supplies to production |
| areas."                                                               |
+-----------------------------------------------------------------------+

In aseptic processing, there are various areas of operation which
require separation and control, with each area having different degrees
of air quality depending on the nature of the operation. Area design is
based upon satisfying microbiological and particulate standards defined
by the equipment, components, and products exposed, as well as the
particular operation conducted, in the given area.

Critical and support areas of the aseptic processing operation should be
classified and supported by microbiological and particulate data
obtained during qualification studies. While initial clean room
qualification includes some assessment of air quality under as-built and
static conditions, the final room or area classification should be
derived from data generated under dynamic conditions, i.e., with
personnel present, equipment in place, and operations ongoing. The
aseptic processing facility monitoring program should assess conformance
with specified clean area classifications under dynamic conditions, on a
routine basis.

The following table summarizes clean area air classifications (Ref. 1).

#  {#section-8 .TOC-1}

**TABLE 1- Air Classifications^a\ ^**

+-------------+-------------+-------------+-------------+-------------+
| Clean Area  | [\>]{       | [\>]{       | Micr        |             |
| Cla         | .underline} | .underline} | obiological |             |
| ssification | 0.5 um      | 0.5 um      | Limit^b^    |             |
|             | part        | par         |             |             |
|             | icles/ft^3^ | ticles/m^3^ | cfu/10      |             |
|             |             |             | ft^3^       |             |
|             |             |             | cfu/m^3^    |             |
+-------------+-------------+-------------+-------------+-------------+
| 100         | 100         | 3,500       | \<1^c^      | \<3^c^      |
+-------------+-------------+-------------+-------------+-------------+
| 1000        | 1000        | 35,000      | [\<]{       | [\<]{.      |
|             |             |             | .underline} | underline}7 |
|             |             |             | 2           |             |
+-------------+-------------+-------------+-------------+-------------+
| 10,000      | 10,000      | 350,000     | [\<]{       | [\<]{       |
|             |             |             | .underline} | .underline} |
|             |             |             | 5           | 18          |
+-------------+-------------+-------------+-------------+-------------+
| 100,000     | 100,000     | 3,500,000   | [\<]{.u     | [\<]{       |
|             |             |             | nderline}25 | .underline} |
|             |             |             |             | 88          |
+-------------+-------------+-------------+-------------+-------------+

a.  All classifications based on data measured in the vicinity of
    exposed articles during periods of activity.

b- Alternate microbiological standards may be established where
justified by the nature of the operation. c- Samples from class 100
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)**

A critical area is one in which the sterilized drug product, containers,
and closures are exposed to environmental conditions designed to
preserve sterility. Activities conducted in this area 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 the product is not processed further in
its immediate container and is vulnerable to contamination. In order to
maintain product sterility, the environment in which aseptic operations
are conducted should be of appropriate quality throughout operations.
One aspect of environmental quality is the particulate content of the
air. Particulates are significant because they can enter a product and
contaminate it physically or, by acting as a vehicle for microorganisms,
biologically. Particle content in critical areas should be minimized by
effective air systems.

Air in the immediate proximity of exposed sterilized containers/closures
and filling/closing operations is of acceptable particulate quality when
it has a per-cubic-foot particle count of no more than 100 in a size
range of 0.5 micron and larger (Class 100) when counted at
representative locations normally not more than one foot away from the
work site, within the airflow, and during filling/closing operations.
Deviations from this critical area monitoring parameter should be
documented as to origin and significance.

Measurements to confirm air cleanliness in aseptic processing zones
should be taken with the particle counting probe oriented in the
direction of oncoming airflow and at specified sites where sterilized
product and container-closure are exposed. Regular monitoring should be
performed during each shift. Nonviable particulate monitoring with a
remote counting system is generally less invasive than the use of
portable particle counting units and provides the most comprehensive
data. See Section X.D, \"Particulate Monitoring.\"

Some powder filling operations can generate high levels of powder
particulates 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 noise\" levels of powder particles from air contaminants.
In these instances, air should be sampled in a manner that, to the
extent possible, characterizes the true level of extrinsic particulate
contamination to which the product is exposed. Initial certification of
the area under dynamic conditions without the actual powder filling
function should provide some baseline information on the non-product
particle generation of the operation.

Air in critical areas should be supplied at the point of use as HEPA
filtered laminar flow air at a velocity sufficient to sweep particulate
matter away from the filling/closing area and maintain laminarity during
operations. The velocity parameters established for each processing line
should be justified, and appropriate to maintain laminarity and air
quality under dynamic conditions within a defined space (Ref. 2).[^3]

Proper design and control should prevent turbulence or stagnant air in
the aseptic processing line or clean zone. Once relevant parameters are
established, airflow patterns should be evaluated for turbulence. Air
pattern or "smoke" studies demonstrating laminarity and sweeping action
over and away from the product under dynamic conditions should be
conducted. The studies should be well-documented with written
conclusions. Videotape or other recording mechanisms have been found to
be useful in assessing airflow initially as well as facilitating
evaluation of subsequent equipment configuration changes. However, even
successfully qualified systems can be compromised by poor personnel,
operational, or maintenance practices.

Active air monitoring of critical areas should normally yield no
microbiological contaminants. Contamination in this environment should
receive investigative attention.

## **B. Supporting Clean Areas**

Supporting clean areas include various classifications and functions.
Many support areas function as zones in which non-sterile components,
formulated product, in‑process materials, equipment, and
container/closures are prepared, held, or transferred. These
environments should be designed to minimize the level of particulate
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 should
determine its classification. An area classified at Class 100,000 would
be used for less critical activities (such as initial equipment
preparation). The area immediately adjacent to the aseptic processing
line should, at a minimum, meet Class l0,000 standards (see Table 1)
under dynamic conditions. Depending on the operation, manufacturers can
also classify this area as Class 1000 or maintain the entire aseptic
filling room at Class 100.

## **C. Clean Area Separation** 

Adequate separation is necessary between areas of operation to prevent
contamination (211.42). In order to maintain air quality in areas of
higher cleanliness, it is important to achieve a proper airflow and a
positive pressure differential relative to adjacent less clean areas.
Rooms of higher classification should have a positive pressure
differential relative to adjacent lower classified areas of generally at
least 0.05 inch of water (with doors closed). When doors are open,
outward airflow should be sufficient to minimize ingress of
contamination (Ref. 3). Pressure differentials between clean rooms
should be monitored continuously throughout each shift and frequently
recorded, and deviations from established limits investigated.

An adequate air change rate should be established for a cleanroom. For
Class 100,000 supporting rooms, airflow sufficient to achieve at least
20 air changes per hour is typically acceptable.

Facility monitoring systems should be established to rapidly detect
atypical changes that can compromise the facility's environment.
Operating conditions should be restored to established, qualified levels
before reaching action levels. For example, pressure differential
specifications should enable prompt detection (i.e., alarms) of any
emerging low pressure problem in order to preclude ingress of
unclassified air into a classified room.

## **D. Air Filtration** 

### 1. Membrane (Compressed Gases)

A compressed gas should be of appropriate purity (e.g., free from oil
and water vapor) and its microbiological and particulate quality should
be equal to or better than air in the environment into which the gas is
introduced. Compressed gases such as air, nitrogen, and carbon dioxide
are often used in clean rooms and are frequently employed in operations
involving purging or overlaying.

Membrane filters allow for the filtration of compressed gases to meet an
appropriate high quality standard, and can be used to produce a sterile
compressed gas. A sterile-filtered gas is used when the gas contacts a
sterilized material. Certain equipment also should be supplied with a
sterile-filtered gas. For example, sterile bacterial retentive membrane
filters should be used for autoclave air lines, lyophilizer vacuum
breaks, vessels containing sterilized materials, and hot air sterilizer
vents. Sterilized tanks or liquids should be held under continuous
overpressure to prevent microbial contamination. Safeguards should be in
place to prevent a pressure change that can result in contamination due
to back flow of non-sterile air or liquid.

Gas filters (including vent filters) should be dry. Condensate in a gas
filter can cause blockage or microbial contamination. Frequent
replacement, heating, and use of hydrophobic filters prevent moisture
residues in a gas supply system. These filters also should be integrity
tested upon installation, and periodically thereafter (e.g., including
at end of use). Integrity test failures should be investigated.

### 2. High Efficiency Particulate Air (HEPA)[^4]

An essential element in ensuring aseptic conditions is the maintenance
of HEPA filter integrity. Integrity testing should be performed at
installation to detect leaks around the sealing gaskets, through the
frames or through various points on the filter media. Thereafter,
integrity 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 needed when air quality is found to be
unacceptable, or as part of an investigation into a media fill or drug
product sterility failure. Among the filters that should be integrity
tested are those installed in dry heat depyrogenation tunnels commonly
used to depyrogenate glass vials.

One recognized method of testing the integrity of HEPA filters is use of
a dioctylphthalate (DOP) aerosol challenge. However, alternative
aerosols may be acceptable. Poly-alpha-olefin can also be used, provided
it meets specifications for critical physicochemical attributes such as
viscosity. Some alternative aerosols are problematic because they pose a
risk of microbial contamination of the environment being tested. Firms
should ensure that any alternative does not promote microbial growth.

An intact HEPA filter is capable of retaining at least 99.97 percent of
particulates greater than 0.3 micron in diameter. It is important to
ensure that the aerosol used for the challenge has a sufficient number
of particles of this size range. Performing an integrity test without
introducing particles of known size upstream of the filter is
ineffective for detecting leaks. The DOP challenge should introduce the
aerosol upstream of the filter in a concentration of 80 to l00
micrograms/liter of air at the filter\'s designed airflow rating. The
downstream side of the filter is then scanned with an appropriate
photometer probe at a sampling rate of at least one cubic foot per
minute. Scanning 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. While
vendors often provide these services, the drug manufacturer is
responsible for ensuring that these essential certification activities
are conducted satisfactorily. A single probe reading equivalent to 0.01
percent of the upstream challenge should be considered as indicative of
a significant leak and should result in replacement of the HEPA filter
or perhaps repair in a limited area. A subsequent confirmatory re-test
should be performed in the area of any repair.

There is a major difference between filter integrity testing and
efficiency testing. The purpose of regularly scheduled integrity testing
is to detect leaks from the filter media, filter frame and seal. The
challenge is a polydispersed aerosol usually composed of particles
ranging in size from one to three microns. The test is done in place and
the filter face is scanned with a probe; the measured downstream leakage
is taken as a percent of the upstream challenge. The efficiency test, on
the other hand, is a test used only to determine the rating of the
filter.[^5]

HEPA filter integrity testing alone is not sufficient to monitor filter
performance. This testing is usually done only on a semi-annual basis.
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 generally increase the possibility of
contamination, as these changes (e.g., velocity reduction) can have an
effect on the laminarity of the airflow. Airflow velocities are measured
six inches from the filter face or at a defined distance proximal to the
work surface for each HEPA filter. For example, velocity monitoring as
frequently as weekly may be appropriate for the clean zone in which
aseptic processing is performed. HEPA filters should be replaced when
inadequate airflow (e.g., due to blockage) or non-uniformity of air
velocity across an area of the filter is detected.

## **E. Design**

+-----------------------------------------------------------------------+
| Section 211.42 requires that aseptic processing operations be         |
| "performed within specifically defined areas of adequate size. There  |
| shall be separate or defined areas for the firm's operations to       |
| prevent contamination or mixups."                                     |
|                                                                       |
| Section 211.42 states that "flow of components, drug products         |
| containers, closures, labeling, in-process materials, and drug        |
| products through the building or building shall be designed to        |
| prevent contamination." HEPA filtered air as appropriate, as well as  |
| "floors, walls and ceilings of smooth, hard surfaces that are easily  |
| cleanable" are some additional requirements of this section.          |
|                                                                       |
| Section 211.63 states that equipment "shall be of appropriate design, |
| adequate size, and suitably located to facilitate operations for its  |
| intended use and for its cleaning and maintenance."                   |
|                                                                       |
| Section 211.65 states that "equipment shall be constructed so that    |
| surfaces that contact the 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."       |
|                                                                       |
| Section 211.68 includes requirements for "automatic, mechanical and   |
| electronic equipment."                                                |
|                                                                       |
| Section 211.113 states that "appropriate written procedures, designed |
| to prevent microbiological contamination of drug products purporting  |
| to be sterile, shall be established and followed."                    |
+-----------------------------------------------------------------------+

An aseptic process is designed to minimize exposure of sterile articles
to dynamic conditions and potential contamination hazards presented by
the operation. Limiting the duration of open container exposure,
providing the highest possible environmental control, and designing
equipment to prevent entrainment of lower quality air into the Class 100
zone are essential to this goal (Ref. 3).

Any intervention or stoppage during an aseptic process can increase the
risk of contamination. Personnel and material flow should be optimized
to prevent unnecessary activities that 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 flow of
personnel should be designed to limit the frequency with which entries
and exits are made to and from the aseptic processing room and, more
significantly, its critical area. In order to prevent changes in air
currents that introduce lower quality air, movement adjacent to the
critical area should be limited. For example, personnel intervention can
be reduced by integrating an on-line weight check device, thus
eliminating a repeated manual activity within the critical zone. It is
also important to minimize the number of personnel in the aseptic
processing room.

Transfer of products should be performed under appropriate clean room
conditions. For example, lyophilization processes include transfer of
aseptically filled product in partially-sealed containers. To prevent
contamination, partially-closed sterile product should be staged and
transferred only in critical areas. Facility design should assure that
the area between a filling line and the lyophilizer, and the transport
and loading procedures, provide Class 100 protection.

The sterile product and container-closures should also be protected from
activities occurring adjacent to the line. Carefully designed curtains,
rigid plastic shields, or other barriers should be used in appropriate
locations to partially segregate the aseptic processing line.

Airlocks and interlocking doors facilitate better control of air balance
throughout the aseptic processing area. Airlocks should be installed
between the aseptic processing area entrance and the adjoining
uncontrolled area. Other interfaces such as personnel entries, or the
juncture of the aseptic processing room and its adjacent room, are also
appropriate locations for air locks.

Clean rooms are normally designed as functional units with specific
purposes. A well-designed clean room is constructed with material that
allows for 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 are constructed
of smooth, hard surfaces that can be easily cleaned (211.42). Ceilings
and associated HEPA filter banks should be designed to protect sterile
materials from contamination. Clean rooms also should not contain
unnecessary equipment, fixtures, or materials.

Processing equipment and systems should be equipped with sanitary
fittings and valves. Drains are not considered appropriate for rooms in
classified areas of the aseptic processing facility.

When applicable, equipment must be suitably designed for ease of
sterilization (211.63). The effect of equipment layout and design on the
clean room environment should be addressed. Flat surfaces or ledges that
accumulate dust and debris should be avoided. Equipment should not
obstruct airflow and, in critical zones, its design should not perturb
airflow.

# V. PERSONNEL TRAINING, QUALIFICATION, & MONITORING

+-----------------------------------------------------------------------+
| Sections 211.22 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."                                          |
|                                                                       |
| Section 211.113(b) addresses the procedures designed to prevent       |
| microbiological contamination, stating that "appropriate written      |
| procedures, designed to prevent microbiological contamination of drug |
| products purporting to be sterile, shall be established and           |
| followed."                                                            |
|                                                                       |
| Section 211.25, Personnel Qualifications requires that "each person   |
| engaged in 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...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." This       |
| section also requires "an adequate number of qualified personnel to   |
| perform and supervise the manufacture, processing, packing or holding |
| of each drug product." Section 211.25 also requires that continuing   |
| training in CGMP "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." |
| The 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."                                           |
|                                                                       |
| Section 211.28, Personnel Responsibilities 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." It also states that "personnel shall practice good          |
| sanitization and health habits" and specifies that "protective        |
| apparel, such as head, face, hand, and arm coverings, shall be worn   |
| as necessary to protect drug products from contamination." It also    |
| states that "any person shown at any time (either by medical          |
| examination or supervisory examination) 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."                                                       |
|                                                                       |
| This section also addresses restrictions on entry into limited access |
| areas: "Only personnel authorized by supervisory personnel shall      |
| enter those areas of the buildings and facilities designated as       |
| limited‑access areas."                                                |
|                                                                       |
| Section 211.42 requires the establishment of a "system for monitoring |
| environmental conditions."                                            |
+-----------------------------------------------------------------------+

## 

## **A. Manufacturing Personnel**

A well-designed aseptic process minimizes personnel intervention. As
operator activities increase in an aseptic processing operation, the
risk to finished product sterility also increases. It is essential that
operators involved in aseptic manipulations adhere to the basic
principles of aseptic technique at all times to assure maintenance of
product sterility.

Appropriate training should be conducted before an individual is
permitted to enter the aseptic processing area and perform operations.
For example, such training should include aseptic technique, clean room
behavior, microbiology, hygiene, gowning, and patient safety hazard
posed by a non-sterile drug product, and the specific written procedures
covering aseptic processing area operations. After initial training,
personnel should be updated regularly by an ongoing training program.
Supervisory personnel should routinely evaluate each operator's
conformance to written procedures during actual operations. Similarly,
the quality control unit should provide regular oversight of adherence
to established, written procedures and basic aseptic techniques during
manufacturing operations.

Adherence to basic aseptic technique is a continuous requirement for
operators in an aseptic processing operation. Some of these techniques
aimed at maintaining sterility of sterile items and surfaces include:

> 1\. *Contacting sterile materials only with sterile instruments*.
> Sterile instruments (e.g., forceps) are should always be used in the
> handling of sterilized materials. Between uses, instruments should be
> placed only in sterilized containers. These instruments should be
> replaced as necessary throughout the operation.
>
> After initial gowning, sterile gloves should be regularly sanitized to
> minimize the risk of contamination. Personnel should not directly
> contact sterile products, containers, closures, or critical surfaces.
>
> 2\. *Moving slowly and deliberately.* Rapid movements can create
> unacceptable turbulence in the critical zone. Such movements disrupt
> the sterile field, presenting a challenge beyond intended cleanroom
> design and control parameters. The principle of slow, careful movement
> should be followed throughout the cleanroom.
>
> 3\. *Keeping the entire body out of the path of laminar air*. Laminar
> airflow design is used to protect sterile equipment surfaces,
> container-closures, and product. Personnel should not disrupt the path
> of laminar flow air in the aseptic processing zone.
>
> 4\. *Approaching a necessary manipulation in a manner that does not
> compromise sterility of the product.* In order to maintain sterility
> of nearby sterile materials, a proper aseptic manipulation should be
> approached from the side and not above the product (in vertical
> laminar flow operations). Also, speaking when in direct proximity to
> an aseptic processing line is not an acceptable practice.

Personnel who have been qualified and permitted access to the aseptic
processing area should be appropriately gowned. An aseptic processing
area 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. Gowns need to be sterile and
non-shedding, and should cover the skin and hair. Face masks, hoods,
beard/moustache covers, protective goggles, elastic gloves, clean room
boots, and shoe overcovers are examples of common elements of gowns. An
adequate barrier should be created by the overlapping of gown components
(e.g., gloves overlapping sleeves). If an element of the gown is found
to be torn or defective, it should be changed immediately.

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 sterile quality of the gown after
performance of gowning procedures. Gowning qualification should include
microbiological surface sampling of several locations on a gown (e.g.,
glove fingers, facemask, forearm, chest, other sites). Following an
initial assessment of gowning, periodic requalification should monitor
various gowning locations over a suitable period to ensure the
consistent acceptability of aseptic gowning techniques. Semi-annual or
yearly requalification is acceptable for automated operations where
personnel involvement is minimized.

To protect exposed sterilized product, personnel are expected to
maintain sterile gown quality and aseptic method standards in a
consistent manner. Written procedures should adequately address
circumstances under which personnel should be retrained, requalified, or
reassigned to other areas.

## 

## **B. Laboratory Personnel**

The basic principles of training, aseptic technique, and personnel
qualification in aseptic manufacturing are equally applicable to those
performing aseptic sampling and microbiological laboratory analyses.
Processes and systems cannot be considered to be in control and
reproducible if there is any question regarding the validity of data
produced by the laboratory.

## 

## **C. Monitoring Program** 

Personnel can have substantial impact on 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 aseptic processing
operator\'s gloves on an at least a daily basis, or in association with
each batch. This sampling should be accompanied by an appropriate
sampling frequency for other strategically selected locations of the
gown (Ref. 7). 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 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 may include increased sampling, increased observation,
retraining, gowning requalification, and in certain instances,
reassignment of the individual to operations outside of the aseptic
processing area. Microbiological trending systems, and assessment of the
impact of atypical trends, are discussed in more detail under Section
XI., Laboratory Controls.

# VI. COMPONENTS AND CONTAINER/CLOSURES

## **A. Components**

+-----------------------------------------------------------------------+
| Section 210.3(b)(3) defines a \"component\" as "any ingredient        |
| intended for use in the manufacture of a drug product, including      |
| those that may not appear in such drug product."                      |
|                                                                       |
| Section 211.80, General Requirements, requires, in part, the          |
| establishment of written procedures "describing in sufficient detail  |
| the receipt, identification, storage, handling, sampling, sampling,   |
| testing, and approval or rejection of components and drug product     |
| containers and closures...Components and drug product containers and  |
| closures shall at all times be handled and stored in a manner to      |
| prevent contamination."                                               |
|                                                                       |
| Section 211.84, Testing and approval or rejection of components, drug |
| product containers, and closures, requires that "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." |
+-----------------------------------------------------------------------+

A drug product produced by aseptic processing can become contaminated by
use of one or more components (e.g., active ingredients, excipients,
Water for Injection) that are contaminated with microorganisms or
endotoxins. It is important to characterize the microbial content of
each component liable to contamination and establish appropriate
acceptance/rejection limits based on information on bioburden. Knowledge
of bioburden is critical in assessing whether the sterilization process
is adequate.

In aseptic processing, each component is individually sterilized or
several components are combined, with the resulting mixture
sterilized.[^6] There are several methods to sterilize 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
USP Water For Injection (WFI). 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 involves subjecting the filtered
solution to aseptic crystallization and precipitation 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. If a component is not adversely affected by heat, and
is soluble, it may be made into a solution and subjected to steam
sterilization, typically in an autoclave or a pressurized vessel.

Dry heat sterilization is a suitable method for components that are heat
stable and insoluble. However, carefully designed heat penetration and
distribution studies should be performed for powder sterilization
because of the insulating effects of the powder.

Ethylene oxide (EtO) exposure is often used for surface sterilization.
Such methods should be carefully controlled and validated if used for
powders to evaluate whether consistent penetration of the sterilant is
achieved and to minimize residual ethylene oxide and by-products.

Parenteral products are intended to be non-pyrogenic. 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 endotoxin specifications should be rejected.

# 

## **B. Containers/Closures**

+-----------------------------------------------------------------------+
| Section 211.94 (drug product containers and closures) 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." It also 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."                                                            |
|                                                                       |
| Section 211.113(b) requires "validation of any sterilization process" |
| as part of designing procedures "to prevent microbiological           |
| contamination of drug products purporting to be sterile."             |
+-----------------------------------------------------------------------+

### 1. Preparation 

Containers and closures should be rendered sterile and, for parenteral
drug products, pyrogen-free. The type of processes used will depend
primarily on the nature of the material comprising the container and/or
closure. The validation study for any such process should be adequate to
demonstrate its ability to render materials sterile and pyrogen-free.
Written procedures should specify the frequency of revalidation of these
processes as well as time limits for holding sterile, depyrogenated
containers and closures.

Presterilization preparation of glass containers usually involves a
series of wash and rinse cycles. These cycles serve an important role in
removing foreign matter. Rinse water should be of high purity so as not
to contaminate containers. For parenteral products, final rinse water
should meet the specifications of Water for Injection, USP.

The adequacy of the depyrogenation process can be assessed by spiking
containers or closures with known quantities of endotoxin, followed by
measuring endotoxin content after depyrogenation. The challenge studies
should be performed with a reconstituted endotoxin solution applied
directly onto the surface being tested and air-dried. 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% (3 logs).

Glass containers are generally subjected to dry heat for sterilization
and depyrogenation. Validation of dry heat sterilization/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.

Pyrogen on plastic containers can be generally removed by multiple WFI
rinses. Plastic containers can be sterilized with an appropriate gas,
irradiation or other suitable means. For gases such as EtO, 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. Biological indicators are of special importance in
demonstrating the effectiveness of EtO and other gas sterilization
processes.

Rubber closures (e.g., stoppers and syringe plungers) are 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 Purified Water USP of minimal endotoxin
content, followed by final rinse(s) with WFI for parenteral products.
Normally, depyrogenation is achieved by multiple rinses of hot WFI. The
time between washing and sterilizing should be minimized because
moisture on the stoppers can support microbial growth and the generation
of endotoxins. Because rubber is a poor conductor of heat, extra
attention should be given to the validation of processes that use heat
to sterilize rubber stoppers. Validation data should also 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 be
rendered sterile and should not have an adverse effect on the safety,
quality, or purity of the drug product.

See Section VIII for discussion of the need to establish production time
limits for the holding of sterilized containers and closures.

Contract facilities that perform sterilization and depyrogenation of
containers and closures are subject to the same CGMP requirements as
those established for in-house processing. The finished dosage form
manufacturer is responsible for the review and approval of the
contractor\'s validation protocol and final validation report.

# 

### 2. Inspection of Container-Closure System

A container-closure system that permits penetration of air, or
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 non-sterility. Equipment suitability problems or incoming
container or closure deficiencies have caused loss of container-closure
system integrity. As examples, failure to detect vials fractured by
faulty machinery, or 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 should be investigated in accord with
Section 211.192.

#  {#section-14 .TOC-1}

# VII. ENDOTOXIN CONTROL

+-----------------------------------------------------------------------+
| Section 211.63, equipment design, size, and location, states that     |
| equipment "shall be of appropriate design, adequate size, and         |
| suitably located to facilitate operations for its intended use and    |
| for its cleaning and maintenance.\"                                   |
|                                                                       |
| Section 211.65, equipment construction requires, in part, that        |
| "equipment shall be constructed so that surfaces that contact the     |
| 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."                          |
|                                                                       |
| Section 211.67, equipment cleaning and maintenance requires, 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."                                            |
|                                                                       |
| Section 211.94 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."                       |
|                                                                       |
| Section 211.167 states: "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 be 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 need for appropriate CGMP controls to prevent
generation of endotoxin. Drug product components, container-closures,
equipment, and storage time limitations are among the concerns to
address in establishing endotoxin control.

Adequate cleaning, drying, and storage of equipment provides for control
of bioburden and prevents contribution of endotoxin load. Equipment
should be designed to be easily assembled and disassembled, cleaned,
sanitized, and/or sterilized. Endotoxin control should be exercised for
all product contact surfaces both prior to and after sterile filtration.

Endotoxin on equipment surfaces is inactivated by high temperature dry
heat, or removed from equipment surfaces by validated 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. Sterilizing filters and moist heat
sterilization have not been shown to be effective in removing
endotoxins. Processes that are designed to achieve depyrogenation should
demonstrate a 3-log reduction of endotoxin.

# VIII. TIME LIMITATIONS

  -----------------------------------------------------------------------
  Section 211.111 (time limitations on production) states: "When
  appropriate, time limits for the completion of each phase of production
  shall be established to assure the quality of the drug product."

  -----------------------------------------------------------------------

Time limits should be established for each phase of aseptic processing.
Time limits should include, for example, the period between the start of
bulk product compounding and its filtration, filtration processes,
product exposure while on the processing line, and storage of sterilized
equipment, containers and closures. Maintenance of in-process quality at
different 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 in order to prevent microorganisms from penetrating
the filter. Such a time limit should also prevent a significant increase
in upstream bioburden and endotoxin load. Sterilizing filters should
generally be replaced following each manufactured lot. 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. PROCESS VALIDATION AND EQUIPMENT QUALIFICATION 

+-----------------------------------------------------------------------+
| Section 211.113(b) (control of microbiological contamination) states: |
| "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."                                           |
|                                                                       |
| Sections 211.63, 211.65, and 211.67 address, respectively,            |
| "Equipment, design, size, and location," "Equipment construction,"    |
| and "Equipment cleaning and maintenance."                             |
|                                                                       |
| Section 211.84(c)(3) states that "sterile equipment and aseptic       |
| sampling techniques shall be used when necessary."                    |
+-----------------------------------------------------------------------+

The following sections primarily discuss routine qualification and
validation study expectations. Change control procedures are only
briefly addressed, but are an important part of the quality systems
established by a firm. A change in equipment, process, test method, or
systems requires evaluation through the written change control program,
and should trigger an evaluation of the need for revalidation or
requalification.

## **A. Process Simulations**

In order to ensure the sterility of products purporting to be sterile,
both sterilization and aseptic filling/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, the container and the closure) are brought together
under conditions that contaminate those elements. Similarly, product
sterility is compromised when the product elements are non-sterile at
the time they are assembled.

The validation of an aseptic processing operation should include the use
of a microbiological growth nutrient medium in place of product. This
has been termed a "media fill" or "process simulation." The nutrient
medium is exposed to product contact surfaces of equipment, container
systems, critical environments, and process manipulations to closely
simulate the same exposure that the product itself will undergo. The
sealed containers filled with the media are then incubated to detect
microbial contamination. The results are interpreted to determine the
potential for any given unit of drug product to become contaminated
during actual operations (e.g., start-up, sterile ingredient additions,
aseptic connections, filling, closing). Environmental monitoring data is
integral to the validation of an aseptic processing operation.

### 

### 1. Study Design 

A validation protocol should detail the overall strategy, testing
requirements, and acceptance criteria for the media fill. Media fill
studies should simulate aseptic manufacturing operations as closely as
possible, incorporating a \"worst-case\" approach. A media fill study
should address applicable issues such as:

a\) factors associated with the longest permitted run on the processing
line

b\) ability to produce sterile units when environmental conditions
impart a greater risk to the product

c\) number and type of normal interventions, atypical interventions,
unexpected events (e.g., maintenance), stoppages, equipment adjustments
or transfers

d\) lyophilization, when applicable

e\) aseptic assembly of equipment (e.g., at start-up, during processing)

f\) number of personnel and their activities

g\) number of aseptic additions (e.g., charging containers and closures
as well as sterile ingredients)

h\) shift changes, breaks, and gown changes (when applicable)

i\) number and type of aseptic equipment disconnections/connections

j\) aseptic sample collections

k\) line speed and configurations

l\) manual weight checks

m\) operator fatigue

n\) container-closure systems (e.g., sizes, type, compatibility with
equipment)

o\) consideration of temperature and humidity set point extremes

p\) specific provisions of aseptic processing related Standard Operating
Procedures (conditions permitted before line clearance is mandated,
etc.).

A written batch record, documenting conditions and activity simulated,
should be prepared for each media fill run. The same vigilance should be
observed in both media fill and routine production runs. Media fills
cannot be used to \"validate\" an unacceptable practice.

### 2. Frequency and number of runs 

When a processing line is initially validated, separate 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. A minimum of three consecutive separate
successful runs should be performed during initial line qualification.
Subsequently, routine semi-annual revalidation runs should be conducted
for each shift and processing line to evaluate the state of control of
the aseptic process. All personnel who enter the aseptic processing
area, including technicians and maintenance personnel, should
participate in a media fill at least once a year

Each change to a product or line change should be evaluated using a
written change control system. Any changes or events that appear 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 modification, line
configuration change, significant changes in personnel, anomalies in
environmental testing results, container-closure system changes or, end
product sterility testing showing contaminated products may be cause for
revalidation of the system.

Where a media fill's data indicates the process may not be in control, a
comprehensive documented investigation should be conducted to determine
the origin of the contamination and the scope of the problem. Once
corrections are instituted, multiple repeat process simulation runs
should be performed to confirm that deficiencies in practices and
procedures have been corrected and the process has returned to a state
of control. However, when an investigation fails to reach
well-supported, substantive conclusions as to the cause of the media
fill failure, three consecutive successful runs and increased scrutiny
(i.e., extra supervision, monitoring) of the production process should
be implemented.

### 

### 3. Size and Duration of runs 

The duration of aseptic processing operations is a major consideration
in determining the size of the media fill run. Although the most
accurate simulation model would be the full batch size and duration
because it most closely simulates the actual production run, other
appropriate models can be justified. In any study protocol, the duration
of the run and the overall study design should adequately mimic
worst-case operating conditions and cover all manipulations that are
performed in the actual processing operation. Adequate batch sizes are
needed to simulate commercial production conditions and accurately
assess the potential for commercial batch contamination. The number of
units filled should be sufficient to reflect the effects of potential
operator fatigue, as well as the maximum number of interventions and
stoppages. The run should be large enough to accurately simulate
production conditions and sensitive enough to detect a low incidence of
contaminated units. For batches produced over multiple shifts or
yielding an unusually large number of units, the media fill protocol
should adequately encompass conditions and any potential risks
associated with the larger operation.

While conventional manufacturing lines are highly automated, often
operate at relatively high speeds, and are designed to limit operator
intervention, there are some processes that 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 in order to best simulate operator fatigue.

For simulation of lyophilization operations, unsealed containers should
be exposed to pressurization and partial evacuation of the chamber in a
manner that is representative of process stresses. Vials should not be
frozen, as this may inhibit the growth of microorganisms.

### 4. Line Speed 

The media fill program should adequately address the range of line
speeds (e.g., by bracketing all vial sizes and fill volumes) employed
during production. In some cases, more than one line speed should be
evaluated in the course of a study.

Each individual media fill run should evaluate a single worst-case line
speed and the speed chosen for each batch during a study should be
justified. For example, use of high line speed is justified for
manufacturing processes characterized by frequent interventions or a
significant degree of manual manipulation. Use of slow line speed is
justified for manufacturing processes characterized by prolonged
exposure of sterile components in the aseptic area

### 5. Environmental Conditions 

Media fills should be conducted under environmental conditions that
simulate normal as well as \"worst case\" conditions of production. 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, it is crucial
that media fills should include rigorous challenges in order to support
the validity of these studies.

### 

### 6. Media 

In general, a microbiological growth medium such as soybean casein
digest medium should be used. Use of anaerobic growth media (such as
Fluid Thioglycollate Medium) is appropriate in special circumstances.
Media selected should be demonstrated to promote growth of USP \<71\>
indicator microorganisms as well as isolates that have been identified
by environmental monitoring, personnel monitoring, and positive
sterility test results. Positive control units should be inoculated with
a \<100 CFU challenge and incubated. For those instances in which the
growth promotion testing fails, the origin of any contamination found
during the simulation should nonetheless be investigated and the media
fill should be promptly repeated.

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 and swirled) and permit visual detection of
microbial growth.

Some drug manufacturers have expressed concern over the possible
contamination of the facility and equipment with the 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.

### 7. Incubation and Examination of Media Filled Units 

### 

Media units should be incubated for a sufficient time (a period of not
less than 14 days) at a temperature adequate to enhance detection of
organisms that can otherwise be difficult to culture.

Each media filled unit should be examined for contamination by personnel
with appropriate education, training and experience in microbiological
techniques. There should be direct quality control unit oversight
throughout any such examination. Clear containers with otherwise
identical physical properties should be used as a substitute for amber
or other opaque containers to allow visual detection of microbial
growth.

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.[^7] 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 batch, because the incubation of
the units simulates release to the market. Any decision to exclude such
incubated units (i.e., non-integral) from the final batch 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, these intervention units do not need to be incubated during
media fills. Where procedures lack specificity, there would be
insufficient justification for exclusion of units removed during an
intervention from incubation. As an example, if a production procedure
requires removal of ten 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, which can result in
removal of a positive unit caused by an unrelated event or intervention.
If unavoidable, appropriate study provisions should be made to
compensate in such instances.

Appropriate criteria should be established for yield and accountability.
Batch record reconciliation documentation should include an accurate
accounting and description of units rejected from a batch.

### 8. Interpretation of Test Results 

The process simulation run should be observed, and contaminated units
should be reconcilable with the approximate time and the activity being
simulated during the media fill. Videotaping of a media fill has been
found to be useful in identifying personnel practices which could
negatively impact on the aseptic process.

Any contaminated unit should be considered as objectionable and fully
investigated. The microorganisms should be identified to species level.
In the case of a media fill failure, a comprehensive investigation
should be conducted, surveying all possible causes of the contamination.
The impact on commercial drugs produced on the line since the last
successful media fill should also be assessed.

Whenever contamination exists in a media fill batch, it should be
considered as indicative of a potential production problem. The use of
statistics has limitations for media fill evaluation in that 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 show, with a high degree of confidence, 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)
and should normally yield no media fill contamination. For example, a
single contaminated unit in a 10,000 unit media fill batch should be
fully investigated, but is normally not considered on its own to be
sufficient cause for line revalidation. However, intermittent incidents
at this media fill contamination level can be indicative of a persistent
low level contamination problem. Accordingly, any pattern of media fill
batches with such low level contamination should be comprehensively
investigated and would be cause for line 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 non-sterile unit. The purpose of
an aseptic process is to prevent any contamination. A manufacturer is
fully liable for the shipment of any non-sterile unit, an act that is
prohibited under the FD&C Act. FDA also recognizes that there might be
some scientific and technical limitations on how precisely and
accurately validation can characterize a system of controls intended to
exclude contamination.

As with any validation batch, it is important to note that
\"invalidation\" of a media fill run should be a rare occurrence. A
media fill lot 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. An
appropriate sterilizing grade filter is one which reproducibly removes
all microorganisms from the process stream, producing a sterile
effluent. Such filters usually have a rated porosity of 0.2 micron or
smaller. Whatever filter or combination of filters is used, validation
should include microbiological challenges to simulate \"worst case\"
production conditions regarding the size of microorganisms in the
material to be filtered and integrity test results of the filters used
for the study. The microorganisms should be small enough to both
challenge the nominal porosity of the filter and simulate the smallest
microorganism that may occur in production. The microorganism
*Brevundimonas diminuta* (ATCC 19146) when properly grown, harvested and
used, can be satisfactory in this regard because it is one of the
smallest bacteria (0.3 micron mean diameter). Bioburden of unsterilized
bulk solutions should be determined, in order to trend the
characteristics of potentially contaminating organisms. In certain
cases, when justified as equivalent or better than use of *Brevundimonas
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 have potential to allow passage of
microorganisms (Ref. 9). The probability of such passage is considered
to increase as the number of organisms (bioburden) in the material to be
filtered increases (Ref. 10). A challenge concentration of at least
10^7^ organisms per cm^2^ of effective filtration area of *B. diminuta*
is generally used. A commercial lot\'s actual influent bioburden should
not include microorganisms of a size and/or concentration that would
present a challenge beyond that considered by the validation study.

Direct inoculation into the drug formulation 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 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, the 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 which can affect filter performance normally
include: (1) viscosity 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. 11). 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 used in commercial
production should be evaluated in filter 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 filter
replacements (membrane or cartridge) used in production runs will
perform in the same manner. Sterilizing filters should be routinely
discarded after processing of a single batch. Normally, integrity
testing of the filter is performed after the filter unit is assembled
and sterilized prior to use. It is important that the integrity testing
be conducted after filtration in order 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
acceptable integrity tests. A production filter's integrity test
specification should be consistent with data generated during filtration
efficacy studies.

## **C. Sterilization of Equipment and Container/Closures**

In order to maintain sterility, equipment surfaces that contact
sterilized drug product or sterilized container/closure surfaces must be
sterile so as not to alter purity of the drug (211.63 and 211.113).
Those surfaces that are in the vicinity of sterile product or
container-closures, but do not directly contact product should also be
rendered sterile where reasonable contamination potential exists. It is
as important in aseptic processing to properly validate the processes
used to sterilize such critical equipment as it is to validate processes
used to sterilize the drug product and its container/closure. Moist heat
and dry heat sterilization are most widely used and the primary
processes discussed in this document. It should be noted that many of
the heat sterilization principles discussed in this document are also
applicable to other sterilization methods.

Sterility of aseptic processing equipment (e.g., stopper hoppers) should
be maintained by batch-by-batch sterilization. Following sterilization
of equipment, containers, or closures, any transportation or assembly
needs to be performed in a manner in which its sterile state is
protected and sustained, with adherence to strict aseptic methods.

### 1. Sterilizer Qualification and Validation

Validation studies should be conducted demonstrating the efficacy of the
sterilization cycle. Requalification studies should also be performed on
a periodic basis. For both the validation studies and routine
production, use of a specified load configuration should be documented
in the batch records.

Unevacuated air's insulating properties prevent moist heat from
penetrating or heating up materials, and achieving the lethality
associated with saturated steam. Consequently, there is a far slower
thermal energy transfer and rate of kill from the dry heat in insulated
locations in the load. It is important to remove all of the air from the
autoclave chamber during the sterilization cycle. Special attention
should be given to the nature or type of the materials to be sterilized
and the placement of biological indicator within the sterilization load.
D-value of the biological indicator can vary widely depending on the
material (e.g., glass versus Teflon) to be sterilized. Difficult to
reach locations within the sterilizer load and specific materials should
be an important part of the evaluation of sterilization cycle efficacy.
Thereafter, requalification/revalidation should continue to focus on
load areas identified as the most difficult to penetrate or heat (e.g.,
worst-case locations of tightly wrapped or densely packed supplies,
securely fastened load articles, lengthy tubing, the sterile filter
apparatus, hydrophobic filters, stopper load).

The formal program providing for regular (i.e., semiannual, annual)
revalidation should consider the age of the sterilizer and its past
performance. Change control procedures should adequately address issues
such as a load configuration change or a modification of the sterilizer.

a\) Qualification: Empty Chamber

> Temperature distribution studies evaluate numerous locations
> throughout an empty sterilizing unit (e.g., steam autoclave, dry heat
> oven) or equipment train (e.g., large tanks, immobile piping). It is
> important that these studies assess temperature uniformity at various
> locations throughout the sterilizer to identify potential \"cold
> spots\" where there can be insufficient heat to attain sterility.
> These heat uniformity or \"temperature mapping\" studies should be
> conducted by placing calibrated temperature measurement devices in
> numerous locations throughout the chamber.
>
> b\) Validation: Loaded Chamber
>
> Heat penetration studies should be performed using the established
> sterilizer load(s). 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 "cold spots" where there
> is insufficient heat to attain sterility. The placement of biological
> indicators (BI) at numerous positions in the load, including the most
> difficult to sterilize places, is a direct means of demonstrating the
> efficacy of any sterilization procedure. In general, the thermocouple
> (TC) is placed adjacent to the BI so as to assess the correlation
> between microbial lethality and thermal input. Validation of
> sterilization can be performed using a partial or half-cycle approach.
> In some cases, the "bioburden" based cycle is used for sterilization
> validation. For further information on validation using moist heat
> sterilization, please refer to FDA guidance, "Guideline for the
> Submission of Documentation for Sterilization Process Validation in
> Applications for Human and Veterinary Drug Products" (November, 1994).
>
> Sterilization cycle specifications are based upon the delivery of
> adequate thermal input to the slowest to heat locations. When
> determining which articles are most difficult to sterilize, special
> attention should be given to the sterilization of filters. For
> example, some filter installations in piping cause a significant
> pressure differential across the filter, resulting in a significant
> temperature drop on the downstream side. Biological indicators should
> be placed at appropriate downstream locations of this equipment to
> determine if the drop in temperature affects the thermal input at
> these sites. Established load configuration should be part of batch
> record documentation. A sterility assurance level of 10^-6^ or better
> should be demonstrated for the sterilization process.

### 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 these devices are maintained in a calibrated
state. For example:

-   Temperature monitoring devices for heat sterilization should be
    calibrated at suitable intervals, as well as before and after
    validation runs.

-   Devices used to monitor dwell time in the sterilizer should be
    periodically calibrated.

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-   The microbial count and D-value of a biological indicator should be
    confirmed before a validation study.

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-   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.

Sterilizing equipment should be properly maintained to allow for
consistently satisfactory function. Evaluation of sterilizer performance
attributes such as equilibrium ("come up") time studies should be
helpful in assessing if the unit continues to operate properly.

# X. LABORATORY CONTROLS

+-----------------------------------------------------------------------+
| Section 211.160 (General Requirements) states "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."                                                 |
|                                                                       |
| Sections 211.165 and 211.194 require that validation of test methods  |
| be established and documented.                                        |
|                                                                       |
| Sections 211.22 (c) states that "the quality control unit shall have  |
| the responsibility for approving or rejecting all procedures and      |
| specifications impacting on the identity, strength, quality, and      |
| purity of the drug product."                                          |
|                                                                       |
| Section 211.42 requires, for aseptic processes, the establishment of  |
| a "system for monitoring environmental conditions."                   |
|                                                                       |
| Section 211.56 requires, "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." The "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." Section 211.113 (b) requires that    |
| "appropriate written procedures, designed to prevent microbiological  |
| contamination of drug products purporting to be sterile, shall be     |
| established and followed."                                            |
|                                                                       |
| Section 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."                                  |
+-----------------------------------------------------------------------+

## **A. Environmental Monitoring** 

### 

### 1. General Written Program

## 

In aseptic processing, one of the most important laboratory controls is
the establishment of an environmental monitoring program. This
monitoring provides meaningful information on the quality of the aseptic
processing environment when a given batch is being manufactured as well
as environmental trends of the manufacturing area. An adequate program
identifies 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 validated methods.
The monitoring program should cover all production shifts and include
air, floors, walls, and equipment surfaces, including the critical
surfaces in contact with product and container/closures. Written
procedures should include a list of locations to be sampled. Sample
timing, frequency, and location should be carefully selected based upon
its relationship to the operation performed. Samples should be taken
throughout the aseptic processing facility (e.g., aseptic corridors;
gowning rooms) using appropriate, scientifically sound sampling
procedures, standards, and test limits.

Locations posing the most microbiological risk to the product are a
critical part of the program. It is especially important to monitor the
microbiological quality of the aseptic processing clean zone to
determine whether or not aseptic conditions are maintained during
filling/closing activities. Critical surfaces which contact sterile
product should be sterile. Critical surface sampling should be performed
at the conclusion of the aseptic processing operation to avoid direct
contact with sterile surfaces during processing. Air and surface samples
should be taken at the actual working site and at locations where
significant activity or product exposure occurs during production.

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 of the likelihood of false
negatives, consecutive growth results are only one type of adverse
trend. Increased incidence of contamination over a given period in
comparison to that normally detected is an equally significant trend to
be tracked.

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 areas 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 limits; and (7) appropriate response to
deviations from alert or action limits.

### 2. Establishing Limits and a Trending Program

Microbiological monitoring limits should be established based on the
relationship of the sampled location to the operation. The limits 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 procedure studies in
developing monitoring limits.

Microbiological environmental monitoring should include both alert and
action limits. Each individual sample result should be evaluated for its
significance by comparing to the alert or action limits. Averaging of
results can mask unacceptable localized conditions. A result at the
alert limit 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, identification of contaminants, 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, lot,
room, operator, or other search parameters. The quality control unit is
responsible for producing specialized data reports (e.g., a search on a
particular atypical isolate over a year period) in order to investigate
results beyond established limits and identify any appropriate follow-up
actions. In addition to microbial counts beyond alert and action limits,
the presence of any atypical microorganisms in the cleanroom environment
should be investigated, with any appropriate corrective action promptly
implemented.

Written procedures should define the system whereby the most responsible
managers are regularly informed and updated on trends and
investigations.

### 3. Sanitization Efficacy

The suitability, efficacy, and limitations of sanitization agents should
be assessed with their implementation for use in clean areas. The
effectiveness of these sanitization procedures should be measured by
their ability to ensure that potential contaminants are adequately
removed from surfaces (i.e., via obtaining samples before and after
sanitization).

Upon preparation, disinfectants should be rendered sterile, and used for
a limited time, as specified by written procedures. Disinfectants should
retain efficacy against the normal microbial flora and be effective
against spore-forming microorganisms. Many common sanitizers are
ineffective against spores, for example, 70% isopropyl alcohol is not
effective against *Bacillus,* spp*.* spores. A sporicidal agent should
be used regularly to prevent contamination of the manufacturing
environment with otherwise difficult to eradicate spore forming bacteria
or fungi.

After the initial assessment of sanitization procedures, ongoing
sanitization efficacy should be frequently monitored through specific
provisionsin the environmental monitoring program, with a defined course
of action in the event samples are found to exceed limits.

### 4. Monitoring Methods

Acceptable methods of monitoring the microbiological quality of the
environment include:

> a\. [Surface Monitoring-]{.underline}
>
> Environmental monitoring should include testing of various surfaces
> for microbiological quality. For example, product contact surfaces,
> floors, walls, ceilings, and equipment should be tested on a regular
> basis. Routinely used for such tests are touch plates, swabs, and
> contact plates. Other surfaces in controlled areas should be tested to
> show the adequacy of cleaning and sanitizing procedures.
>
> b\. [Active Air Monitoring-]{.underline}
>
> The method of assessing the microbial quality of air should involve
> the use of \"active\" devices such as slit to agar samplers, , those
> using liquid impingement and membrane filtration, or centrifugal
> samplers. Each device has certain advantages and disadvantages,
> although all allow a quantitative testing of the number of organisms
> per volume of air sampled. The use of such devices in aseptic areas is
> considered an essential part of evaluating the environment during each
> production shift, at carefully chosen critical locations.
> Manufacturers should be aware of a device\'s air monitoring
> capabilities, and should determine suitability of any new or current
> devices with respect to sensitivity and limit of quantification.
>
> c\. [Passive Air Monitoring (Settling Plates)-]{.underline}
>
> Another method is the use of passive air samplers such as settling
> plates (petri dishes containing nutrient growth medium exposed to the
> environment). These settling plates lack value as quantitative air
> monitors because only microorganisms that settle onto the agar surface
> will be detected. Their value as qualitative indicators in critical
> areas is enhanced by positioning plates 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** 

The environmental monitoring program should include routine
characterization of recovered microorganisms. Monitoring of critical and
immediately surrounding 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 has revealed migration of
microorganisms into the aseptic processing room from either uncontrolled
or lesser-controlled areas. To detect such trends, an adequate program
of differentiating microorganisms in lesser-controlled environments
(e.g., Class 100,000) should be in place. At minimum, the program should
require species (or, where appropriate, genus) identification of
microorganisms in 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). 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 the associated investigation.

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 35^o^C
for 48 to 72 hours. Total combined yeast and mold count is generally
obtained by incubating at 20 to 25^o^C for 5 to 7 days.

Incoming lots of environmental monitoring media should include positive
and negative controls. 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 clean room
disinfectants.

## **C. Pre-filtration Bioburden**

For any parenteral manufacturing process, pre-filtration bioburden
should be minimal. In addition to increasing the challenge to the
sterilizing filter, high bioburden can contribute endotoxin or other
impurities to the drug formulation. An in-process limit for bioburden
level for each formulated product (generally sampled immediately
preceding sterile filtration) should be established.

## **D. Particulate Monitoring**

Routine particle monitoring is useful in detecting significant
deviations in air cleanliness from qualified processing norms (e.g.,
clean area classification). A result outside the established
specifications at a given location should be investigated consistent
with the severity of the \"excursion.\" Appropriate corrective action
should be implemented to prevent future deviations.

See Section IV.A for additional guidance on particulate monitoring.

# XI. STERILITY TESTING

+-----------------------------------------------------------------------+
| Section 211.167 (Special Testing Requirements) states: "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."                                       |
|                                                                       |
| Section 211.165 states "For each batch of drug product, there shall   |
| be appropriate laboratory determination of satisfactory conformance   |
| to final specifications for the drug product ...prior to release."    |
|                                                                       |
| Section 211.165(e) requires methods for testing to be validated as    |
| reliable and reproducible (e.g., bacteriostasis/fungistasis, method   |
| robustness, etc.), stating: "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 Sec. 211.194(a)(2)."                  |
|                                                                       |
| Section 211.110 requires, in part, that sampling procedures are       |
| established in order to ensure batch uniformity: The \"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.\"                           |
|                                                                       |
| Section 211.160 requires the establishment of sound and appropriate   |
| sampling plans which are representative of the batch.                 |
|                                                                       |
| Section 210 defines "representative sample" as one based on rational  |
| criteria that provide an "accurate portrayal" of the material or      |
| batch being sampled.                                                  |
|                                                                       |
| Section 211.180 states a review of, \"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.\" |
| Investigations conducted under Section 211.192 for each drug product  |
| are required to be addressed within this annual review.               |
+-----------------------------------------------------------------------+

Certain aspects of sterility testing are of particular importance,
including control of the testing environment, understanding the test
limitations, and the investigation of manufacturing systems following a
positive test.

The testing laboratory environment should employ facilities and controls
comparable to those used for filling/closing operations. Poor or
deficient sterility test facilities or controls can result in a high
rate of test failures. If production facilities and controls are
significantly better than those for sterility testing there is the
danger of attributing the cause of a positive sterility test result to
the faulty laboratory even when the product tested could have, in fact,
been non-sterile. Therefore, some manufacturing deficiency may go
undetected. The use of isolators to perform sterility testing is a
well-established means for minimizing false positives.

## **A. Choice of Methods** 

Sterility testing methodologies are required to be accurate and
reproducible, in accord with 211.194 and 211.165. The methodology
selected should present the lowest potential for yielding a false
positive. The USP specifies membrane filtration as the method of choice,
when feasible.

As a part of methods validation, appropriate bacteriostasis/fungistasis
testing should be conducted. Such testing should demonstrate
reproducibility of the method in recovering each of a panel of
representative microorganisms. Study documentation should include
evaluation of whether microbial recovery from inoculated controls and
product samples is comparable throughout the incubation period. If
growth is inhibited, modifications (e.g., increased dilution, additional
membrane filter washes, addition of inactivating agents) in the
methodology should be implemented to optimize recovery. Ultimately,
methods validation studies should demonstrate that the methodology does
not provide an opportunity for \"false negatives.\"

## **B. Media**

It is essential that the media used to perform sterility testing be
rendered sterile and demonstrated as growth promoting.

## **C. Personnel**

Personnel performing sterility testing should be qualified and trained
for the task. A written program should be in place to regularly update
training of personnel and confirm acceptable sterility testing
practices.

## **D. Sampling and Incubation**

Sterility tests are limited in their ability to detect low levels of
contamination. For example, statistical evaluations indicate that the
USP sterility test sampling plan has been described by USP as \"only
enabling 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. 12). To further illustrate, if a 10,000 unit lot with a 0.1%
contamination level was sterility tested using 20 units, there is a 98%
chance that the batch would pass the test.

This limited sensitivity makes it necessary to ensure that for batch
release purposes an appropriate number of units are tested and that the
samples uniformly represent the:

> \(1\) Entire batch-
>
> Samples should be taken at the beginning, middle, and end of the
> aseptic processing operation;
>
> \(2\) Batch processing circumstances-
>
> Samples should be taken in conjunction with processing interventions
> or excursions.

Because of the limited sensitivity of the test, any positive result is
considered a serious CGMP issue and should be thoroughly investigated.

## **E. 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 to be
non‑sterile. It is inappropriate to attribute a positive result to
laboratory error on the basis of a retest that exhibits no growth.[^8]
The evaluation of a positive sterility test result should include an
investigation to determine whether the growth observed in the test arose
from product contamination or from laboratory error.

Although it is recognized that such a determination may not be reached
with absolute certainty, it is usually possible to acquire persuasive
evidence showing that causative laboratory error is absent. When
available evidence is inconclusive, batches should be rejected as not
conforming to sterility requirements.

It would be difficult to support invalidation of a positive sterility
test. Only if conclusive and documented evidence clearly shows that the
contamination occurred as part of testing should a new test be
performed.

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 upon at least the following:

1.  [Identification (speciation) of the organism in the sterility
    test]{.underline}. Identification of the sterility test isolate(s)
    should be 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.

2.  [Record of laboratory tests and deviations.]{.underline} Review of
    trends in laboratory findings can help to eliminate or implicate the
    laboratory as the source of contamination. If the organism is seldom
    found in the laboratory environment, then product contamination is
    likely. If the organism is found in laboratory and production
    environments, it can 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.

Deviation and sterility test positive trends should be evaluated
periodically (e.g., quarterly, annually) to provide an overview of
operations. A sterility positive result can be viewed as indicative of
production or laboratory problems and should be investigated globally
since such problems often can extend beyond a single batch.

In order to more accurately monitor potential contamination sources, it
is useful to keep separate trends by product, container type, filling
line, and personnel. Where the degree of sterility test 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. See Section IX.A, Process Simulations,
which includes similar issues that are investigated as part of a media
fill failure investigation.

Microbial monitoring of the laboratory environment and personnel over
time can also reveal trends that are informative. Upward trends in the
microbial load in 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 help remove 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 the laboratory
> has a poor track record, firms should not assume that the
> contamination is automatically more attributable to the error in
> laboratory and consequently overlook a genuine production problem.
> Accordingly, all sterility positives should be thoroughly
> investigated.

3.  [Monitoring of production area environment.]{.underline} Of
    particular importance is trend analysis of microorganisms in the
    critical and immediately adjacent area. Trends are an important tool
    in investigating the product as the possible source of a sterility
    failure. Consideration of environmental microbial loads 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 trend analysis.

4.  [Monitoring of Personnel]{.underline}. Daily personnel monitoring
    data and associated trends should be reviewed and can in some cases
    strongly indicate a route of contamination. The adequacy of
    personnel practices and training should also be considered.

5.  [Product pre-sterilization bioburden.]{.underline} Trends in product
    bioburden should be reviewed (counts and identity). Adverse
    bioburden trends occurring during the time period of the test
    failure should be considered in the investigation.

6.  [Production record review]{.underline}. Complete batch and
    production control records should be reviewed to detect any signs of
    failures or anomalies which could have a bearing on product
    sterility. For example, the investigation should evaluate batch and
    trending data that indicate whether utility/support systems (e.g.,
    HVAC, WFI) are functioning properly. Records of air quality
    monitoring for filling lines should show a time at which there was
    improper air balance, an unusual high particulate count, etc.

7.  [Manufacturing history.]{.underline} The manufacturing history of
    the 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.

# XII. BATCH RECORD REVIEW: PROCESS CONTROL DOCUMENTATION

+-----------------------------------------------------------------------+
| Sections 211.100, 211.186, and 211.188 address documentation of       |
| production and control of a batch, including recording various        |
| production and process control activities at the time of performance. |
| Section 211.100 (b) requires a documented record and evaluation of    |
| any deviation from written procedures.                                |
|                                                                       |
| Section 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.\"                         |
+-----------------------------------------------------------------------+

Maintaining process and environmental control is a daily necessity for
an aseptic processing operation. 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 batch calls for an overall
review of process and system performance for that given cycle of
manufacture. All in-process data must be included with the batch record
documentation per Section 211.188. Review of environmental monitoring
data as well as other data relating to the 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), should be viewed as 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 general, there is a correlation between
product (or container-closure) dwell time in the aseptic processing zone
and the probability of contamination. Sterility failures can be
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/container-closures 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, during aseptic
processing is a manufacturing deviation and must be included in batch
records (211.100, 211.192).

# [APPENDIX 1:]{.underline} ASEPTIC PROCESSING ISOLATORS

An emerging aseptic processing technology uses isolation systems to
minimize the extent of personnel involvement and to separate the
external cleanroom environment from the aseptic processing line. A well
designed positive pressure barrier isolator, supported by adequate
procedures for its maintenance, monitoring, and control, appears to
offer an advantage over classical aseptic processing, including fewer
opportunities for microbial contamination during processing. However,
users should not adopt a "false sense of security" with these systems.
Manufacturers should be also aware of the need to establish new
procedures addressing issues unique to these systems.

## **A) Maintenance**

### 1. General

Isolator systems have a number of special maintenance requirements.
While no isolator unit forms an absolute seal, very high integrity can
be achieved in a well-designed unit. However, a leak in any of certain
components of the system can constitute a significant breach of
integrity. The integrity of gloves, half-suits, seams, gaskets, and
seals require daily attention and a comprehensive preventative
maintenance program. Replacement frequencies should be established in
written procedures that require changing parts before they breakdown or
degrade.

### 2. Glove Integrity

A faulty glove or sleeve (gauntlet) assembly represents a route of
contamination and a critical breach of isolator integrity. The choice of
durable glove materials, coupled with a well-justified replacement
frequency, are two aspects of good manufacturing practice that should be
addressed. With every use, gloves should be visually evaluated for any
macroscopic physical defect. Mechanical integrity tests should also be
performed routinely. This attentive preventative maintenance program is
necessary to prevent use of gloves lacking integrity that would place
the sterile product at risk. When such a breach is discovered, the
operation should be terminated.

Due to the potential for microbial migration through microscopic holes
in gloves and the lack of a highly sensitive glove integrity test, the
inner part of the installed glove should be sanitized regularly and the
operator should also wear a second pair of thin gloves.

## **B) Design** 

### 1. Airflow

The design of an aseptic processing isolator normally employs
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 zone once, and then is
systematically exhausted. Air handling systems should employ HEPA and/or
ULPA filters in series.

### 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 sterilization. For
example, rigid wall construction incorporating stainless steel and glass
materials is widely used.

### 3. Pressure Differential 

Isolators that include an open exit portal represent a potential
compromise in achieving complete physical separation from the external
environment. A positive air pressure differential adequate to achieve
this full 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 0.07\" to
0.2\" water gauge. The appropriate minimum pressure differential
specification established by a firm will be dependent on the system's
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 appropriate
protection at the product egress point(s) in order to overcome the
potential for ingress of any airborne particles from the

external environment by induction. Induction can result from local
turbulent flow causing air swirls or pressure waves that can push
extraneous particles into the isolator. Local Class 100 protection at an
opening can provide a further barrier to induction of outside air into
the isolator.

### 4. Clean Area Classifications

The interior of the isolator should, at minimum, meet Class 100
standards. The classification of the environment surrounding the
isolator should be based on the design of the product interfaces, such
as transfer ports and discharge points, as well as the number of
transfers into and out of the isolator. A Class 10,000 or Class 100,000
background is appropriate depending on isolator design and manufacturing
situations. The area surrounding the isolator should be justified. An
isolator should not be located in an unclassified room.

## **C) Transfer of Materials/Supplies**

The ability to maintain integrity and sterility of an isolator is
impacted by the design of transfer ports. Various adaptations, of
differing capabilities, allow for the transfer of supplies into and out
of the isolator.

### 1. Introduction: 

> Multiple material transfers are generally made during the processing
> of a batch. Frequently, transfers are performed via direct interface
> with a decontaminating transfer isolator or dry heat depyrogenation
> tunnel with balanced airflow. Such provisions, if well designed, help
> ensure that microbiological ingress does not result from the
> introduction of supplies. Properly operated RTPs (rapid transfer
> ports) are also generally considered to be an effective transfer
> mechanism. The number of transfers should be kept to a minimum because
> the risk of ingress of contaminants increases with each successive
> material transfer.
>
> Some transfer ports can have significant limitations, including
> marginal decontaminating capability (e.g., ultraviolet) or a design
> that would compromise isolation by allowing ingress of air from the
> surrounding room. In the latter case, localized HEPA-filtered laminar
> airflow cover in the area of such a port should be implemented.

### 2. Discharge: 

> Isolators often include a \"mousehole\" or other exit port through
> which product is discharged, opening the isolator to the outside
> environment. The mousehole represents a potential route of
> contamination. 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

> Written procedures for decontamination of the isolator should be
> established. The decontamination process should provide full exposure
> of all isolator surfaces to the chemical agent. For example, in order
> to facilitate contact with the sterilant, the glove apparatus should
> be fully extended with glove fingers separated during the
> decontamination cycle.

### 2. Efficacy

> A decontamination method should be developed which renders the inner
> surfaces of the isolator free of viable microorganisms.
> Decontamination can be accomplished using a number of vaporized
> agents, although these agents possess limited capability to penetrate
> obstructed or covered surfaces. 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. An appropriate, quantified BI challenge should be
> placed on various materials 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. For most
> production applications, demonstration of a six-log reduction of the
> challenge BI is recommended.
>
> The uniform distribution of the defined concentration of
> decontaminating agent should also be evaluated concurrent with these
> studies. Chemical indicators may also be useful as a qualitative tool
> to show that the decontaminating agent reached a given location.

### 

### 3. Frequency

> While isolators vary widely in design, their interior and content
> should be designed to be frequently decontaminated. If an isolator is
> to be used for multiple days between decontamination cycles, the
> frequency adopted should include a built-in safety margin and be well
> justified. This frequency, established during validation studies,
> should be reevaluated and increased if production data indicate any
> deterioration of the microbiological quality of the isolator
> environment.
>
> A breach of isolator integrity (e.g., power failure, glove/seam tear,
> other air leaks, valve failure, out of specification pressure) should
> lead to a decontamination cycle. Breaches of integrity should be
> investigated and any product that may have been impacted by the breach
> rejected.

## **E) Filling Line Sterilization**

In order to ensure sterility of product contact surfaces from the start
of each operation, the entire path of the sterile liquid stream should
be sterilized. In addition, loose materials or equipment to be used
within the isolator should be chosen based on their ability to withstand
steam sterilization (or equivalent method). It is expected that any
materials that can be subjected to a steam sterilization cycle will, in
fact, be autoclaved.

## **F) Environmental Monitoring**

An appropriate environmental monitoring program should be established
which routinely ensures acceptable microbiological quality of air,
surfaces, and gloves (or half-suits) as well as particulate levels,
within the isolator. Air quality should be monitored periodically during
each shift. As an example, the exit port should be monitored for
particulates to detect any unusual results.

## **G) Personnel**

While cleanroom apparel requirements are generally reduced, the
contribution of human factor to contamination should not be overlooked.
Isolation processes generally include periodic or even frequent use of
one or more gloves for aseptic manipulations and handling of component
transfers into and out of the isolator. Contaminated gloves can lead to
product non-sterility. This concern is heightened because locations on
gloves, sleeves, or half suits can be among the more difficult to reach
places during surface sterilization. Meticulous aseptic technique
standards must be observed (211.113).

# [APPENDIX 2:]{.underline} 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 of ophthalmics and, less frequently, for injectables. This
section 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 be applied to Blow
Fill Seal technology.

## **A) Equipment Design and Air Quality**

A BFS machine operates by 1) heating a plastic polymer resin; 2)
extruding it to form a parison (a tubular form of the hot resin); 3)
cutting the parison with a high temperature knife; 4) moving the parison
under the blow-fill needle (mandrel); 5) inflating it to the shape of
the mold walls; 5) filling the formed container with the liquid product;
6) removing the mandrel; 7) sealing. 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 which pose
greatest potential for exposure to particle contamination and/or
surrounding air are those in which: the parison is cut; the parison is
moved under the blow-fill mandrel; and the mandrel is removed (just
prior to sealing).

BFS machinery and its surrounding barriers should be designed to prevent
potential for extraneous contamination. As with any aseptic processing
operation, it is critical that contact surfaces be sterile. A validated
steam-in-place cycle should be used to sterilize the equipment path
through which the product is conveyed. In addition, any other surface
(e.g., above or nearby) that has potential to contaminate the sterile
product needs to be sterile.

The classified environment surrounding BFS machinery should generally
meet Class 10,000 standards, but special design provisions (e.g.,
isolation technology) can justify an alternate classification.
HEPA-filtered or sterile air provided by membrane filters is necessary
in the critical zone in which sterile product or materials are exposed
(e.g., parison formation, container molding/filling steps). Air in the
critical zone should meet Class 100 microbiological standards. A
well-designed BFS system should also normally achieve Class 100
particulate levels.

Equipment design should incorporate specialized measures to reduce
particulate levels. In contrast to non-pharmaceutical applications using
BFS machinery, control of air quality (i.e., particulates) is critical
for sterile drug product manufacture. Particles generated during the
plastic extrusion, cutting, and sealing processes provide a potential
means of transport for microorganisms into open containers prior to
sealing. Provisions for carefully controlled airflow could protect the
product by forcing generated particles outward while preventing any
ingress from the adjacent environment. Furthermore, designs separating
the filling zone from the surrounding environment are important in
ensuring product protection. Barriers, pressure vacuums,
microenvironments, and appropriately directed high velocities of sterile
air have been found useful in preventing contamination (Ref. 13). Smoke
studies and multi-location particulate data are vital when performing
qualification studies to assess whether proper particulate control
dynamics have been achieved throughout the critical area.

In addition to suitable design, an adequate preventative maintenance
program should be established. For example, because of its potential to
contaminate the sterile drug product, the integrity of the boiling
system (e.g., mold plates, gaskets) should be carefully monitored and
maintained.

## **B) Validation/Qualification**

Advantages of BFS processing are known to include rapid
container/closure processing and minimized interventions. However, a
properly functioning process is necessary to realize these advantages.
Equipment qualification/requalification and personnel practices should
be given special attention. Equipment sterilization, media fills,
polymer sterilization, endotoxin removal, product-plastic compatibility,
forming/sealing integrity, and unit weight variation are among the key
issues that should be covered by validation/qualification studies.

Appropriate data should ensure that BFS containers are sterile and
non-pyrogenic. This can generally be achieved by validating that
time-temperature conditions of the extrusion process destroy the
worst-case endotoxin load on the polymeric material.

The plastic polymer material chosen should be pharmaceutical grade,
safe, pure, and pass USP criteria for plastics. Polymer suppliers should
be qualified and monitored for raw material quality.

## **C) Batch Monitoring and Control**

In-process monitoring should include various control parameters (e.g.,
container weight variation, fill weight, leakers, air pressure, etc.) to
ensure ongoing process control. Environmental monitoring is particularly
important. Samples should be taken during each shift at specified
locations under dynamic conditions. Due to the generation of high levels
of particles near the exposed drug product, 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 necessary for the operation to be designed and set-up
to uniformly manufacture leak-proof units. As a final measure,
inspection of each unit of a batch should employ a reliable, sensitive
final product examination capable of detecting a defective unit (e.g.,
"leakers"). Significant defects due to heat or mechanical problems, such
as mold thickness, container/closure interface deficiencies, poorly
formed closure, or other deviations should be investigated in accord
with Sections 211.100 and 211.192.

# [APPENDIX 3:]{.underline} PROCEsSING PRIOR TO FILLING/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 from early in the manufacturing
process, or that require aseptic processing through the entire
manufacturing process, due to their inability to be sterilized. 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**

Due to their nature, some products undergo aseptic processing at some or
all manufacturing steps preceding the final product closing step. There
is a point in the process after which a product can no longer be
rendered sterile by filtration, and the product is handled aseptically
in all subsequent steps. Some products are formulated aseptically
because the formulated product cannot be sterilized by filtration. 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 early steps, 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 are carefully controlled at each step of
the process to maintain sterility of the product.

Procedures that expose the product or product contact equipment surfaces
to the environment, such as aseptic connections, should be performed
under unidirectional airflow in a Class 100 environment. The environment
of the room surrounding the Class 100 environment should be class 10,000
or better. Microbiological and particulate 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 should be designed to incorporate all
conditions, product manipulations, and interventions that could impact
on the sterility of the product during manufacturing. The process
simulation, from early process steps, should demonstrate that controls
over the process 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, 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
product or transport to other manufacturing areas. For instance, there
should be assurance of bulk vessel integrity for specified holding
times. The transport of bulk tanks or other containers should be
simulated as part of the media fill. Please refer to Section IX.A for
more guidance on media simulation studies. Process simulation studies
for the formulation stage should be performed at least twice per year.

## 

## **B) Aseptic processing of cell-based therapy products (or of products intended for use as cell based therapies)** 

Cell-based therapy products represent a subset of the products for which
aseptic manipulations are used throughout the process. Where possible,
closed systems should be used during production. Cell-based therapy
products often have short processing times at each manufacturing stage,
even for the final product. Often, it is appropriate for these products
to be 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,
especially after the last manipulation of the product prior to
administration. Other tests that may indicate microbial contamination,
such as microscopic examination, gram stains, and endotoxin testing
should be performed prior to product release.

# REFERENCES

# 

l\. ISO 14644-1: Cleanrooms and Associated Controlled Environments,
Classification of Air Cleanliness.

2\. Technical Order 00‑25‑203, Contamination Control of Aerospace
Facilities, U.S. Air Force, December l, l972.

3\. Ljungqvist, B., and Reinmuller, B., [Clean Room Design: Minimizing
Contamination Through Proper Design]{.underline}; Interpharm Press,
1997.

4\. NASA Standard for Clean Room and Work Stations for Microbially
Controlled Environment, Publication NHB 5340.2 (August l967)

5\. 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.

6\. Grandics, Peter, "Pyrogens in Parenteral Pharmaceuticals,"
Pharmaceutical Technology, April 2000.

7\. Lord, A. and John W. Levchuk, \"Personnel Issues in Aseptic
Processing,\" Biopharm, 1989.

8\. Technical Report No. 36, "Current Practices in the Validation of
Aseptic Processing,\" Parenteral Drug Association, Inc., 2002.

9\. Leahy, Timothy J. and Mary J. Sullivan "Validation of Bacterial ‑
Retention Capabilities of Membrane Filters,\" Pharmaceutical Technology,
Nov. l978.

10\. Pall, David B. and Erwin 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.

11\. Technical Report No. 26, \"Sterilizing Filtration of Liquids,\"
Parenteral Drug Association, Inc., 1998.

12\. Pharmacopoeial Forum, July‑August l980, p. 354, \"Commentary on the
Sterility Tests and Sterilization Chapters of the U.S. Pharmacopoeia,\"
Aubrey S. Outschoorn, Sr. Scientist, U.S.P. Drug Standards Division.

13\. Price, Jeff, "Blow-Fill-Seal Technology: Part I, A Design for
Particulate Control," Pharmaceutical Technology, February, 1998.

14\. United States Pharmacopoeia

# RELEVANT GUIDANCE DOCUMENTS

Some relevant FDA guidances include:

-   Guidance for the Submission of Documentation for Sterilization
    Process Validation in Applications for Human and Veterinary Drug
    Product, 1994

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-   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, 1987

-   Guide to Inspections of Lyophilization of Parenterals, 1993

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-   Guide to Inspections of High Purity Water Systems, 1993

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-   Guide To Inspections of Microbiological Pharmaceutical Quality
    Control Laboratories, 1993

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-   Guide To Inspections of Sterile Drug Substance Manufacturers, 1994

-   Pyrogens: Still a Danger; 1979 (Inspection Technical Guide)

-   Bacterial Endotoxins/Pyrogens; 1985 (Inspection Technical Guide)

-   Heat Exchangers to Avoid Contamination; 1979 (Inspection Technical
    Guide)

*For more information on FDA guidance, see our website at*
[www.fda.gov](http://www.fda.gov/).

# 

# DRAFT GLOSSARY

[Air lock-]{.underline} 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 Limit]{.underline}- An established microbial or particulate level
giving early warning of potential drift from normal operating conditions
and which trigger appropriate scrutiny and follow-up to address the
potential problem. Alert Limits are always lower than Action Limits.

[Action Limit]{.underline}- An established microbial or particulate
level which when exceeded should trigger appropriate investigation and
corrective action based on the investigation.

[Aseptic Processing Facility]{.underline}- Building containing
cleanrooms in which air supply, materials, and equipment are regulated
to control microbial and particulate contamination.

[Aseptic Processing Room]{.underline}- A room in which one or more
aseptic activities or processes is performed.

[Asepsis]{.underline}- 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]{.underline}- Total number of microorganisms associated with
a specific item prior to sterilization.

[Barrier]{.underline}- Physical partition that affords aseptic
manufacturing zone protection by partially separating it from the
surrounding area.

[Biological Indicator (BI)]{.underline}- A population of microorganisms
inoculated onto a suitable medium (e.g., solution, container/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]{.underline}- An area with defined particulate and
microbiological cleanliness standards (e.g., Class 100, Class 10,000 or
Class 100,000).

[Cleanroom-]{.underline} A room designed, maintained, and controlled to
prevent particulate and microbiological contamination of drug products.
Such a room is assigned and reproducibly meets an appropriate air
cleanliness classification.

[Clean Zone]{.underline}- See Clean Area.

[Component]{.underline}- 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)-]{.underline} A microbiological term which
describes the formation of a single macroscopic colony after the
introduction of one (or more) microorganism(s) to microbiological growth
media. One colony forming unit is expressed as 1 CFU.

[Critical areas]{.underline} ‑ Areas designed to maintain sterility of
sterile materials. Sterilized product, container/closures, and equipment
may be exposed in critical areas.

[Critical surfaces]{.underline} ‑ Surfaces which may come into contact
with or directly impact on sterilized product or containers/closures.
Critical surfaces are rendered sterile prior to the start of the
manufacturing operation and sterility is maintained throughout
processing.

[Decontamination-]{.underline} A process which eliminates viable
bioburden via use of sporicidal chemical agents.

[Depyrogenation-]{.underline} A process used to destroy or remove
pyrogens (e.g., endotoxin).

[D value]{.underline} ‑ The time (in minutes) of exposure to a given
temperature that causes a one-log or 90% reduction in the population of
a specific microorganism.

[Dynamic]{.underline}- Conditions relating to clean area classification
under conditions of normal production.

[Endotoxin]{.underline}- 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]{.underline}- Program which 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]{.underline}- High Efficiency Particulate Air filter with
minimum 0.3 micron particle retaining efficiency of 99.97%.

[HVAC-]{.underline} Heating, Ventilation, and Air Conditioning.

[Intervention]{.underline}- An aseptic manipulation or activity that
occurs at the critical zone.

[Isolator]{.underline} - A decontaminated unit, supplied with HEPA or
ULPA filtered air, which provides uncompromised, continuous isolation of
its interior from the external environment (e.g., surrounding clean room
air and personnel).

[Laminarity-]{.underline} Unidirectional air flow at a velocity
sufficient to uniformly sweep particulate matter away from a critical
processing or testing area.

[Operator]{.underline}- 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]{.underline} ‑ A process that is
sufficient to provide at least a 12 log reduction of microorganisms
having a minimum D value of 1 minute.

[Pyrogen-]{.underline} Substance which induces a febrile reaction in a
patient.

[Sterilizing grade filter-]{.underline} A filter which, when
appropriately validated, will remove all microorganisms from a fluid
stream, producing a sterile effluent.

[Terminal sterilization]{.underline}- 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 non-sterile unit of greater than one in
a million).

[ULPA filter]{.underline}- Ultra-Low Penetration Air filter with minimum
0.3 micron particle retaining efficiency of 99.999 %.

[Validation-]{.underline} Establishing documented evidence which
provides a high degree of assurance that a specific process will
consistently produce a product meeting its predetermined specifications
and quality attributes.

[Worst case-]{.underline} A set of conditions encompassing upper and
lower processing limits and circumstances, including those within
standard operating procedures, which pose the greatest chance of process
or product failure (when compared to ideal conditions). Such conditions
do not necessarily induce product or process failure.

[^1]: Due to their nature, certain products are aseptically processed
    from an earlier stage in the process, or in their entirety.
    Cell-based 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]: Nearly all drugs recalled due to Non-sterility or Lack of
    Sterility Assurance in the period spanning 1980-2000 were produced
    via aseptic processing.

[^3]: A velocity from 90 to 100 feet per minute is generally
    established, with a range of plus or minus 20% around the setpoint.
    Higher velocities may be appropriate in operations generating high
    levels of particulates.

[^4]: The same broad principles can be applied to ULPA filters.

[^5]: The efficiency test uses a monodispersed aerosol of 0.3 micron
    size particles, relates to filter media, and usually requires
    specialized testing equipment. Downstream readings represent an
    average over the entire filter surface. Therefore, the efficiency
    test is not intended to test for leakage in a filter.

[^6]: See Appendix III for discussion of certain biologic components
    that are aseptically handled from the start of the process.

[^7]: Separate incubation of certain categories of rejected units may
    nonetheless provide valuable information with respect to
    contamination that may arise from container/closure integrity
    deficiencies.

[^8]: Underscoring this regulatory standard, USP XXV, section \<71\>,
    states that an initial positive test is invalid only in an instance
    in which \"microbial growth can be without a doubt ascribed to\"
    laboratory error (as described in the monograph).