VMP Nih

Bethesda: 1 April 2009
Pharmaceutical Development Section
Validation Master Plan
First Draft (For review) 1 April 2009

Begin NIH Review 2 April 2009
Signature Adoption
Effective Date
Pharmacy Department
10 Center Drive, Room 1N257
Bethesda, Maryland 20892-1196
This Page Intentionally Left
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National Institutes of Health, Inc.
Signature Page
Document: Revision Identification: Original Origination

Validation Master Plan 1 Apr 2009
Facility: Location: This Revision

National Institutes of Health Pharmacy Department Date:
Department of Pharmacy 10 Center Drive, Room 1N257
Bethesda, Maryland 20892-1196
Complete Title:
Validation Master Plan
Objective:
The objective of this Validation Master Plan is to define the Facility, Equipment, and Process
Validation requirements, methodologies, and acceptance criteria for qualification of the Pharmacy
PDS liquid and solid dose manufacturing area.
Document Document
Written By: Narlin Beaty Approved By: ______________________
Title: Contractor - Qualification Process
Solutions, LLC Title: _____________________________
Signature: Signature:
Date: Date:
Document Document
Approved By: ______________________ Approved By: ______________________
Title: _____________________________ Title: _____________________________
Signature: Signature:
Date: Date:
Blank
TABLE OF CONTENTS BY SECTIONS
1. Introduction, Policy and Scope ............................................................................................................. 8
2. Concept of Qualification / Validation ................................................................................................. 12
3. Organizational Responsibilities for Validation .................................................................................... 26
5. Calibration Program ............................................................................................................................ 41
6. Preventative Maintenance Program ................................................................................................... 47
7. Key Standard Operating Procedures requiring Validation .................................................................. 51
8. Guidelines for Specific Equipment and Systems ................................................................................. 57
9. Process Validations ............................................................................................................................. 86
10. Environmental Monitoring Program I – Physical Tests ................................................................. 123
11. Microbiological Testing ................................................................................................................. 142
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NATIONAL INSTITUTES OF HEALTH
Document Number:
Title: Validation Master Plan for Drug Manufacturing Operations
Effective Date: Supersedes / Date: Section 1
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Contents
1. Introduction, Policy and Scope ............................................................................................................. 8
1.1. Project Description........................................................................................................................ 8
1.2. Scope ............................................................................................................................................. 8
1.3. Definition of the Term Validation ............................................................................................... 10
1.4. Living Document ......................................................................................................................... 10
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1. Introduction, Policy and Scope
1.1. Project Description
The objective of this Validation Master Plan is to define the Facility, Equipment, and Process Validation
requirements, methodologies, and acceptance criteria for qualification of the Pharmacy located at the
National Institutes of Health Pharmacy Department (NIH/PHAR) 9000 Rockville Pike Building 10,
Pharmacy, Bethesda, MD 20892.
Where appropriate this document makes reference to suitable internal policies and procedures,
identified by document number. Every attempt is made to generate a consistent uniform policy,
however due to the revision lag that occurs when this document is updated, conflicts may occur
from time to time (such as in issuing updated SOPs to reflect new policy). Where conflicts exist, the
most recent document that has undergone signature review must prevail.
1.2. Scope
The Validation Master Plan (VMP) covers all of the FDA regulated cGMP items that need to be
validated in order to comply with Part 21: 211 of the Code of Federal Regulations. The VMP is not
intended to be an exact prescription for the validation of processes or equipment. Instead it serves
as an internal guidance document that lays out the policies of NIH/PHAR as they apply to validation.
This Master Plan is limited to the NIH/PHAR and its Drug Manufacturing Operations, its
manufacturing equipment, and dedicated or shared utility systems. The following systems /
equipment / processes are addressed by this plan:
Equipment
Number Equipment Name
201 HPLC Unit(s)
202 Mass Spectrometer
203 Polarimeter
DSC differential scanning
204 calorimeter
CZE capillary zone
205 electrophoresis
206 IR infra-red spectrophotometer
207 Dissolution Testing Apparatus
UV ultra-violet
208 spectrophotometer
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401 Vial Washer

402 Autoclave
403 Depyrogenation Oven
404 Pass Thru
405 Lyophilizer
406 Bio-Safety Cabinet
407 Laminar Flow Hood
408 Dishwasher
409 Glatt, fluid bed dryer
410 Bio-Safety Cabinet
411 Dishwasher
412 Capsule Machine
413 "V" Blender
414 Tablet Press
HVAC
WFI
Pure Steam
RODI
Compressed Air
Nitrogen
Chilled Water
In addition to the IQ/ OQ / PQ of equipment, the plan includes processes that must be validated.
These are processes that are performed by NIH/PHAR personnel and for which the validation is
proving not only the process, but especially that specific personnel can conduct the process.
Products used for process validation should be manufactured using the same production
equipment, methods and procedures that will be used in routine production.
Some of the Processes Covered by the VMP are listed in the following table.
Section 9 Title
1 Fill Volume Qualification
2 Thawing of Frozen Product (pre-fill)
3 Filter Sterilization
4 Product Sterile Sampling
5 Media Fills
6 Visual Inspection
7 Product Lyophilization
8 Holding Period for Clean Items Prior to Use
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9 Holding Period for Sterile Items Prior to Use

10 General Hold Time Studies
11 Gowning Procedure Validation
12 Sterile Filter Integrity Testing
13 Washing of components other than vials
14 Sterilization of Loads (Steam or Dry Heat) other than vials
15 Facility Cleaning and Sanitization
16 Environmental Monitoring (non viable)
17 Environmental Monitoring (viable)
1.3. Definition of the Term Validation
Validation is a system of inter-related practices and procedures which, in combination with routine
production methods and quality control techniques, provides a high degree of assurance that a
system is performing as intended and will consistently produce a product meeting its pre-
determined specifications and quality systems.
1.4. Living Document

To be of any use, the VMP must be reviewed regularly and changed as needed. It should serve
as an organizational history and all changes should be included in an appendix with an
explanation of what changed and why. In the beginning it is expected that many fundamental
changes may occur as the organization evolves CFR 21 compliance. Later, the primary changes
may include additions of equipment and processes.
Section 3 – “Organizational Responsibilities for Validation” lays out the specific responsibilities
of three groups. These include the Validation Department, the Operations Department, and the
Quality Control Personnel. The assigned responsibilities for these groups facilitates keeping the
document and keeping it as a living document.
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Contents
2. Concept of Qualification / Validation ................................................................................................. 12
2.1. Definitions ................................................................................................................................... 12
2.2. Validation Life Cycle & Revalidation ........................................................................................... 14
2.3. System Impact Assessment – What processes must be validated? ........................................... 15
2.4. Elements of a Process Validation Protocol ................................................................................. 15
2.5. Elements of an Equipment Validation Protocol .......................................................................... 16
2.5.1. User Requirement Statement ............................................................................................. 17
2.5.2. Start Up ............................................................................................................................... 17
2.5.3. Commissioning .................................................................................................................... 17
2.5.4. Qualifications run by the Equipment Vendor ..................................................................... 17
2.5.5. Standard Operating Procedures for Equipment ................................................................. 18
2.5.6. Calibrated Measurement Equipment ................................................................................. 18
2.5.7. Documentation Use ............................................................................................................ 18
2.5.8. Installation Qualification ..................................................................................................... 18
2.5.9. Operational Qualification .................................................................................................... 19
2.5.10. Performance Qualification .................................................................................................. 20
2.5.11. Product Process Validation ................................................................................................. 21
2.6. Documentation Format for Qualification Programs ................................................................... 22
2.6.1. Installation Qualification Protocol Format.......................................................................... 22
2.6.2. Operational Qualification Protocol Format ........................................................................ 23
2.6.3. Performance Qualification Protocol Format ....................................................................... 23
2.6.4. SOP on SOPs ........................................................................................................................ 24
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2. Concept of Qualification / Validation
The regulations define process validation as establishing by objective evidence that a process
consistently produces a result or product meeting its predetermined specifications. The requirement for
process validation appears in section CFR 21: 211.68. The goal of a quality system is to consistently
produce products that are fit for their intended use. Process validation is a key element in assuring that
these principles and goals are met.
The process validation requirements stated in the regulations and the guidance offered here have
general applicability to manufacturing processes for pharmaceutical drugs. Many technologies are used
in the production of pharmaceuticals. The details of process validation will vary according to the nature
of the drug (e.g., sterile or non-sterile) and the nature and complexity of the process being validated.
The process specifications, hereafter called parameters, are derived from the specifications for the
drug(s), component(s) or other entity to be produced by the process. The process is developed such that
the required parameters are achieved. To ensure that the output of the process will consistently meet
the required parameters during routine production, the process is validated.
The basic principles for validation may be stated as follows:
• Establish that the process equipment has the capability of operating within required
parameters;
• Demonstrate that controlling, monitoring, and/or measuring equipment and instrumentation
are capable of operating within the parameters prescribed for the process equipment;
• Perform replicate cycles (runs) representing the required operational range of the equipment to
demonstrate that the processes have been operated within the prescribed parameters for the
process and that the output or product consistently meets predetermined specifications for
quality and function; and
Monitor the validated process during routine operation. As needed, re-qualify and recertify the
equipment.
2.1.Definitions
Installation Qualification
Installation Qualification (IQ) is the process that ensures that all equipment used in the
manufacturing process meets the specified requirements and is constructed, placed, and installed in
accordance with pre-approved specifications, to facilitate maintenance, adjustment, cleaning and
use. The Installation Qualification includes the verification of the installation requirements, the
verification of the equipment specifications, and the verification of the installation.
Operational Qualification
Operational Qualification (OQ) is the process that establishes confidence, through testing, that the
equipment used in the manufacturing process is capable of functioning within the anticipated range
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of operations. The Operational Qualification is normally conducted following the successful

completion of the Installation Qualification.
Performance Qualification
Performance Qualification (PQ) is the process that establishes through appropriate testing that the
equipment or system can consistently perform as specified. The Performance Qualification is
normally conducted following the successful completion of the Operational Qualification.
Process Validation
The process that establishes confidence through appropriate testing that the manufacturing
process, inclusive of all related equipment, personnel and practices, will produce a product meeting
all the pre-determined requirements for functionality and safety.
Validation Protocol
The protocol will be a written and approved plan stating how the validation will be conducted,
including test parameters, product characteristics, production equipment and decision points in
what constitutes acceptable results.
Prospective Validation
The validation process conducted prior to the distribution of either a new product or a product
made under a revised manufacturing process, where revision may affect the product’s
characteristics.
Static Operation
Operational state wherein normal cleaning, sanitization, and maintenance are conducted, but no
manufacturing processes are operating.
Dynamic Operation
Normal operating conditions with manufacturing (or simulated manufacturing) in progress, with the
normal number of personnel present.
CIP
Clean In Place – Cleaning of fixed equipment by use of an automated cleaning system.
COP
Clean Out of Place – Cleaning of non-fixed equipment by use of a semi-automated COP tank.
Manual Cleaning
Cleaning performed by individuals, per codified procedure, using manual methods (e.g. using wipes,
brushes, etc.)
Process By-Product
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Products produced by the manufacturing process that are not the desired product. Also included
are products that have been altered, modified, or denatured by the cleaning process.
Direct Product Contact Equipment

Such equipment will be defined as any surface in direct contact with the product, either fluids or
solid components, or in direct contact with product contact components / equipment.
Indirect Product Contact Equipment

Defined as any surface NOT in direct contact with product fluids / solids (e.g. in the product
transport path), but having the potential to become soiled, and when grossly soiled, have the
possibility to contaminate the manufactured product (e.g. formulation areas / isolators, where
residual particulate matter could contaminate direct contact surfaces).
Dedicated Equipment
Relative to cleaning validation, defined as product contact equipment that is used in the
manufacturing of a single Product, or Product Family (e.g. multiple strengths of the same active,
using the same excipients). Dedicated Equipment is appropriately labeled, and is stored and
controlled procedurally such that use with other products is precluded.
Multi-Use Equipment
For purposes of cleaning validation, is defined as any product contact equipment used for more than
one Product or Product Family. Equipment used for both Product manufacture and Media Fills is
considered Multi-use.
Visually Clean
For purpose of cleaning validation, is defined as visually free of particulates and soils, when carefully
examined under normal lighting conditions.
Pyrogens / Endotoxins
Bacterial components (typically lipopolysaccharides) that can deleteriously elevate body
temperature when introduced into the bloodstream are called Pyrogens. Endotoxins are
lipopolysaccharides that behave as Pyrogens.
2.2.Validation Life Cycle & Revalidation

As long as the process operates in a state of control and no changes have been made to the process or
output product, the process does not have to be revalidated. Whether the process is operating in a
state of control is determined by analyzing day-to-day process control data and any finished device
testing data for conformance with specifications and for variability. When changes or process deviations
occur, the process must be reviewed and evaluated, and revalidation must be performed where
appropriate. Review, evaluation, and revalidation activities must be documented. Processes may be
routinely validated on a periodic basis; however, periodic validation may not be adequate. More
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important is appropriate monitoring so that if problems develop or changes are made, the need for
immediate revalidation is considered.
2.3.System Impact Assessment – What processes must be validated?

Where process results cannot be fully verified during routine production by inspection and test, the
process must be validated according to established procedures. In the case of equipment used in the
manufacture of a drug product, the process must be validated for reproducibility regardless of its
obvious or not state of operation.
When any of the conditions listed below exist, process validation is the only practical means for assuring
that processes will consistently produce products that meet their predetermined specifications:
• Routine end-product tests have insufficient sensitivity to verify the desired safety and efficacy of
the finished products;
• Clinical or destructive testing would be required to show that the manufacturing process has
produced the desired result or product.
• Routine end-product tests do not reveal all variations in safety and efficacy that may occur in
the finished products.
• The process capability is unknown, or it is suspected that the process is barely capable of
meeting the drug specifications.
• Specific cGMP regulations require validation, for example CFR 21: 211.68.
2.4.Elements of a Process Validation Protocol

Careful planning of a validation study is essential to ensure that the process is adequately validated. The
plan should include design reviews. The plan for the validation study is documented in the validation
protocol. Planning for the validation should include the following elements as well as any other relevant
issues that must be addressed to conduct the validation study:
• Protocol Approval
• Identification of the process to be validated;
• Identification of all personnel participating in the validation;
• Identification of the use of any equipment or process in manufacturing;
• Criteria for a successful study;
• Length and duration of the study;
• Assumptions (shifts, operators, equipment, components);
• Identification of equipment to be used in the process;
• Identification of utilities for the process equipment and quality of the utilities;
• Complete description of the process – may be done by reference to another document;
• Relevant specifications including those for the product, components, manufacturing materials,
the environment, etc.;
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• Any special controls or conditions to be placed on preceding processes during the validation;
• Process parameters to be controlled and monitored, and methods for controlling and
monitoring;
• Product characteristics to be monitored and method for monitoring;
• Any subjective criteria used to evaluate the product – may require a discussion of how the
subjective criteria are to be evaluated;
• Definition of what constitutes nonconformance for both measurable and subjective criteria;
• Statistical methods for data collection and analysis ;
• Consideration of maintenance and repairs ;
• Statement of conditions that may indicate that the process should be revalidated;
• Stages of the study where design review is required;
• A place to record deviations from the protocol.
2.5.Elements of an Equipment Validation Protocol

The equipment validation is called a qualification and will be carry one of several adjectives. At NIH, the
types of equipment qualifications that will be performed include only installation qualification and
operational qualification. Performance qualifications fall into the broad category of “process
validations” and were discussed above.
After process equipment is designed or selected, it should be installed, reviewed, calibrated, challenged,
and evaluated to ensure that it is capable of operating within established limits and tolerances as well as
throughout all anticipated operating ranges. Installation and operation qualification studies establish
confidence that all equipment used in the manufacturing process meets specified requirements and is
appropriately designed, constructed, placed, and installed to facilitate maintenance, adjustment,
cleaning, and use.
The installation and operation qualification phases of process validation include:
• examining equipment design and supplied documentation;
• determining installation requirements;
• establishing any needed environmental controls and procedures;
• assuring that the work area has sufficient space to perform the processing and associated
activities;
• installing the equipment;
• verifying correct installation;
• establishing manufacturing procedures for the monitoring, operation, and control of the
equipment including the minimum number of operators;
• determining calibration, cleaning, maintenance, adjustment, and expected repair requirements;
• identifying important elements of the equipment that could affect the output or finished device;
• verifying that the system or subsystem performs as intended throughout all anticipated
operating ranges; and
• documenting the above information.
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2.5.1. User Requirement Statement
User Requirement Specifications (URS) may exist for major equipment or other installations. When such
documents exist, they can be used as a guide in drafting equipment validation protocols.
2.5.2. Start Up
“Start up” of equipment and facilities means to turn it on, apply power, start it up and otherwise
operate it for the first time. Start up may be performed by the vendor, the customer, or a third party
contractor. NIH/PHAR does not require a formal document in conjunction with start up. However,
evidence and verification of start-up activity should be presented to the customer. Start-up activities
are not part of validation. At start up, it is common to assure that the equipment is safe to operate, at
which time, it may be tagged “out of service” pending commissioning work.
2.5.3. Commissioning
Commissioning of equipment and facilities means to apply power and demonstrate that the equipment
comes on and superficially appears to operate in the manner that is intended. Commissioning is often
performed by the vendor under purchase obligations, but may be performed by external contractors or
company personnel. NIH/PHAR may require a formal document in conjunction with commissioning
when it is done by third parties. Otherwise, documentation is limited to qualification programs as
described in this Validation Master Plan.
Some of the commissioning may be performed at the time of a factory acceptance test (FAT) and more
can be done in conjunction with site acceptance testing (SAT). Such testing is often well documented,
the results being used as part of the installation qualification process. These FAT’s and SAT’s do not
negate the need for completion of IQ forms.
2.5.4. Qualifications run by the Equipment Vendor
Equipment fabricators may perform qualification runs at their facilities and analyze the results to
determine that the process equipment is ready for delivery to the buyer. If such vendor qualification
has occurred, then NIH/PHAR should request copies of the suppliers' qualifications studies to use as
guides, to obtain basic data, and to supplement their own qualification studies. However, it is usually
insufficient to rely solely upon the representations and studies of the equipment supplier. NIH/PHAR is
ultimately responsible for evaluating, challenging, and testing the equipment and deciding whether the
equipment is suitable for use in the manufacture of a specific drug. The evaluations may result in
changes to the equipment or process.
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2.5.5. Standard Operating Procedures for Equipment
Installation and operation qualifications should include establishing pertinent methods, procedures, and
schedules for calibration, cleaning, and maintenance, and establishing a repair parts list for each piece of
equipment. Planning for eventual maintenance and repairs can reduce or prevent confusion during
emergency repairs which could lead to improper repairs such as the use of the wrong replacement part.
Post-repair cleaning, calibration, and re-start requirements should be established if necessary to prevent
inadvertent manufacture of nonconforming products. The objective is to assure that all repairs can be
performed in a way that will not affect the characteristics of material processed or products
manufactured after repairs.
2.5.6. Calibrated Measurement Equipment
Monitoring equipment (validation instruments) should be calibrated at the beginning of the validation
study, and the calibration should be checked at the end of the study to establish confidence in the
validation of the process. Equipment found out of calibration at the end of a process validation study
may indicate that the process has not been operating in a state of control and cannot be considered
validated. More frequent calibration or more robust equipment may be necessary. Whenever possible,
monitoring equipment should be separate from the instrumentation provided as part of the equipment
being validated. In that way, it is possible to have parallel datasets, one provided by the equipment
being validated and the other provided from validation monitoring equipment.
2.5.7. Documentation Use
It is important to document installation and operation qualification studies. Such documentation can
substitute for part of the re-qualification of equipment in future process validation studies. When
equipment is moved to a new location, installation and operation should be re-qualified. By comparing
data from the original installation and operation qualification and the re-qualification, the manufacturer
can determine whether there have been any changes in equipment performance as a result of the
move. Changes in equipment performance should be evaluated to determine whether it is necessary to
revalidate the process.
2.5.8. Installation Qualification
The purpose of installation qualification is to assure the system / equipment has been installed properly,
utilities are connected, suitable documents have been received, procedures have been drafted,
calibrations are complete, training has been conducted, and equipment is in a ready to operate
condition.
The following will be obtained and verified during the installation qualification. Where documents are
unavailable, scientific rationale will explain why their omission is acceptable.
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• Operational and maintenance documents are available and either copies are included with the
IQ, or an exact reference is made to their location. These documents will reflect As-Built
conditions. Mark-up / Red-line documents are suitable.
• Product contact surfaces are finished to pharmaceutical standards and clean.
• Support utilities and lubricants are reviewed and compliant.
• Installation, hookup is complete and satisfactory.
• Software interfaces and controllers are documented and connected.
• Instrument calibration is complete. Non-critical gauges/instruments possess a “non-critical” or
“Do Not Calibrate” label. All other gauges/instruments possess a “Calibration” sticker.
• Preventative maintenance requirements are documented and complete.
• Equipment Safety has been evaluated, and found acceptable.
• Deficiency reports are reviewed and closed out.
• An IQ final report has been written and appended to the beginning of the IQ documentation.
Documents that can be included within the Installation Qualification include the following. Where
applicable for these documents, the NIH/PHAR location where this information is stored will be
indicated.
• Process and Instrumentation Diagrams. P&IDs must be available for large equipment such as
Ovens, Sterilizers, HVAC units, and Water Systems. Smaller “lab” equipment and “catalog”
items from vendors may contain electronic schematics but not P&ID.
• Flow Diagrams. Flow Diagrams are applicable to product process equipment as well as suites of
operating rooms. Lab testing equipment, temperature storage devices, and other non-product
processing equipment may not have flow diagrams.
• User Requirements Specifications. Much scientific equipment is purchased “as supplied” by a
catalog vendor and the customer will not have a written User Requirements Specification. In all
instances where a URS exists, it should be appended.
• Manuals.
• Product Specification Sheets.
• Assembly Documentation (weld maps, certificates)
• Cleaning Documentation (passivation, decontamination)
• Material Certificates
• Calibration Certificates
2.5.9. Operational Qualification
The purpose of the operational qualification of the equipment / system is to assure the key components
and features that may impact product / processes integrity have been tested and measured. Limit and
Boundary Tests will be conducted and all hi/lo alarm phase conditions will be identified. Any
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exceptional conditions, which may impact the product quality or process integrity, will be investigated
and appropriate corrective actions will be generated.
The following will be obtained and verified during the operational qualification. Where items cannot be
included, scientific rationale will be provided about why their omission is acceptable.
• Utilities and services have been installed and qualified.

• Equipment control function tests have been qualified.
• Identification of critical process parameters (CPP) has been established and all CPP related tests
have passed.
• Final commissioning trials under operational conditions are satisfactory and all tests have
passed.
• Standard operating procedures have been written.
• Safety conditions, alarms, and controls have been qualified.
• Deficiency reports have been closed out and reviewed.
• An OQ final report has been written an appended to the front of the OQ documentation.
2.5.10. Performance Qualification
Performance Qualification of equipment is often synonymous with Process Validation for product. The
purpose of process performance qualification is to rigorously test the process to determine whether it is
capable of consistently producing an output or in-process or finished product which meets
specifications. In entering the process performance qualification phase of validation, it is understood
that the:
• product, packaging, and process specifications have been established, documented, and
essentially proven acceptable through engineering, laboratory or other verification methods;
and
• process and ancillary equipment and the environment have been judged acceptable on the basis
of installation and operation qualification studies.
Challenges to the process should simulate conditions that will be encountered during actual production.
Challenges should include the range of conditions allowed in written standard operating procedures and
should be repeated enough times to assure that the results are meaningful and consistent. Challenges
may need to include forcing the preceding process to operate at its allowed upper and lower limits.
Process and product data should be analyzed to determine what the normal range of variation is for the
process output. Knowledge about the normal variation of the output is crucial in determining whether a
process is operating in a state of control and is capable of consistently producing the specified output.
Process and product data should also be analyzed to identify any variation due to controllable causes.
Depending on the nature of the process and its sensitivity, controllable causes of variation may include:
• temperature,
• humidity,
• variations in electrical supply,
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• vibration,
• environmental contaminants,
• purity of process water,
• light, and
• inadequate employee training.
Appropriate measures should be taken to eliminate controllable causes of variation. For example,
extreme variations in temperature can be eliminated by installing heating and air conditioning.
Employee training can be improved and conducted more frequently, and employees can be monitored
more closely to assure that they are properly performing the process. Eliminating controllable causes of
variation will reduce variation in the process output and result in a higher degree of assurance that the
output will consistently meet specifications.
After routine production begins, data derived from monitoring the process and output product can be
analyzed for variation and compared to the normal range of variation. Such analyses can detect when
the process output is shifting so that corrections can be made before, or soon after, nonconforming
product is produced.
2.5.11. Product Process Validation
The purpose of product performance qualification (process validation) is to demonstrate that the
process has not adversely affected the finished product and that the product meets its predetermined
specifications and quality attributes. Product performance qualification and design validation of initial
finished devices are closely related. According to the design control requirements, process validation
shall be performed under defined operating conditions on initial production units, lots, or batches, or
their equivalents. Products used for process validation should be manufactured using the same
production equipment, methods and procedures that will be used in routine production. Otherwise, the
product used for design validation may not be representative of production units and cannot be used as
evidence that the manufacturing process will produce a product that meets pre-determined
specifications and quality attributes.
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2.6.Documentation Format for Qualification Programs

2.6.1. Installation Qualification Protocol Format
General:
Protocol Page Header / Footer
Company Name
Protocol Name and Type
Document Number
Page Number at footer
Protocol Contents
Protocol Approval: Consists of the Name, Designation, Signature and date of the person(s)
responsible for the Preparation, Checking, and Approval of the Protocol.
System Description & Plan: Brief description of the system. Execution Team - Name, Designation,
and Signatures of the Execution team responsible for the execution of
the protocol.
Statement of Purpose: Define the objective of the validation study for each item of equipment
/ critical system and state validation acceptance criteria.
SOPs: Identify or draft SOPs for Operation, Cleaning, and Preventive
Maintenance of systems, utilities, and process's. This can be one, two,
or three SOPs as determined by equipment complexity.
Calibration List: List of components needing calibration. i.e. gauges, scales.
Installation Check List: List of major components and verification of their installation.
Inspection Check List: Inspection of each part and the sub part of the installed equipment in
detail.
Documentation Check List: All the relevant drawings will be attached in this section (P&IDs, General
Arrangement, Utilities Lay-Out, Isometric Diagrams, Schematics)
Material of Construction: All documents detailing material of construction.
Support Utilities: List the supporting utilities against P&ID and record whether or not they
are properly connected and identified.
Manufacturers Certification: Review and attach all manufacturers certificate(s) like: Commissioning /
Installation Report / FAT / Inspection Report, Calibration Certifications,
etc.
Safety Assessment: Review of equipment and identification of safety issues.
Deficiencies Report: Description of deficiency and date observed, Person responsible for
corrective action and date assigned, corrective action taken and date
resolved
Final Report: Summary of validation activities, analysis / evaluation, deviation-
resolutions, report approval and signatures
Appendix: Abbreviations and Definitions, Reference Documents
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2.6.2. Operational Qualification Protocol Format
General:
Company Name
Document Number
Protocol Contents
Protocol Approval: Consists of the Name, Designation, Signature and date of the persons
responsible for the Preparation, Checking, and Approval of the Protocol
System Description and Plan: Brief description of the system. Execution Team - Name, Designation,
the protocol.
SOPs: Verification of the SOPs for Operation, cleaning, and preventive
maintenance
Key Functionality: The purpose of these procedures is to demonstrate that the unit
provides the proper functionality as specified by the manufacturer.
Operating Parameters: Identify the Operating parameters used to demonstrate that the unit
provides the proper functionality as specified by the manufacturer.
Safety Features: Identify the Safety features / hazards of the unit / system / process,
includes alarm and interlock testing.
2.6.3. Performance Qualification Protocol Format
General
Company Name
Document Number
Protocol Contents
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Protocol Approval: Consists of the Name, Designation, Signature and date of the persons
responsible for the Preparation, Checking, and Approval of the Protocol
System Description and Plan: Brief description of the system. Execution Team - Name, Designation,
the protocol.
Validation: The purpose of these procedures is to demonstrate that the unit
repeatedly provides the proper functionality as defined.
2.6.4. SOP on SOPs
Specific:
NIH/PHAR’s
General:
A typical SOP contains the following elements:
A Header which shows the Title of the SOP, Original Issue Date, Revision/Review Date, number of pages
contained in the SOP, who wrote the SOP, and the Approval Signature. The following is a list of
headings which might appear within the SOP.
• Table of Contents
• Related SOPs
• Purpose
• Scope
• Responsibility
• Definitions
• Materials and Equipment needed
• Safety
• Procedure
• Documentation
• Forms
• Revision History
• References
SOPs are reviewed periodically on a schedule set by NIH/PHAR related to the need for review.
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Contents
3. Organizational Responsibilities for Validation .................................................................................... 26
3.1. Validation Department Personnel .............................................................................................. 26
3.2. Operations Department Personnel ............................................................................................. 26
3.3. Quality Control Personnel ........................................................................................................... 26
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3. Organizational Responsibilities for Validation
3.1. Validation Department Personnel
• Manage the Validation Master Plan (VMP): Original VMP is to be generated by a contractor.
• Oversee execution of the IQ/OQ/PQ protocols as specified within the VMP (Vendor or
Contractor supplied IQ/OQ/PQ protocols may be used).
• Approve Standard Operating Procedures (SOPs) for validated equipment and processes. The
source of SOPs will be knowledgeable personnel or others.
• As needed, provide technical training to personnel involved in the Validation execution.
• Approve Final Summary Reports upon completion of the IQ/OQ/PQ protocols as specified within
the VMP.
3.2. Operations Department Personnel
• Review and approve the VMP

• Review and approve all protocols generated pursuant to the VMP.
• Review and approve SOPs for validated equipment and processes.
• Provide personnel to assist in execution; provide review and approval; installation, start-up and
prequalification inspection of systems addressed by this VMP.
3.3. Quality Control Personnel
• Review and approval of associated protocols and related documentation to assure compliance to
NIH/PHAR’s procedures and internal standards.
• Archive all completed validation documentation and related documentation in a manner
consistent with approved procedures.
• Aid in the development of the specific cleaning assays, and to provide trained sampling personnel
and / or recovery methods training of personnel, during the execution of this master plan and the
specific cleaning protocols.
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Table of Contents
4. Description of the Area and Equipment Covered by this VMP ........................................................... 28

4.1. Facility Use .................................................................................................................................. 29
4.1.1. Filling Area Description ....................................................................................................... 29
4.1.2. Tablet Area Description ...................................................................................................... 29
4.1.3. Capsule Manufacture .......................................................................................................... 29
4.1.4. Capacities ............................................................................................................................ 29
4.1.4.1. Filling Capacity ............................................................................................................ 30
4.1.4.2. Tablet Capacity ............................................................................................................ 30
4.1.4.3. Capsule Capacity ......................................................................................................... 31
4.1.4.4. Fluid Bed Dryer............................................................................................................ 31
4.1.4.5. Lyophilization Capacity ............................................................................................... 31
4.1.5. Packaging Capacity .............................................................................................................. 32
4.2. Building ....................................................................................................................................... 32
4.2.1. Floor Plan – People flow ..................................................................................................... 32
4.2.2. Floor Plan – Product flow .................................................................................................... 33
4.2.3. Floor Plan – Waste flow ...................................................................................................... 33
4.2.4. Floor Plan – Room Classification ......................................................................................... 33
4.2.5. Floor Plan – Room Pressurization ....................................................................................... 33
4.2.6. Routine Operation – Description ........................................................................................ 33
4.3. Utilities ........................................................................................................................................ 33
4.3.1. Plant Utilities ....................................................................................................................... 33
4.3.1.1. Chilled Water .............................................................................................................. 33
4.3.1.2. Plant steam ................................................................................................................. 34
4.3.1.3. Humidification ............................................................................................................. 34
4.3.1.4. Sanitary waste ............................................................................................................. 34
4.3.1.5. Compressed air ........................................................................................................... 34
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4.3.1.6. Nitrogen System .......................................................................................................... 34

4.3.1.7. Purified Water ............................................................................................................. 34
4.3.1.8. Water for Injection ...................................................................................................... 34
4.3.1.9. Purified Steam ............................................................................................................. 35
4.3.2. Electrical .............................................................................................................................. 35
4.3.2.1. Lighting ........................................................................................................................ 35
4.3.2.2. Outlets ......................................................................................................................... 35
4.3.2.3. Switches ...................................................................................................................... 35
4.3.2.4. Power Distribution ...................................................................................................... 35
4.3.2.5. Fire Alarm .................................................................................................................... 35
4.3.2.6. Emergency Power ....................................................................................................... 35
4.3.3. Architectural Finishes .......................................................................................................... 35
4.3.4. Structural............................................................................................................................. 36
4.3.5. HVAC ................................................................................................................................... 36
4.3.5.1. Supply System ............................................................................................................. 36
4.3.5.2. Supply Equipment ....................................................................................................... 37
4.3.5.3. HEPA Filters ................................................................................................................. 37
4.3.5.4. Dampers ...................................................................................................................... 37
4.3.5.5. Controls ....................................................................................................................... 37
4.3.5.6. Redundancy................................................................................................................. 37
4.3.5.7. Emergency Power ....................................................................................................... 37
4.3.5.8. Monitoring .................................................................................................................. 37
4.3.6. Class Air Designations ......................................................................................................... 38
4.3.6.1. Grade A ....................................................................................................................... 38
4.3.6.2. Grade B........................................................................................................................ 38
4.3.6.3. Grade C & D ................................................................................................................. 38
4.3.7. Terminal HEPA Filtration for Grades A, B, and C ................................................................. 38
Description of the Area and Equipment Covered by this VMP

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4.1.Facility Use
4.1.1. Filling Area Description
The Pharmacy is located at National Institutes of Health Pharmacy Department (NIH/PHAR) 9000
Rockville Pike Building 10, Pharmacy, Bethesda, MD 20892. The manufacturing operations area is
generally referred to as the “Atrium Infill. The new Pharmacy area comprises approximately 15,000 ft2 of
interior space including manufacturing, quality control testing, pharmaceutical processing, packaging and
clerical support/administrative activities.
The Pharmacy is intended to be a multi-product manufacturing facility for aseptically filled sterile
products either in liquid or lyophilized form as well as a site for the production of capsules, tablets,
creams, and ointments. Fill volumes range between 2 to 20 cc in vials. The Pharmacy clean areas are
supplied with clean air through HEPA filters with air classes of class 100 and class 10,000.
The aseptic core area consists of one custom designed 10 foot wide laminar flow hood and a 8 foot wide
biological safety cabinet, both within a class 10,000 HEPA filtered room. Both the custom laminar flow
hood and the bio-safety cabinet are by Nuaire.
Glass is sterilized in clean prep room 1C166A5A using a Despatch depyrogenation oven, model LLC2-
14-3PT. Other items are sterilized by way of a pass through steam sterilizer, Beta-Star model N2020.
There is a lyophilizer door access to the class 10,000 area. From time to time, drug products may be
placed into the lyophilizer and dried. Aseptic technique for this procedure has to be addressed.
4.1.2. Tablet Area Description
Chemicals are weighed in either of rooms 1C166A7 or 1C166A4 and as needed transferred to room
1C166A7d for blending in a Patterson-Kelley/Harsco "V" Blender - twin shell 1 cu ft. After blending the
material is transferred to room 1C166A7E and made into a tablet with a Pressima tablet press.
4.1.3. Capsule Manufacture
For capsule manufacture the weighed chemical components are introduced to a GPCG2 Lab System fluid
bed dryer by Glatt in room 1C166A7AB. Subsequently, the processed components are placed into
capsules in room 1C166A7E.
4.1.4. Capacities
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4.1.4.1. Filling Capacity
Filling is done in a laminar flow hood or a biological safety cabinet, by hand using a volumetric delivery
automatic pipette. Sterile, RS West system stoppers are applied using sterile tweezers under the laminar
flow air. The maximum fill lot size is approximately 1500 units. There is no limitation on the size of the
vial filled. However, as the vial size increases the number of units filled necessarily decreases to keep the
total filling time under about six hours.
4.1.4.2. Tablet Capacity

Pressima is a force feed tablet press for R&D applications, clinical trial batches and small scale
production.
MAIN CHARACTERISTICS
Interchangeable turret.
Special turret version with both B and D type tooling is available.
Pre-pressure and main pressure rollers are designed for forces similar to production
machines ensuring a good scale-up.
Compression area completely accessible from all sides for quick and easy cleaning.
Mechanical area fully accessible for maintenance operations by simply opening the external
casing.
Average pressure displayed without instrumentation for R&D
Machine operated by the control panel and fitted with hand wheels to adjust fill
depth, pre-pressure and main pressure. All values are displayed on the control
panel.
The machine instrumented for R&D can also display the following values: upper
and lower compression force, ejection force, pre-compression force, tablet scraper
force, upper and lower punch displacement.
Press Type 8(4x4) 8 13 16 19

Number of 4–B 8 13 16 19
Punches 4–D
Tool Type EU-TSM D-B EU-TSM D-B EU-TSM D-B
Max tablet 25 -18 25 25 16 13
Diameter (mm)
Max die filling 17 17 17 17 17
(mm)
Pitch circle 180 180 180 180 180
diameter (mm)
Tablets/hr (min) 2400 4800 7800 9600 11400
Tablets/hr (max) 16800 33600 54600 67200 79800
Die table rpm 10 to 70 10 to 70 10 to 70 10 to 70 10 to 70
Pre-comp force 10 10 10 10 10
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kN
Compression Max 50 50 50 50 50
kN
4.1.4.3. Capsule Capacity

Zanasi (IMA) 6/12 F are capsule filling machines able to dose powders, pellets, liquids and tablets into
hard gelatin capsules. The powder and pellet dosing units feature dosators and pistons that move down in
the powder bowl, picking up the product by means of vacuum. The liquid dosing group makes use of an
extremely precise volumetric dosing system; finally the tablet dosing unit is fitted with feeding tubes and
blades that transfer the tablets into the capsule body. The machines are equipped with a statistic weight
checking unit for production monitoring.
Model Zanasi/IMA 6/12 F model 6F

Number of Capsules per Cycle 2
Capsule Size 000-5, Supro A-E, DB, DBAA
Maximum Output 6000/hr
Aspiration 3,850 liters/minute – 2,500 mm H2O
Compressed Air 30 dm3/min – 6 bar
Vacuum 40 m3/h– 3 mbar
Standard Volatage 230/440 V (-10%) – 50/60 Hz
Weight 1000 kgs
4.1.4.4. Fluid Bed Dryer

The fluid bed dryer is a CPCG 2 Fluid Bed Lab System from Glatt. The optimal batch size is from 0.3
kg to 0.5 kg.
Batch fluid bed: The moist starting product is fed batch-by-batch into the material container of the dryer
for drying. Here it is mixed vigorously in the upwards-pointing heated gas stream and held in suspension.
In doing so, it dries to the required end moisture content with high heat and material transition
coefficients. The optimum gas speed depends essentially on the particle size and density. Heat is fed in
via the process air. The temperature of the air can be varied during the drying process. After drying, the
product can be cooled before the material container is emptied and the next batch starts.
Batch fluid bed granulation: For Agglomeration/Granulation in batch mode, the dry starting product is
placed in the product container. Here it is mixed vigorously in the heated gas stream, held in suspension
and agglomerated/granulated by spraying with a suitable bonding material. The product is then dried to
the required end moisture content with high heat and material transition coefficients.
Fluid bed coating: With fluid bed coating, particles are fluidized and the coating fluid sprayed on and
dried. Small droplets and a low viscosity of the spray medium ensure an even product coating.
4.1.4.5. Lyophilization Capacity

The lyophilizer has 4.59 square ft of shelf space over 3 shelves. Each shelf is 10.75 inches across and
20.5 inches deep. In making the calculation for the number of vials, each tray of 10.5 x 20.5 inch OD has
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been assumed to be 10 x 20 ID. The tray walls are substantially thinner than 0.5 inches, thus the number
of units that will go into a tray is underestimated.
The 35 Liter condenser capacity would allow for all trays (10.5” x 20.5”) to be filled 1 inch deep and still
have ample capacity for bulk lyophilization.
Capacity Height Body OD

Units per Shelf
mL mm mm
2 33 15 627
2 86 12 578
5 40 22 264
5 47 23 252
10 50 24 220
10 54 26 198
20 63 29 152
20 58 33 119
30 63 37 91
50 73 43 66
100 95 52 45
125 107 54 40
4.1.5. Packaging Capacity
The packaging line consists of a room for the hand packaging operation of all products. All vial products
are visually inspected for particulates against a black and white background.
Labels are printed on demand immediately prior to use. Label accountability is controlled through paper
records. Labels that are not used are destroyed immediately at the end of the labeling operation.
4.2.Building
4.2.1. Floor Plan – People flow
The People Flow Diagram - NIH/PHAR.1006.01.DWG depicts the normal flow of people during routine
operations.
Only the minimum number of personnel required should be present in clean areas; this is particularly
important during aseptic processing. The number of people present in the area is defined by the
environmental monitoring program validation.
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4.2.2. Floor Plan – Product flow
The Material Flow Diagram - NIH/PHAR.1007.01.DWG depicts the normal flow of materials during
routine operations.
4.2.3. Floor Plan – Waste flow
The Waste Flow Diagram – NIH/PHAR.1007.01.DWG depicts the normal flow of waste during routine
operations.
4.2.4. Floor Plan – Room Classification
The Room Classification Diagram – NIH/PHAR.1008.DWG depicts the room classifications during
routine operations.
4.2.5. Floor Plan – Room Pressurization
The Room Pressurization Diagram – see (Appendix 2) depicts the room pressurization during routine
operations. Numbers in the table are in inches of water.
Grade A – Target : > 0.05 above adjacent areas (approx. class 100)
Grade C – Target : > 0.05 above adjacent areas (approx. class 10,000)
Grade D – Target : > 0.05 above adjacent areas (approx. class 100,000)
4.2.6. Routine Operation – Description
Routine operations are those operations where validated processes are conducted. This includes the
necessary operations to maintain the facility in a validated state. Operations such as equipment or facility
shut-downs for periodic maintenance, calibration, or repair are non-routine and require a verification of
appropriate critical operational parameters through an approved SOP or protocol.
4.3.Utilities
4.3.1. Plant Utilities
4.3.1.1. Chilled Water

The chilled water will be supplied using a permanent system at 42°F from a central facility supply. The
local capacity flow is at least 447 GPM. The chilled water system is a continuous process. The primary
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use of chilled water will be the summer cooling requirements of the HVAC. A secondary user of chilled
water will be for the process equipment. The air-handling units were designed for a chilled water supply
temperature of 42°F and a return temperature of 52°F. Other uses of chilled water include the WFI
condenser, cold room condenser, various point of use heat exchangers, the lyophilizer, and the clean
steam generator.
4.3.1.2. Plant steam

NIH supplies steam from an existing main on the B2 level at 165 psi. Plant steam is used to generate hot
water, supply heat for purified steam, heat the water for injection (WFI), , and keep the jacket hot on the
autoclave.
4.3.1.3. Humidification
Humidification to the HVAC system is derived from a special purpose humidifier which uses plant steam
as a source of humidity. The steam available is 1040 lb/hr.
4.3.1.4. Sanitary waste

Waste designated as sanitary waste is gravity drained to an existing sanitary sewer for the building.
4.3.1.5. Compressed air

The compressed air system will supply -40°F dew point to the utilities and production users. The system
will consist of a compressor, a refrigerant type air dryer, and a receiver tank. Air will be designated as
instrument air and process air. There will be no functional difference between these designations as all air
will come from the same source. Air will be supplied at a pressure of at least 100 psig and will be
regulated by individual regulators at equipment locations.
4.3.1.6. Nitrogen System

The high-purity nitrogen system distributes nitrogen from cylinders to the lyophilizer, the vial fillers, and
process utilities. The system consists of a multi-cylinder switchover manifold and distribution piping to
the users. Equipment will be supplied by a suitable vendor according the owner’s URS documents.
Distribution system will be of suitable design according to the mechanical engineer.
4.3.1.7. Purified Water

A RODI Water System is available to feed the WFI pure steam generator. Sanitization of the RODI
water system may be performed with peracetic acid / hydrogen peroxide solutions. An example product
is Minncare® from Mar Cor. Directions should be followed from the product manufacturer. The
chemical sterilant may be used on the RO membranes as well as associated piping and components.
4.3.1.8. Water for Injection

The WFI Production System consists of a pure steam generator and condenser, sanitary storage tank,
distribution system, and point of use heat exchangers. The WFI System is used for equipment cleaning
and compounding. System will be located in the “pent-house” (level 3). The generation system uses
water from RODI to produce pure steam that is then condensed and collected in a 400 gallon jacketed
tank and maintained at 80° C during recirculation throughout the manufacturing area.
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4.3.1.9. Purified Steam

The Pure Steam System consists of a MECO Pure Steam Generator and distribution piping throughout the
building. It supplies Pure Steam at suitable pressure for process use and as steam for the autoclaves, and
vial washer. RODI Water Loop supplies water to the Pure Steam Generator. The Pure Steam Generator
heating source is plant steam at 100 -125 psig.
4.3.2. Electrical
The following electrical systems serve the facility
4.3.2.1. Lighting
The lighting for the NIH/PHA project is based on providing 70foot-candles (fc) in the process areas. The
light fixtures in the process area are rated for Grade C clean room applications, meaning that they can be
panel cleaned. The corridors and warehouse foot-candle levels comply with National Electrical Code
guidelines. Emergency lighting is provided by battery-operated emergency fixtures in the mechanical
and electrical area, and by the use of battery-operated ballast in the light fixtures in the process area.
Light Fixtures are Static transfer with flush frame, sealed housing, diverted prismatic acrylic lens, and
electronic ballast. Fixtures contain multiple bulb 32W lamps. The exit sign light fixtures are white face
with 8” high red letters stencil, battery pack, test switch, and down light component.
4.3.2.2. Outlets
Three prong electrical outlets will be located within all rooms within the facility. They will be protected
with suitable covers to allow periodic washing of rooms with approved sanitizers. Outlets within sterile
hoods are of suitable vendor design to allow safe cleaning and operation.
4.3.2.3. Switches
Light switches will be located within all rooms of the facility. They will be protected with suitable covers
to allow periodic washing of rooms with approved sanitizers.
4.3.2.4. Power Distribution

National Institutes of Health has its own power plant and distribution service. The facility management
group is responsible for providing suitable power to all locations.
4.3.2.5. Fire Alarm

Conform to current NFPA and ADA requirements.
4.3.2.6. Emergency Power

Suitable emergency power will be brought online at a later time.
4.3.3. Architectural Finishes
The architectural layout is designed around the people and product flows for the facility. Consideration is
given to the provided building code-required means of egress. The following finishes shall be furnished
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by suitable vendor(s). All applications will be verified by the vendor(s) and approved by NIH/PHAR
consultants. Finishes are durable and resistant to chemical cleaning and decontamination. Finishes in the
Grade C areas must be compatible with alkaline phenolic compounds, acidic phenolic compounds and
low concentration chlorine bleach to permit repeated contact with disinfection agents.
Floors are seamless, chemical-resistant poured epoxy. Grade C ceiling systems are full drywall and
incorporate HEPA filter units.
Finish details are designed to facilitate cleaning and decontamination and minimize the accumulation of
dust and dirt. Floor finishes are turned up the wall to make a coved base in all clean areas. Wall finishes
within classified clean areas are designed so that doorframes are flush with the wall surface. Doors and
frames are hollow metal and are finished with a two part epoxy paint system. Door hardware is suitable
for clean room use and compatible with approved sanitizers. Vision panels and frames are flush mounted
with a gasket and seal.
Example sanitizers which must be approved by NIH/PHAR prior to use include: LpH-se phenolic
(Steris); Vesphene® IIse Phenolic Disinfectant (Steris); Spor-Klenz (Steris); 1% hypochlorite (Clorox).
Sprinkler heads will be concealed drop-down style.
Architectural materials and finishes have been reviewed and are consistent with the ISPE Baseline Guide,
Volume 3 Sterile Manufacturing Facilities, Edition 1999. The principal architectural aspects of the
facility are that the primary function of the room is to provide an enclosure to contain the defined activity
and its associated equipment. Finished materials are non-shedding, non-porous, and resistant to
sustaining microbial growth. Such finishes exclude the use of wood. Surfaces are smooth and easy to
clean, with minimal ledges, joints, and with corners that are difficult to access, particularly near the
product and process equipment. Finishes are capable of withstanding repeated cleaning and sanitization
with various chemicals and resist surface oxidization.
4.3.4. Structural
Structural steel will be provided to support HVAC equipment. An access mezzanine will be provided
above strategic portions of the facility to provide for servicing of HVAC, WFI, and related utility
installations. Use of wood in structural / mezzanine areas is not allowed.
4.3.5. HVAC
The HVAC system for the Pharmacy is provided to ensure the proper environmental conditions including
temperature, pressure, particulate, and viable conditions are achieved.
4.3.5.1. Supply System

The supply system consists of three primary air-handling units and two secondary fans. One primary unit
serves the office areas while the other two AHUs are built in parallel for redundant supply to the lab and
process areas.
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4.3.5.2. Supply Equipment

The system is capable of maintaining a discharge temperature of 55oF (adjustable) when the outside
temperature varies between 15oF and 95oF. Heating hot water coils and chilled water coils located in the
system will temper the air stream before entering the distribution system. Humidity control is provided in
this system. The relative humidity must stay between 30 and 60%.
4.3.5.3. HEPA Filters

The HVAC system will be supplied with 95% filters within the primary AHUs and Terminal HEPA
filters within all classified rooms. Terminal HEPAs will be installed in suitable housings with self-
contained dampers for final adjustment, and test ports for certification. Filters will be appropriately
designed for in-situ filter testing. Provisions are made in the filter units for DOP testing.
4.3.5.4. Dampers
Dampers will be low leakage design with handle for manual adjustments during balancing and
recertification.
4.3.5.5. Controls
The controls for the HVAC shall be through a Siemens control system. The building management system
incorporates all inputs, outputs, set points, sequence of operations, programming, alert alarms, operator
interfaces and graphical displays as necessary to provide for a fully functional and operational system.
4.3.5.6. Redundancy
The supply and exhaust air system will not be provided with totally redundant capabilities. Each
recirculation loop will be provisioned to operate independently. Primary air-handling unit and Supply
Fan will operate independently. Production will not be allowed to continue if there is a failure of any
major components of the equipment.
4.3.5.7. Emergency Power

All HVAC is to be configured for emergency power backup capability. The HVAC system is designed to
operate continuously in the event of a power outage maintaining facility cleanliness and integrity.
4.3.5.8. Monitoring
The HEPA filters (hoods and HVAC supply air) are re-certified twice a year. In between, the Phar/NIH
performs regular particulate monitoring in appropriate areas to assure compliance.
Clean areas for the manufacture of sterile products (bio safety cabinets) are classified according to the
required characteristics of the environment. Each manufacturing operation requires an appropriate
environmental cleanliness level in the operational state in order to minimize the risks of particulate or
microbial contamination of the product or materials being handled.
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4.3.6. Class Air Designations
In order to meet “in operation” conditions these areas (class C rooms and bio safety cabinets or laminar
flow hoods) should be designed to reach certain specified air-cleanliness levels in the “at rest” occupancy
state. The “at-rest” state is the condition where the facility is installed and operational, complete with
production equipment but with no operating personnel present. The “in operation” state is the condition
where the installation is functioning in the defined operating mode with the specified number of personnel
working.
The “in operation” and “at rest” states should be defined for each clean room or or laminar flow hood.
The following four grades are defined (although grade B is not used):
4.3.6.1. Grade A
Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open vials, making
aseptic connections. Normally such conditions are provided by a laminar air flow work area. Laminar air
flow systems should provide a homogenous air speed in a range of 0.36-0.54 m/s at the working position
in the open clean room applications. The maintenance of laminarity should be demonstrated and
validated. A uni-directional air flow and lower velocities may be used in closed isolators and glove
boxes.
4.3.6.2. Grade B
These areas include the sterile storage, sterile gowning and the air lock between the fill room and sterile
storage.
4.3.6.3. Grade C & D

These are clean areas for carrying out less critical stages in the manufacture of sterile products and for the
regular manufacturing of creams, tablets, capsules, and ointments.
At Rest In Operation (after “clean up

period”)
Grade Maximum permitted number of particles / m3 equal to or above
0.5 µm 5 µm 0.5 µm 5 µm
A 3500 1 3500 1
B 3500 1 350000 2000
C 350000 2000 3500000 20000
D 3500000 20000 Not defined Not defined
4.3.7. Terminal HEPA Filtration for Grades A, B, and C

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Grade A – Aseptic preparation and filling

Grade B – Storage of Sterilized items.
Grade C – Preparation of solutions to be filtered
Grade D – Pre-gowning and background air
Recommended limits for microbial contamination
Grade Air sample cfu/m Settle plates Contact plates Glove print 5
(diameter 9.0 (diameter 55 fingers cfu /
mm), cfu/4 hours mm), cfu / plate glove
A <1 <1 <1 <1
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -
Components after washing should be handled in a grade C environment. Handling of sterile starting
materials and components, unless subject to sterilization or filtration through a micro-organism filter later
in the process, should be done in a grade A environment with grade C background.
Preparation of solutions which are to be sterile filtered during the process should be done in a grade C
environment; if not filtered, the preparation of materials and products should be done in a grade A
environment with a grade C background.
Handling and filling of aseptically prepared products should be done in a grade A environment with a
grade C background.
Prior to the completion of stoppering, transfer of partially closed containers, as used in freeze drying
should be done in a grade A environment with a grade B background or in sealed transfer trays in a grade
B environment.
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Contents
5. Calibration Program ............................................................................................................................ 41
5.1. Introduction ................................................................................................................................ 41
5.2. Equipment Selection ................................................................................................................... 42
5.3. Procedures .................................................................................................................................. 42
5.4. Program Management ................................................................................................................ 42
5.5. Calibration Records ..................................................................................................................... 43
5.6. Schedules .................................................................................................................................... 43
5.7. Standards .................................................................................................................................... 44
5.8. Calibration Environment and External (Contractor) Calibration ................................................ 44
5.9. Assessment Procedure for Inclusion ........................................................................................... 45
5.10. Summary of Validation Policy Applied to Calibration ............................................................. 45
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5. Calibration Program
5.1. Introduction
When a medical drug manufacturer uses a contract calibration laboratory, FDA expects the manufacturer
to have evidence that the equipment was calibrated according to the GMP requirements.
The drug manufacturer can do this by:

• requiring and receiving certification that the equipment was calibrated under controlled
conditions using traceable standards;
• maintaining an adequate calibration schedule;
• maintaining records of calibration; and
• Periodically auditing the contractor to assure appropriate and adequate GMP procedures are being
followed. For example, the contractor should have:
o written calibration procedures;
o records of calibration;
o trained calibration personnel; and
o Standards traceable to NIST or other independent reproducible standards.
Certification notes and data should include accuracy of equipment when received by the lab to facilitate
remedial action by the finished device manufacturer, if necessary. Certification should also include
accuracy after calibration, standards used, and environmental conditions under which the equipment was
calibrated. The certification should be signed and dated by a responsible employee of the contract lab.
If in-house standards are used by a contractor to calibrate device-related measuring equipment, these
standards shall be documented, used, and maintained the same as other standards.
NIH/PHAR recognizes three means by which equipment may obtain calibration. These are Outside
Contractor, External Calibration, and Internal Calibration. The specific designations are explained below.
Outside Contractor: A qualified (by training and/or experience) and approved organization or individual
who provides professional services for the calibration of equipment, systems, instruments, gauges or
testing devices and facilities.
External Calibration: the process of testing equipment, systems, instruments, gauges or testing devices,
as performed by an outside contractor, to ensure that the equipment is performing within its approved
limits of accuracy and precision. This procedure may include remedial actions, performed by the
contractor, to return the equipment’s function back to its approved limits of accuracy and precision.
Internal Calibration: The process of testing equipment, systems, instruments, gauges or testing devices,
as performed by NIH/PHAR employees, to ensure that the equipment is performing within its approved
limits of accuracy and precision this procedure may include remedial actions, performed by NIH/PHAR
employees, to return the equipment’s function back to its approved limits of accuracy and precision.
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5.2. Equipment Selection

NIH/PHAR maintains procedures to ensure that purchased and otherwise received equipment and
associated supplies conform to specified requirements. The purchase of stable and accurate measuring
equipment can reduce the frequency of calibration and increase confidence in NIH/PHAR's metrology
program. Where economically feasible, equipment with more accuracy than needed for various
measurements can be used longer without recalibration than equipment that marginally meets the desired
accuracy requirements. Delicate instruments, however, that are "pushing the state-of-the-art" should not
be used for routine measurements unless no other approach is feasible.
5.3. Procedures
There are a number of sources of information from which calibration procedures can be developed.
Instrumentation manufacturers often include calibration instructions with their instruction manuals.
Although these instructions alone may not be adequate for the SOP, they usually contain instructions for
the actual calibration process. In some cases, voluntary standards exist such as those by the American
Society for Testing and Materials (ASTM), the American National Standards Institute (ANSI), and the
Institute of Electrical and Electronic Engineers (IEEE).
Information contained in calibration procedures should be adequate to enable qualified personnel to
properly perform the calibrations.
A typical equipment calibration procedure includes:
• purpose and scope;
• frequency of calibration;
• equipment and standards required;
• limits for accuracy and precision;
• preliminary examinations and operations;
• calibration process description;
• remedial action for product; and
• documentation requirements.
5.4. Program Management

The selection and training of competent calibration personnel is an important consideration in establishing
an effective metrology program. Personnel involved in calibration should ideally possess the following
qualities:
• technical education and experience in the area of job assignment;
• basic knowledge of metrology and calibration concepts;
• an understanding of basic principles of measurement disciplines, data processing steps, and
acceptance requirements;
• knowledge of the overall calibration program;
• ability to follow instructions regarding the maintenance and use of measurement equipment
and standards; and
• Mental attitude which results in safe, careful, and exacting execution of his or her duties.
Initial Calibrations are performed prior to the Installation Qualification / Operational Qualification
process. Equipment that has not been calibrated or has an expired calibration will be locked and tagged
out of service. Internal calibration procedure will be performed with equipment htat has been calibrated
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to NIST Standards. Calibration SOPs will include procedure, frequency of testing, standards to be used to
perform tests, limits of precision and acceptance criteria. The SOP may include remedial actions to be
performed to return the equipment’s function to its acceptable limits.
5.5. Calibration Records

Calibration of each piece of equipment shall be documented to include as a minimum:
• equipment identification,
• the calibration date,
• the calibrator, and
• The date the next calibration is due.
Calibration Reports will include equipment identification, serial number of standards used, specifications,
results, pass/fail status and if required, the remedial action required to return the equipment back to its
acceptable limits. Original Certificates of Calibration and/or Calibration Reports will be maintained by
the Validation Department. Outside Contractors should provide procedures to the Validation Department,
including standards to be used in the performance of testing, limits of accuracy, acceptance criteria and if
necessary, remedial action taken to return the equipment’s function back to its acceptable limits.
Certificates of Calibration from outside contractors will be reviewed and approved by the Validation
Department prior to using the equipment so certified. In addition, a system is used where each device has
a decal or tag which contains the date of calibration, by whom calibrated, and date the next calibration is
due. An example decal is shown.
CALIBRATION
NOT REQUIRED
CALIBRATE
BEFORE
EACH USE
Other decals may be appropriate.
5.6. Schedules
Measuring instruments should be calibrated at periodic intervals established on the basis of stability,
purpose, and degree of usage of the equipment. Intervals between calibrations should be shortened as
required to assure prescribed accuracy as evidenced by the results of preceding calibrations. Intervals
should be lengthened only when the results of previous calibrations indicate that such action will not
adversely affect the accuracy of the system, i.e., the quality of the finished product.
Calibration decals on the measuring equipment are a quick check to verify that equipment is calibrated.
These labels are not used to set the calibration schedule. The Validation Department schedule will be
maintained to determine when routine calibration on equipment is due.
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5.7. Standards
Where practical, NIH/PHAR requires that standards used to calibrate equipment be traceable to the
National Institute of Standards and Technology (NIST), or other recognized national or international
standards. Traceability also can be achieved through a contract calibration laboratory which in turn uses
NIST services. The meaning of traceability to NIST is not always self-evident. Two general methods
commonly used to establish and maintain traceability to NIST are:
• NIST calibration of standards or instruments: When this method is used, private standards are
physically sent to NIST for calibration and returned.
• Standard Reference Materials (SRM's): NIST provides reference materials to be used in a user's
calibration program. These SRM's are widely used in the chemical, biological, medical, and
environmental fields.
Information can be obtained from the "Catalog of NIST Standard Reference Materials," available free
from the National Institute of Standards and Technology, Office of Standard Reference Materials,
Gaithersburg, MD 20899, phone: (301)975-2016.
When in-house standards are used, they should be fully described in the pertinent SOP. Independent or
in-house standards should be given appropriate care and maintenance and should be used according to a
written procedure as is required for other calibration activities.
5.8. Calibration Environment and External (Contractor) Calibration

As appropriate, environmental controls should be established and monitored to assure that measuring
instruments are calibrated and used in an environment that will not adversely affect the accuracy required.
Consideration should be given to the effects of temperature, humidity, vibration, and cleanliness when
purchasing, using, calibrating, and storing instruments.
When NIH/PHAR uses a contract calibration laboratory, NIH/PHAR must obtain from the contractor
evidence that the equipment was calibrated according to the GMP requirements. The must require of the
contractor:
• written certification that the equipment was calibrated under controlled conditions using traceable
standards;
• written verification (or company audit) that the contractor maintains an adequate calibration
schedule for their instruments;
• Periodically submit to an audit by NIH/PHAR to assure appropriate and adequate GMP
procedures are being followed. For example, the contractor should have:
o written calibration procedures;
o records of calibration;
o trained calibration personnel; and
o Standards traceable to NIST or other independent reproducible standards.
Certification notes and data should include accuracy of equipment when received by the lab to facilitate
remedial action by the finished device manufacturer, if necessary. Certification should also include
accuracy after calibration, standards used, and environmental conditions under which the equipment was
calibrated. The certification should be signed and dated by a responsible employee of the contract lab.
If in-house standards are used by a contractor to calibrate device-related measuring equipment, these
standards shall be documented, used, and maintained the same as other standards.
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5.9. Assessment Procedure for Inclusion
Some equipment is for informational use only or is solely for use during maintenance of the equipment.
Such devices should be identified during the IQ/OQ procedures for the equipment and tagged
accordingly. A typical tag might say “non-critical” or “For information only”
Most dials, gauges, digital readouts, etc. have a purpose in assuring the correct operation of critical
functions. NIH/PHAR assumes that all of its instruments and their associated components must be
maintained in a state of calibration. Exceptions should be noted during IQ/OQ and instruments that do
not need calibration can be so tagged. An explanation for why the instrument does not require calibration
should be included with the IQ for the device.
5.10. Summary of Validation Policy Applied to Calibration
Equipment and systems carrying out operational functions as part of the production process must be
validated. Instruments and Components of the equipment that serve only to verify an operation and/or
generate data, whether or not that data is recorded, are calibrated, but not validated.
During the IQ process, those components of the system that will obtain calibration only, will be so noted.
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Table of Contents
6. Preventative Maintenance Program .................................................................................................... 47

6.1. Service Personnel ........................................................................................................................ 47
6.2. Service Requirements ................................................................................................................. 47
6.3. External Contractors Performing Service ................................................................................... 47
6.4. Service Equipment ...................................................................................................................... 48
6.5. Maintenance Procedures ............................................................................................................. 48
6.6. Service Reports ........................................................................................................................... 49
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6. Preventative Maintenance Program
6.1. Service Personnel
Service shall be conducted by appropriately trained and experienced service personnel in order to:
• assure, to the extent feasible, that the process of device handling, diagnosis, and repair will not
compromise the determination of the root cause of any product failure;
• identify and correct failures;
• correctly report the service information; and
• identify and report data related to a serious incident or adverse event.
The repair diagnosis should also try to determine, and/or provide adequate data to assist analysts in
determining, the actual failure mechanism to the objective level necessary to correct or reduce the
problem. Thus, service personnel must be trained to adequately perform their assigned maintenance,
repair, and reporting responsibilities. Such training shall be documented. The training is also performed
in accordance with the instructions and procedures established for performing and verifying that servicing
meets the specified requirements. Because servicing must be verified, service personnel must be made
aware of defects and errors that may be encountered as part of their job functions. This training
requirement usually does not require separate or additional training because basic training to perform
repairs emphasizes the identification of defects and errors.
6.2. Service Requirements
Not all equipment must be serviced. However, it would be very unusual for equipment that is subject to
validation to not require service. If it is judged that a piece of equipment does not justify service, then it
should be given an expiration period, tagged, and discarded or replaced at the end of its expiration.
Where servicing is a specified requirement, NIH/PHAR shall establish and maintain instructions and
procedures for performing and verifying that the servicing meets the specified requirements.
Service reports shall be documented and shall include:
• The name of the device serviced;
• Any device identification(s) and control number(s) used;
• The date of service;
• The individual(s) servicing the device;
• The service performed; and
• The test and inspection data.
6.3. External Contractors Performing Service
When NIH/PHAR contracts with another supplier to perform a service, then the service (or service
contractor) must comply with the same rules as if NIH/PHAR were doing the service themselves.
NIH/PHAR shall establish and maintain procedures to ensure that all purchased or otherwise received
services conform to specified requirements.
NIH/PHAR shall:
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• Evaluate and select potential contractors on the basis of their ability to meet specified
requirements, including quality requirements.
• Define the type and extent of control to be exercised over the contractors based on the evaluation
results.
• Establish and maintain records of acceptable contractors.
6.4. Service Equipment
NIH/PHAR shall make adequate provisions for resources, including the assignment of trained personnel,
for management, performance of work, and assessment. As appropriate, adequate resources include
service instructions, service procedures, supporting drawings, and service equipment. Service equipment
includes equipment to perform the repair and to verify the proper performance of the serviced devices.
Service equipment may include complex apparatus; however, it also includes any simple jigs, test cables,
special hand tools, etc., as needed to meet the service needs of pharmaceutical manufacturing equipment.
Maintenance requires verifying that the equipment meets acceptance criteria. Therefore, appropriate and
calibrated test equipment should be used. NIH/PHAR will ensure that all inspection, measuring, and test
equipment, including mechanical, automated, or electronic inspection and test equipment, is suitable for
its intended purposes and is capable of producing sufficiently accurate and precise results. NIH/PHAR
shall establish and maintain procedures to ensure that equipment is routinely calibrated, inspected,
checked, and maintained. The procedures shall include provisions for handling, preservation, and storage
of equipment, so that its accuracy and fitness for use are maintained.
6.5. Maintenance Procedures
Maintenance needs, schedules, and procedures are developed in conjunction with the equipment IQ/OQ.
Some preventive maintenance tasks and their schedules may result from reliability studies performed
during design development. Maintenance procedures are based in part on service manuals provided by
the equipment manufacturer. Such manuals are likely to be insufficient for long term reliability of the
equipment. Careful servicing procedures should be developed internally by NIH/PHAR.
Identifying defective subassemblies or modules in equipment and replacing them with good modules is a
common servicing practice. The defective assembly is discarded, sent to be investigated, or is repaired at
a designated facility with the necessary environmental conditions and facilities; test equipment and tools;
component availability; trained rework employees; etc. This approach should also be covered by
appropriate procedures.
The development of service procedures includes the development of appropriate service reporting forms.
Service instructions and procedures must be documented. Equipment maintenance documents should
cover the following. Some of this information may be included in vendor manuals or documents.
• What the device does;
• Theory of operation;
• Operating instructions;
• Safety;
• Operating specifications;
• Component specifications, identification, and nomenclature;
• Test apparatus, jigs, and special tools;
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• Typical failure modes and conditions;

• How to identify and isolate failures;
• Test points where specific parameters may be measured;
• Removal and replacement of parts;
• Testing and inspecting (verifying) the repaired device;
• Re-installation procedures, if applicable; and
• Reporting forms.
6.6. Service Reports
Service activities shall be documented by service personnel and filed by NIH/PHAR according to
established procedures. Service reports shall include:
• the name of the device serviced;
• any device identification(s) and control number(s) used;
• the date of service;
• the individual(s) servicing the device;
• the service performed; and
• the test and inspection data.
The device identification should be specific regarding the revision level, modification version, software
version, etc., of the device in order to support analysis of the service data.
The test and inspection data should verify that the servicing meets NIH/PHAR's specified requirements.
That is, the serviced equipment did, or did not, meet the acceptance criteria.
The service reports should also include information such as:
• device owner, address and phone number;
• specific location of the device;
• any unusual environmental conditions;
• any evidence of damage or misuse;
• if the device failed to meet specifications;
• if the device was being used for treatment or diagnosis;
• relationship, if any, of device to a death or serious injury; and
• date of last service and service report number, if known.
The maintenance report is prepared by a person who is not generally trained to evaluate the need for re-
validation following maintenance. When parts are changed during maintenance, then a change control
needs to be opened. The change control procedure (SOP) will help QA determine the need for re-
validation.
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Contents
7. Key Standard Operating Procedures requiring Validation .................................................................. 51
7.1. Overview of SOP Program ........................................................................................................... 51
7.2. SOP requirements by activity and location (chart) ..................................................................... 51
7.3. Shutdown and Startup/Facility Release Procedure ..................................................................... 52
7.4. Water/Steam Sampling Procedures ............................................................................................. 52
7.5. Gasses Sampling Procedure* ...................................................................................................... 53
7.6. Visual Inspection Procedure* ..................................................................................................... 53
7.7. Manual Equipment Cleaning Procedure* ................................................................................... 53
7.8. Equipment Sanitization Procedure* ............................................................................................ 53
7.9. Gown Training* .......................................................................................................................... 53
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7. Key Standard Operating Procedures requiring Validation
7.1. Overview of SOP Program
In addition to system / equipment validation, operating processes and practices governed by SOP
exclusively may require validation depending upon the nature of the process and practice. The
procedures listed within this section have been identified to require a validation of the particular
procedure. Validation will consist of documented trials.
7.2. SOP requirements by activity and location (chart)

The tables in this section show the common SOP’s and cross them against the room to which they apply.
Some SOP’s will be specific within a room and others may be general, applying to all of the rooms where
the column is checked.
Room # Room Name Cleaning Decontamination

1C166
Clean Prep Rm X X
A5A
1C166
Filling Room X X
A7A2
-- Filling Cabinets X X
1C166
AirLock between Clean Prep & Fill X X
A5A1
1C166
Mech Rm (Lyo) No No
A6
1C166
Air Lock entry to Clean Prep X X
A5
1C166
Weigh Room X No
A3
1C166
Weigh Room X No
A4
1C166 A7A1 Air Lock from Wash Rm #1 to Fill X X
1C166 A Main GMP Corridor X No
1C166 A7B Test Lab X No

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Room # Room Name Cleaning Decontamination

1C166 A4 Wash Rm #2 X No
1C166 A7A Wash Rm #1 X No
1C166 A7C Large Batch Rm X X
1C166 AB Interior GMP Corridor X No
1C166 A7D Blending X X
1C166 A7F Liquid Formulation Rm X X
1C166 A7E Tableting X X
1C166 A9 Subdivide and Finish Packaging No No
1C166 A1 Storage No No
1C166 A2 Controlled Temp Raw Material Storage No No
1C230B Quarantine Finished Goods No No
7.3. Shutdown and Startup/Facility Release Procedure
Periodically the facility will be shut down for preventive maintenance, repair, or facility modifications.
This procedure will describe the necessary start-up and shut-down practices and release requirements to
resume operations. The procedure should describe a formal Quality Department release to restart
manufacturing. The Restart procedure itself does not get validated. However, it should cause change
control procedures that were opened during the shutdown to be assessed to see if any of those changes
require re-validation.
7.4. Water/Steam Sampling Procedures
A compendia specification for sterility of bulk WFI used in aseptic pharmaceutical manufacturing
operations does not exist per se. The FDA does expect WFI to be essentially sterile but acknowledges that
sampling in non-sterile areas is not aseptic and sampling errors can result in occasional low-level
microbial counts. Historical FDA guidance suggests less than 10 colony forming units (CFU)/100 mL as
an acceptable action limit. Actual data collected by the NIH/PHAR facility should be reviewed annually
and an “in house” acceptance level established as data permits.
Operational and sampling procedures (SOPs) are verified to be appropriate for the new system during
OQ. Each step in the water or steam purification process must be verified. For a period of two to four
weeks, each pretreatment sample point and WFI use point is sampled to verify that it meets the specified
water quality. This testing may include sampling at multiple times of the day to determine operational
variances such as sanitization or consumption. At the conclusion of the OQ, the SOPs are finalized and
released at which time the system is ready to begin stable operations on a day-to-day basis.
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7.5. Gasses Sampling Procedure*
The only gasses planned for the parenteral fill facility are nitrogen and air. Nitrogen, USP will be
provided with a certificate of analysis and will be sampled and tested for identity prior to release for use.
The exact sampling method will depend on the identity test chosen and will be described in the SOP for
the test. Air supplied to grade A areas should be point of use filtered and should be tested for viable
particulates in line with general viable air monitoring procedures.
7.6. Visual Inspection Procedure*
Visual inspection will be conducted under an approved procedure. Operators involved in this practice
will be qualified through a suitable demonstration. See section 8.5.8. for additional information.
7.7. Manual Equipment Cleaning Procedure*
Manual cleaning practices will be done under SOP. Manual cleaning will be qualified and documented in
an operator training file.
7.8. Equipment Sanitization Procedure*
Equipment sanitization will be done under SOP. Sanitization will be qualified and documented in an
operator training file.
7.9. Gown Training*
The clothing and its quality should be appropriate for the process and the grade of the working area. It
should be worn in such a way as to protect the product from contamination.
Grade D: Hair and, where relevant, beards should be covered. A general protective suit and appropriate
shoes or overshoes should be worn. Appropriate measures should be taken to avoid any contamination
from outside the clean area. It is a good practice to supply employee shoes that are worn only in the GMP
areas and are not removed from the facility at any time.
Grade C: Hair, and where relevant beards, should be covered. A single or two-piece trouser suit,
gathered at the wrists and with high neck and appropriate shoes or overshoes should be worn. They
should shed virtually no fibers or particulate matter.
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Grade A: Headgear should totally enclose hair and, where relevant, beard and moustache; it should be
tucked into the neck of the suite; a face mask should be worn to prevent the shedding of droplets.
Appropriate sterilized, non-powdered rubber or plastic gloves and sterilized or disinfected footwear
should be worn. Trouser-legs should be tucked inside the footwear and garment sleeves into the gloves.
The protective clothing should shed virtually no fibers or particulate matter and retain particles shed by
the body.
Outdoor clothing will be worn under sterile protective gowns by workers who will be sitting in grade C in
front of laminar flow hoods or biological safety cabinets. This procedure is known to be subject to
accidental contamination due to the presence of outdoor clothing. Gowning and glove procedures need
to be especially rigorous and validated.
For every worker in a grade C area that contains a grade A laminar flow hood or biological safety cabinet,
clean sterile (sterilized or adequately sanitized) protective garments should be provided at each work
session. Gloves should be regularly disinfected during operations. Masks and gloves should be changed
at least for every working session. When a worker leaves the area, new, clean and sterile garments are to
be put on prior to re-entry.
Clean area clothing should be cleaned and handled in such a way that it does not gather additional
contaminants which can later be shed. These operations should follow written procedures. Separate
laundry facilities for such clothing are desirable. Inappropriate treatment of clothing will damage fibers
and may increase the risk of shedding of particles.
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Table of Contents
8. Guidelines for Specific Equipment and Systems ................................................................................. 57

8.1. Fill Cabinets & Fill Room – Release for Use Criteria .................................................................... 57
8.1.1. Environmental Compliance for Fill Suites: .......................................................................... 57
8.1.2. Dynamic Particle Counts ..................................................................................................... 58
8.1.3. Temperature and Humidity ................................................................................................ 58
8.1.4. Pressure .............................................................................................................................. 58
8.1.5. Air Velocity .......................................................................................................................... 58
8.1.6. Surface RODAC sampling .................................................................................................... 59
8.1.7. Settling Plate counts ........................................................................................................... 59
8.1.8. Active Air Sampling ............................................................................................................. 59
8.1.9. Swabs .................................................................................................................................. 59
8.2. Fill Suites – Complete Validation Criteria.................................................................................... 60
8.3. Fill Hoods – Complete Validation Criteria ................................................................................... 60
8.4. Utilities ........................................................................................................................................ 60
8.4.1. Building Utility Systems ....................................................................................................... 61
8.4.2. Pure Steam .......................................................................................................................... 61
8.4.3. WFI ...................................................................................................................................... 61
8.4.4. RODI .................................................................................................................................... 62
8.4.5. Nitrogen .............................................................................................................................. 63
8.4.6. Argon ................................................................................................................................... 64
8.4.7. Compressed Air ................................................................................................................... 64
8.4.8. HVAC ................................................................................................................................... 65
8.5. Major Operating Equipment ....................................................................................................... 66
8.5.1. Filter Integrity Tester [Filter Leakage] ................................................................................. 66
8.5.2. Liquid Filling ........................................................................................................................ 66
8.5.3. Autoclave* .......................................................................................................................... 67
8.5.4. Dry Heat Oven ..................................................................................................................... 69
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8.5.5. Vial Washer ......................................................................................................................... 70

8.5.6. Lyophilizer ........................................................................................................................... 71
8.5.7. Production Dishwasher ....................................................................................................... 73
8.5.8. Equipment Pass Thru .......................................................................................................... 74
8.5.9. V-Blender ............................................................................................................................ 74
8.5.10. Capsule Filling Machine ...................................................................................................... 75
8.5.11. Glatt Tablet Coater & Mixer ................................................................................................ 75
8.5.12. Tablet Filling Machine ......................................................................................................... 77
8.6. Laboratory Equipment ................................................................................................................ 78
8.6.1. Environmental Chambers for Product Storage* ................................................................. 78
8.6.2. Refrigerators and Freezers for Product Storage* ................................................................ 79
8.6.3. Sensors ................................................................................................................................ 79
8.6.4.4. Differential Scanning Calorimeter ................................................................................... 81
8.6.4.5. Capillary Zone Electrophoresis ........................................................................................ 81
8.6.4.6. IR Spectrophotometer .................................................................................................... 81
8.6.4.7. Dissolution Testing Devices............................................................................................. 82
8.6.4.8. UV Spec ........................................................................................................................... 82
8.6.5. Laboratory Dishwasher ....................................................................................................... 82
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8. Guidelines for Specific Equipment and Systems
8.1. Fill Cabinets & Fill Room – Release for Use Criteria
8.1.1. Environmental Compliance for Fill Suites:
Validation of the filling room and associated laminar flow hood and biological safety cabinet is a matter
of demonstrating environmental control. The environmental monitoring program is established to call out
specific tests and specifications. Those specifications must be met in both static and dynamic conditions.
Validation of the room for environmental control is a system validation beyond the validation of each of
the mechanical components which measure or control the system. Due to the system complexity, it is
often desirable to “take down” the room (usually meaning to turn off the flow of air or otherwise permit
non-GMP activity to occur in the area) and then return the room to its validated environmental state
without revalidating each of the components that control the room and without revalidating the personnel
and media fills needed for GMP fills within the room.
A specific Fill Suite(s): Environmental Compliance and Release protocol should be generated and used
every time the suite(s) are significantly removed from a state of compliance. Environmental Compliance
alone may be insufficient to “validate” the fill suites for filling. However, environmental compliance is
sufficient to validate that the rooms will maintain their environment in the absence of personnel.
• Static Particle counts have not been out of specification (OOS) for the most recent 24 hours
during which particle counts were validly collected.
Interpretation: Dynamic activity could have occurred in the room during the most recent 24 hours and any
particle counts which may have been collected during the activity would not be considered “static”. If
activity occurred, one would have to have additional recent data in order to obtain 24 hours of data
without activity. In the example below, the 24 hour static results are interrupted by an employee entering
to obtain other sampling data. That period of time in which the employee is in the room cannot be
counted as part of the static monitoring.
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Grade Location (e.g.) Maximum permitted number of particles/m3 equal to

or above
At rest (c) Operational
0.5µm 5µm 0.5µm 5µm
A Fill Cabinets 3500 1(d) 3500 1(d)
B None 3500 1(d) 350,000 2,000
C Fill room 350,000 2,000 3,500,000 20,000
D Background Air 3,500,000 20,000 NS 200,000
8.1.2. Dynamic Particle Counts
Dynamic Particle counts are within specification when personnel are in the room and operating necessary
equipment and/or taking microbial samples.
8.1.3. Temperature and Humidity
Temperature and humidity must be within specifications.

Temperature = 65°F + 5°F
Humidity = 30% to 60% RH
8.1.4. Pressure
Pressure is within specification for all areas. The specifications for pressure in the various areas may be
set by a specific release protocol. Room pressures need to cascade downward between classes and
especially room pressure must not cause reverse flow. However, the initial pressure values that have been
recommended have a significant noise margin that may be difficult to achieve in all circumstances.
Grade A – Target : > 0.05 above adjacent areas

Grade C – Target : > 0.05 above adjacent areas
Grade D – Target : > 0.05 above adjacent areas
8.1.5. Air Velocity
Air velocities at the filter face throughout the suites have been monitored, and a report has been prepared
showing that the velocities are within specification. Some protocols may specifically exclude this
recommendation. If the protocol does not specifically exclude air velocity testing and report, then it is
assumed to be required.
Clean Air Device Air Flow Limits (m/s) Limits (ft/min)

Unidirectional Air Horizontal Air Flow 0.45 + 0.1 90 + 20
Flow Cabinet Vertical Air Flow 0.30 + 0.05 60 + 10
Safety Cabinet Vertical Air Flow 0.25 – 0.50 50 - 100
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Inward Air Flow Not less than 0.4

Unidirectional Air Vertical Air Flow 0.30 – 0.60 60 - 118
Flow Isolator
Clean Rooms (Grades Vertical Air Flow .45 + 0.1 90 + 20
A/B)
8.1.6. Surface RODAC sampling
EC GMP Location At rest (cfu/m3) Operational (cfu/m3)

Grade
A Fill Room fill cabinet <1 <1
Fill Room background <1 5
B Sterile Storage <1 5
C Support Rooms 5 25
D Hallways 25 50
8.1.7. Settling Plate counts
EC Grade Static (cfu) Dynamic (cfu)

A 1 per 2 plates 1 per 2 plates
B <1* 5
C 5 50
D 50 100
8.1.8. Active Air Sampling
EC GMP Grade Location At rest (cfu/m3) Operational (cfu/m3)

A Fill Cabinets <1 <1
B None <1 10
C Fill room 10 100
D Background Air 100 200
8.1.9. Swabs

Grade
Lyophilizer <1 <1
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B Sterile Storage Tables/Racks <1 5

D Hallways 25 50
8.2. Fill Suites – Complete Validation Criteria
In addition to the criteria of section 8.1, the following tests must be performed under a suitable protocol
and with specifications.
• HEPA filter certification by challenge with upstream particulate matter such as dioctyl phthalate.
This is in addition to face velocity.
• Calibration of the pressure and particulate monitoring equipment.
• Calculation from linear air velocity data of the number of air changes per hour in all rooms.
• Validation report on the HVAC system.
• Air Cleanliness testing will include the following:
o Viable Particulates (5) consecutive days
o Viable Particulates Static and Dynamic conditions
o Viable Particulates Locations defined in protocol.
• Surface Monitoring testing will include the following:
o Viable Particulates (5) consecutive days
o Viable Particulates RODAC
o Viable Particulates Static and Dynamic conditions
o Viable Particulates Locations defined in protocol.
• Video Smoke testing to verify air flow patterns.
8.3. Fill Hoods – Complete Validation Criteria

Filling of sterile liquids, toxic materials, and powders is accomplished in laminar flow hoods installed
within the filling suites. Each hood will be certified for its classification and is subject to the operation
and validation criteria of the fill suites. The following hoods are used by production.
• 8ft BSC Hood (NuAire NU S430-800) is used for filling of toxic liquid compounds.
• Horizontal Hood (10 ft) (NuAire NU-301-1048) is used for filling of non-toxic liquid
compounds.
• 6ft BSA Hood (NuAire NU 430-600) is used for filling of toxic and non-toxic powders and
capsules.
8.4.Utilities
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8.4.1. Building Utility Systems
The Building Utility Systems will be commissioned upon start-up and maintained and operated in
accordance with established procedures. Formal validations, beyond commissioning, will not be
conducted on this equipment unless the system has a direct impact on final product quality at a future
date. Where the utility is required or connected, suitability of the utility will be performed on the specific
process equipment or system.
This policy applies to the following systems:
• Drainage and waste neutralization system

• Plant boilers
• Plant chiller water
• Emergency power generation
• Hot water system
• Cold water system
8.4.2. Pure Steam
The following tests must be performed under a suitable protocol and with specifications.
• Temperature Stability:
• Chemical Quality:
o TOC
o Conductivity
o pH
• Microbial Quality:
o Bacterial Endotoxin
o Total Microbial Count
8.4.3. WFI
Under an OQ protocol, WFI testing results should be followed for a period of 2 to 4 weeks (total time
dependent upon satisfactory results) to verify chemical purity and microbiological acceptance.
Upon completion of the operational qualification, the performance qualification (PQ) begins which
consists of once daily testing of each purification stage and each WFI use point. The testing period must
be longer than the water quality testing period during OQ. PQ demonstrates the system consistently
produces the specified water quality under normal conditions while utilizing operating procedures
specified in approved SOPs. This series of tests is typically performed for 30 consecutive days. At the
conclusion of this testing and review of all the available data, the system is available for pharmaceutical
manufacturing.
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During the first year of operation, the WFI system should be sampled and tested at a higher frequency
than may be required subsequently. The final phase of qualification of the water system is a process
validation (PV) to demonstrate consistent operation during the initial year of operation. The intent of PV
is to detect any seasonal changes in city water quality. Critical sites should be monitored daily, with the
remaining sites sampled on a weekly basis. The sites subject to weekly sampling are rotated so that some
sites are sampled each day and by the end of a week all the sites have been sampled. At the conclusion of
the process validation, data is analyzed to determine if the system is robust. The system should provide
consistent operation and water quality capable of responding to seasonal variations. If seasonal
fluctuations in WFI water quality are detected, it must be determined if this is due to changes in city water
conditions or if a system malfunction is the cause – well designed systems are not impacted by seasonal
variations in the city water supply. In addition, scheduled maintenance on an annual or semi-annual basis
will maintain the WFI system in the validated state; although it is worth remembering that as with all
validated systems, any repair, corrective action or modification must be documented and approved by
means of a formal change control system. The microbial and chemical test data of operating systems
should also be reviewed and analyzed on an annual basis to ensure that there are no trends requiring
investigation.
• Temperature Stability: Tank and drops within range

• Chemical Quality:
o TOC
o Conductivity
o pH
• Microbial Quality:
o Bacterial Endotoxin
o Total Microbial Count
• Certification: Filter integrity is maintained
8.4.4. RODI
The RODI system should be sampled and tested like the WFI system. It will have a lesser standard for
microbial content. RODI must meet USP requirements in the distribution system and at the various
drops. Requirements include meeting the following specifications:
• TOC Specification of < 500 ppb (Alert > 250 ppb, Action > 350 ppb).
• Conductivity (On-Line) to meet USP Purified Water requirements adjusted for temperature
(nominal conductivity <1.3 µS/cm at 25ºC to pass Stage 1 requirements).
• Total Viable Organisms ≤ 100 CFU / 100 mL (Alert ≥ 10 CFU / 100 mL, Action ≥ 50 CFU / 100
mL).
•
Best practice is to set specifications for each stage in the process to assure that each individual component
is performing its function. Specification can be partially based on the manufacturers recommendations.
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City Water Source Test for chlorine, hardness,

endotoxin, total organic carbon,
conductivity.
Pre-Filter Decreases very large No test
particulate
Carbon Filter Removes chlorine Test for chlorine
Water Softener Exchanges Na+ for Ca++ Water hardness
Reverse Osmosis Decreases endotoxin Test for endotoxin
Portable Deionizer Raises water resistance [Ω] Measure conductivity
RODI Tank Vent Filter Prevents internal Safety feature not tested
Pres/Vacuum
RODI Circulation Pump Circulates water Flow rate = turbulent
RODI Ultraviolet Sterilizer Reduces bacteria Bacterial challenge not required.
Change light based on operation time
and manufacturers recommendation.
RODI Final Filter Removes bacteria Integrity test of filter
• Sample ports should be available before and after each component for testing.
• Flow Rate in the Loop should be established via calculation.
8.4.5. Nitrogen
Validation of process gas systems involve documenting the expected system behavior, and verifying that
the system performs as expected. Demonstrate that the process gas delivered at the point-of-use meets the
predetermined user requirements. The nitrogen system is primarily going to be a product contact gas as
overfill for vials with and without lyophilization. Consequently, the user requirement is for USP,
Nitrogen coming from the pipe. It is then passed through an endpoint sterile filter for final sterilization.
The sterile filter is not a part of the “nitrogen system”. The nitrogen coming from the system need only
meet USP specifications.
The “nitrogen system” consists of nitrogen bottles, distribution piping, and some valves. The purchasing
agent must provide documentation of the Nitrogen to meet USP and then perform some release test. The
vendor must be part of a validated vendor program. It may not be practical to perform USP testing for
99% Nitrogen in NIH/PHAR laboratories.
System pressure is critical to the operation. System pressure must be monitored and alarmed.
• Validation of the low pressure alarm condition is expected.

• Vendor validation is expected.
• Filter integrity testing must be performed and the results matched to the final usage of the
nitrogen.
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The following tests may be performed under a suitable protocol and with specifications but may have
vendor audit substituted for on site testing.
• Demonstrate dewpoint of less than -10C

• Hydrocarbons:
o Less than 1 ppm (weight / weight basis)
8.4.6. Argon
Validation of process gas systems involve documenting the expected system behavior, and verifying that
the system performs as expected. Demonstrate that the process gas delivered at the point-of-use meets the
predetermined user requirements. The argon system is primarily going to be a product contact gas as
overfill for vials with and without lyophilization. Consequently, the user requirement is for USP, Argon
coming from the pipe. It is then passed through an endpoint sterile filter for final sterilization. The sterile
filter is not a part of the “argon system”. The argon coming from the system need only meet USP
specifications.
The “argon system” consists of argon bottles, distribution piping, and some valves. The purchasing agent
must provide documentation of the Argon to meet USP and then perform some release test. The vendor
must be part of a validated vendor program. It may not be practical to perform USP testing for 9xx%
Argon in NIH/PHAR laboratories.
System pressure is critical to the operation. System pressure must be monitored and alarmed.
• Validation of the low pressure alarm condition is expected.

• Vendor validation is expected.
• Filter integrity testing must be performed and the results matched to the final usage of the argon.
The following tests may be performed under a suitable protocol and with specifications but may have
vendor audit substituted for on site testing.
• Demonstrate dewpoint of less than -10C

• Hydrocarbons:
o Less than 1 ppm (weight / weight basis)
8.4.7. Compressed Air
• Pressure: 90 – 110 psi - Meets equipment requirements

• Capacity (minimum): >10 scfm –
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• Purity Instrument Air : If air is put into the lyophilizer, then it is product contact and may
require other specifications.
• Particulate: As per room in which it is used - Meet by filtration at point of use as needed. Filter
air into the clean room for instrument use.
• DewPoint: -10F - Test over 1 to 2 weeks for no less than 3 consecutive “in specification” results.
• Hydrocarbon: Non-detectable (e.g. <25 ppm as measured with Draeger 10a/P hydrocarbon tubes.
8.4.8. HVAC
Specifications for the HVAC system and its control are given in the “Environmental Monitoring
Program”. The goal is to maintain a continuous measurement program that results in a continuous state of
validation. The parameters measured are as follows.
• Room particulate for the appropriate grade, either A, B, C, or D.

• Room air velocity for the appropriate grade.
• Microbial load as part of an ongoing monitoring program.
• Pressure differentials for all rooms logged continuously.
• Temperature at many locations throughout the facility.
• Humidity within some rooms and the ductwork.
• HEPA Filter Certification:
o Challenge with Dioctylphthalate or Emery 3004 (10-20 g/L)
o Filter face, seal area, and structural supports scanned with NIST photometer
o No leak greater than 0.01%, repair leaks as required
o Maximum patch size less than 3% of media surface, no repair larger than 1.5 inches in its
longest dimension
o (5) consecutive days
o Static and Dynamic conditions
o <23 C
• Air Change Rates: Within URS requirements
• Pressure:
o Within URS requirements
• Relative Humidity:
o 30-60% RH
• Air Cleanliness:
o Non Viable Particulates - (5) consecutive days
o Non Viable Particulates - Minimum of (3) 1 foot samples taken at each location each day
o Non Viable Particulates - Static and Dynamic conditions
o Non Viable Particulates - Meet cleanliness requirements
• Power Outage Test:
o Document start-up and shut-down
o RODAC monitor facility during outage to determine length of time power can be out
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• Particulate Evaluation < ISO Class 5; Demonstrate compliance once every 6 months
• Particulate Evaluation > ISO Class 5; Demonstrate compliance once every 12 months
• Airflow Velocity: Once every 12 months
• Pressurization: Once every 12 months
• Installed Filter Leakage: Once every 24 months
• Airflow Visualization: Once every 24 months
• Recovery: Once every 24 months
• Containment Leakage: Once every 24 months
8.5. Major Operating Equipment

8.5.1. Filter Integrity Tester [Filter Leakage]
Filter integrity test equipment is highly specialized and available from only a few companies. Those
firms offer complete validation packages and after validation, it is only necessary to maintain the
calibration of certain defined components. The plan is to let the firm from which the specific integrity
tester(s) are purchased do the validation. GAMP guidelines state that the OQ of standard instruments
such as integrity test units can be divided into two parts. A completed OQ1 containing extensive
qualification studies on a reference instrument. A shorter OQ2 protocol on a specific instrument.
Part 11 security is a component of these systems and the validation of that is also provided by the vendor.
8.5.2. Liquid Filling
An optional Watson Marlow Pump (Flexicon Pump or other liquid dispensing pump) will dispense the
various fill volumes into the containers. Other pump types are available at a surcharge under “Options”.
The machine's Programmable Logic Controller (PLC) signals the pumps to initiate the filling cycle
provided a container is present in the fill position. The pumps dispense the solution into the container via
a stationary needle. A Bottom-up fill mechanism is available as an option. The volume dispensed is
adjustable via the pumps’ control panel.
8.5.2.1. Stopper Insertion
Stoppers are manually loaded into a 316L stainless steel vibratory feeder bowl. The bowl orients the
stoppers and transfers them onto the vibratory track. The track transports the stoppers to a position where
the pick and place arm picks them up and inserts them into the filled containers. This station cycles only
when containers are present and can be activated or deactivated via the touch screen.
8.5.2.2. Fill Volume Study

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Initially for water, and subsequently, for each product filled, it is desirable to perform a fill volume study
that shows the tolerance that the filling pumps can maintain when used with the filler. In phase I/II
products, there is usually insufficient product available to perform the study and often the results from the
water study are used to set a fill volume specification.
This study must be performed in the clean room with the actual pumps under conditions that are as close
as possible to those that will be encountered when filling actual product for human use. Pre-tarred empty
vials are seeded into the fill evenly (not random) distributed within a run of greater than 1800 units. At
least 48 pre-tarred vials are needed per fill nozzle to be validated. Fill weights are determined post fill
(without stoppers), and a statistical evaluation is made from the data. A frequency histogram should be
prepared showing on the y axis the number of units that contain a certain weight and on the x axis various
weight distributions. The shape of the histogram should be statistically normal. Additionally, the weight
data should be graphed versus the vial number filled to show that there is no bias (drift) in weight
throughout the fill progress.
No fill weight adjustments on the machine can be made during the study. The study validates fill
tolerance for the number of units between adjustments. Commonly, one runs about 2500 units,
determines the fill volume tolerance and then for the purpose of actual runs, one does weight checks (and
adjustments) before reaching the study number of units (e.g. 2500).
• Component Movement:
o Damage Rate < 0.1%
o Missing Stopper < 0.5%
o Incorrect Stopper Placement < 0.5%
o No-drips
o (3) runs of 5000 units
• Speed Runs correctly: 10-90%
• Fill Volume:
o +-3% of target fill volume
o Limits + 3 σ (as specified by SOP)
o Reject Limits +-3%
o (3) runs of 5000 units
• Cleaning
• Lyo Tray Loading:
o Rejects < 0.1%
(3) runs of 5000 units
8.5.3. Autoclave*
The autoclave must be validated in OQ for those aspects of its operation that are not related directly to a
load. The OQ process is intended to demonstrate that the components of the autoclave operate properly
and that the autoclave is deemed ready for performance or load testing.
Include the following checks.

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• Operational Tests
o Operator/supervisory/maintenance modes
o Doors
o Abort cycle
o Emergency stop (if different from abort)
o Alarms
o Programmable parameters
o Menu navigation,
o Security power-up and shutdown
o Operator interface display checks
o Interlock override control
o Select/start control
o Step advance control
o Switch and interlock tests
• Power Loss Recovery Test
• Source Code Review
• Filter sterilization
• Leak/Air Removal/Steam Penetration/Vacuum Hold Test
o The Bowie Dick test is designed to test air removal, the absence of air leaks and steam
penetration into a porous load. It uses a test pack of fabric with specific dimensions or
there are commercial, use once packs available. It has been widely employed in Europe.
In North America, a Vacuum Hold Test has often been employed. European Standard EN
554 specifies that if a sterilization process includes air removal from the product, a steam
penetration test shall be carried out at the commencement of each day the autoclave is
used. Although a vacuum hold test may be less sensitive than a Bowie Dick test, it is a
satisfactory alternative if strict acceptance criteria are applied. This assumption is based
on steam penetration/lethality in the worst case load items being demonstrated and that the
vacuum hold test therefore demonstrates absence of leaks and that the validated conditions
that resulted in lethality are being met on an ongoing basis.
• The vacuum hold test should achieve a vacuum level of at least 2.5 psia (with vacuum pump).
After a period of stabilization, a leak rate test conducted for a minimum of 10 minutes must verify
that any leak is less than 1.3 mbar/min.
• Saturated Steam Check. The steam is at a temperature corresponding to its vapor pressure. This
requires a calibrated pressure gauge of a capacitance manometer type.
• Empty Chamber Tests.
• Certification: Filter integrity is maintained

• Empty Chamber:
o 121.1ºC
o All temps within a 3ºC band at the sterilization temperature. This means -1ºC below to
+2ºC above the set point.
o (3) cross-sections with 5 thermocouples per cross section
o Maintain temperature for 30 minutes
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o (3) consecutive runs

o Thermocouple Calibration (±0.5ºC accuracy)
• Load Configuration:
o 121.1ºC
o 2ºC stabilization with coldest thermocouple after 15 minutes of stability
o Heat penetration study to determine 10 coldest locations (maximum load)
o Heat penetration study to determine 10 coldest locations (minimum load)
o 1 probe and BI at 10 coldest locations (max and minimum)
o (5) probes outside of load
o BI's are sterile
o Pressure stable within ± 0.5 psi
o Minimum of 15 Fo collected
o +1C, -0.4ºC with coldest thermocouple after 15 minutes of stability
o Each load will be qualified separately
8.5.4. Dry Heat Oven
The LCC/LCD2-14PT-3 SERIES pass-through oven is equipped with sterile and non-sterile doors, for
clean room operation. Safety interlock switches on each door prevents both doors to open at the same
time (these may be manually overridden if required). The operator loads the product from the non-sterile
side, and selects the program to run. At the end of the process, the product is unloaded from the sterile
side. The LCC/LCD2-14PT-3 SERIES pass-through oven offers HEPA filtration for processes where
minimization of contamination is essential. The removable HEPA (High Efficiency Particulate Air) filter
is designed to provide a constant flow of 99.97% clean air to the product being heated. The HEPA filter
with silicone seal provides 99.99% filtration prior to filter burn off. A magnehelic differential pressure
gauge monitors pressure drop across the HEPA filter. The LCC2-14PT-3 Series ovens are rated up to
260°C. The voltages available are 208V, 240V, 415V, and 480V, 3 Phase; with a 16kW heater. The
oven’s main operator interface components are on the hinged control panel located on the non-sterile side.
The power components; fuse blocks and motor starters are located on the equipment panel, behind the
hinged control panel for easy access. The transformer and the heater SSR’s are located in the lower
compartment with the recirculation and exhaust/cooling motors. An EMO (emergency off) switch, and
indicator lights showing what step the process is in, is located on the sterile side control panel. Electrical
components are either touch-proof or are shielded with Lexan to prevent accidental exposure during
maintenance and troubleshooting. The optional Despatch Protocol Manager software is used to enable
customer PC control of an oven. Despatch Protocol Plus controllers may be networked together with a
Modbus communication option when multiple ovens are operated.
A 950 scfm recirculation fan assures rapid convection heating. The depyrogenation oven is a double door
model that sterilizes and depyrogenates vials between a vial preparation and wrap area and the grade C
clean area where they will be used. Vials are covered coming out of the oven and as they are transported
into a laminar flow hood.
Chamber Size: 25.5w x 26d x 37h in. The 25.5 inch width is interior. The Clear opening width is
reduced by 1.5 in. due to 3/4 in. shelf supports on each side.
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Shelves: Although only two shelves are provided with the oven, it is capable of accepting up to 11
shelves on 3” centers. Each shelf can hold up to 50 lbs of glass.
Temperature:
The target temperature for operation and the temperature to validate for operation is 250°C.
The upper range of temperature for the LLC is 260°C. The vendor specification for temperature
uniformity at 260°C is ± 3°C in an empty chamber.
• An empty chamber temperature distribution study should be performed.

• Specific vial loads should be validated for depyrogenation. Sterility is assumed when heat
depyrogenation is proven.
Pressure Differential:
• The LCC Series oven is equipped with a Magnahelic pressure gauge which measures pressure in
front of the HEPA filter. As the filter becomes dirty, pressure will increase. Despatch recommends
changing the filter when the pressure is 1 inch [water column] greater than when the filter was first
installed.
PC Interface: The oven is ready to accept an RS232 output card and cabling for a personal computer for
collection of time and temperature data.
• Empty Chamber: 225.0°C

o 25°C stability with coldest thermocouple after 15 minutes of stabilization.
o (3) cross-sections with 5 thermocouples per cross section
o Maintain temperature for 2 hours
o Thermocouple Calibration (+-0.5ºC accuracy)
• Load Configuration:
o 230.0ºC
o 25ºC stability with coldest thermocouple after 15 minutes of stability
o Heat penetration study to determine 10 coldest locations
o Bacterial Endotoxin Challenge (E. coli w/ 10,000 EU's)
o 1 probe and EI at 10 coldest locations
o Maintain temperature for 2 hours
o Demonstrate 3-Log Reduction
8.5.5. Vial Washer
The vial washer to be validated is a Cozzoli GW-24. The vial washing system consists of a batch washer,
and vial plate sets for individual vial sizes, used to wash vials prior to dry heat depyrogenation. Wash
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media surfaces are constructed of 316 stainless steel, and the base frame is aluminum with 304 stainless
steel exterior surfaces.
Plastic wrapped vial packages are received and logged. The vial packages are placed onto flip trays and
the plastic is removed while retaining the packaging pattern. An additional set of trays are used to flip
and place the vial pack into the vial washer. The vial washer is equipped with a single spray nozzle per
vial.
An operator initiates a washing cycle through a Panel View Display and the vials are washed with an
alternating series of Water For Injection, filtered air, and Pure Steam. Washed vials are then removed and
transferred to trays for dry heat depyrogenation.
The purpose of the vial washing is to remove particulate and incidental contamination from the interior of
the vial. Incidental contamination is defined as larger particles of dust and debris that may have fallen
into the vial during the manufacturers forming and packaging process. Vendor validation supports a
manufacturing process that is well controlled and will not cause gross contamination by endotoxin.
Consequently, the removal of endotoxin is not an expectation of the vial washer. Endotoxin is kept out
by the processes and incidental endotoxin contamination is removed by dry heat and not by the vial
washer. These vials must have been controlled and handled in a manner that would not have caused gross
contamination as the vial washer will not be validated to clean from just any contaminated state. Indeed,
it would be possible to contaminate the vials in a manner that would not be cleanable by the washer.
Duke Scientific is a manufacturer of calibrated solutions of particles that can be used as a particle
contamination substance. (see dukescientific.com or dukescientific-europe.com). A typical procedure is
to add to the vials 0.5 mL of 40,000 particles per mL and then dry them at 60°C. Use USP 24 <788> as a
detection method after washing.
Alternatively, a chemical contamination solution can be used. Add 0.5 mL of 20% sodium chloride to
each vial. Swirl the solution around and then dry the vial for at least 8 hours. Wash, and then test by
USP Sodium chloride assay.
Although endotoxin challenge is not recommended, a procedure that has been used follows. Add
endotoxin in the amount of 1000 to 10,000 EU per vial. Swirl and then dry at 45°C in a vacuum oven for
1 hour or until no visual water is present. Wash and then test for endotoxin. Require a 3 log reduction in
endotoxin. Vial washing is not a procedure that has been successfully validated for the removal of
endotoxin from glass.
8.5.6. Lyophilizer
The Virtis Genesis 35 Super XL lyophilizer is equipped with three shelves for a total of 4.59 square feet.
Clearance between shelves is 4 inches. There is top down stoppering but the machine is not fitted for
steam sterilization. The unit has an external condenser and chamber. The interior construction is 316L
stainless steel. According to the manufacturer’s specifications, the shelf temperature can go to -60°C and
the condenser temperature can reach -70°C. The condenser capacity is listed as 35 liters. There is one
compressor. Shelf temperature control is from -40°C to +65°C with + 0.5°C shelf to shelf variation.
IQ for the lyophilizer will consist of the normal items detailed in Section 2.3.3.
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OQ will contain specific test functions for the following semi-independent systems.
• Chamber:
o Volume determination.
o Leak rate.
o Drainage.
o Cool down time after sterilization.
• Door
o Light function
o Closure mechanisms
o Gasket seal
• Refrigeration:
o Compressor gauge readings and acceptable range for each gauge.
o Record cooling water temperature
o Record cooling water flow rate – measure if possible.
o If possible (rarely), determine and mark the coil position for the start of superheat for
refrigerant gas returning to the compressor head. Do the same for sub-cooling.
o Determine that a saturation chart for the refrigerant is available. If the refrigerant is a
zeotrope (R400 series), then two curves will be needed, one for bubble point and another
for dew point.
o Determine the location of all installed service valves (e.g. King valve) and record their
preferred pressure and range.
o Record the location of sight glasses (for oil) and record (describe) an acceptable
condition. (Bubbles, color/clarity, level). Note these conditions may be subject to change
during operation. Note when to expect changes and what to expect.
o Confirm that all pipes are marked for direction of flow and substance contained within.
o Determine the compressor’s cycle time during primary drying.
o Read (decode) the compressor data plate and confirm that the system amperage,
pressures, etc. are in compliance.
• Shelf Package:
o Level determination. This function is important to proper stoppering of vials as well as
safety of the shelves.
o Flatness determination. This function is important to proper stoppering of vials.
o Range of Motion. The shelves are not expected to go completely closed. Document the
full range of motion in the absence of vials. Confirm the smallest vial that can be
successfully stoppered.
o Temperature uniformity across individual shelves and among different shelves.
• Condenser:
o Time to achieve minimum temperature.
o Minimum temperature attained.
o 24 Hour Ice Capacity (perform Choked Flow Test first. See appendices.)
• Vacuum:
o Ultimate low pressure attainable
o Time versus pressure graph for an empty chamber
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o Time versus pressure graph for a full chamber

o If two pumps, comment on cycling if any.
o Record the correct pump rotation direction.
• Isolation Valve:
o Time to completely close including definition of closed.
o Description of cycle events that result in the valve closing. For example, does it close
during CIP? Is it closed during freezing of product? What is the method by which
operators and later investigators can know if the valve is open or closed at any point in
time?
o Leak determination of the valve.
o Choked Flow Test – See Appendices
• Stoppering:
o Record the ram diameter
o Determine that ram pressure can be read from a gauge or digital output.
o Develop a chart to show ram pressure per vial.
Ram Pressure x ram area = Force
Force / Vials per Shelf = Force/Vial
Vials break at about 25 lbf
Vials fail to stopper at about 4 lbf.
• PLC Control:
o Test the PLC functions independently. Demonstrate that the PLC can be used to run the
lyophilizer or its components without the need of a computer interface.
o Most PLC documentation should be in the IQ. Code and control line drawings (electrical
and pneumatic) must be available.
• Data Capture:
o Confirm data capture for all inputs.
o Confirm data files and test backup procedure
o If installed, confirm SCADA data pass.
o Confirm data output for trending and analysis.
• Shelf Mapping:
o (3) locations per shelf
o (3) temperatures (-40°C, 0°C, 40°C)
o Range not exceed 5.0°C (chamber <1000 um)
o Thermocouple Calibration (+ 0.5ºC accuracy)
• Cleaning
• Leak Rate: Guideline value is < .02 mbar*liter/sec. The sealed chamber leak rate (dry chamber)
shall be determined for baseline purposes, and for use in setting in-process leak rate acceptance criteria.
8.5.7. Production Dishwasher
A Miele Professional, G7883CD dishwasher will be used in the production area for cleaning of
production glassware and reusable materials. The washer is designed to clean, rinse, and dry equipment.
The washer consists of a stainless steel wash chambers, has a direct injection spray nozzles and automatic
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dispensing of cleaning agents (if used). The washing cycles are a balance of wash time, water
temperature, water circulation and spray pattern, and specific cleaning agents. Elimination of reside is
achieved by using multiple heated DI water rinses. An electronic controller is used to operate the
validated wash cycles. This equipment is operated via SOP.
In addition to standard IQ and OQ items, the following tests must be performed under a suitable protocol
• Spray Coverage Test – Using a riboflavin at about 0.5%, materials will be liberally sprayed and
placed within the production dishwasher. An automatic cycle will be run. Following the cycle,
the material will be inspected to verify the soiling has been removed.
8.5.8. Equipment Pass Thru
The Equipment Pass Thru is used for transferring materials from the preparatory area to the filling suite.
The pass thru is constructed of stainless steel with interlocking doors. No specific validation protocol will
be conducted for this equipment. The use of this equipment will be handled by SOP. The procedures
used for cleaning and use of fill suite areas will apply to this equipment.
8.5.9. V-Blender
The 1 cu.ft. Twin Shell SD Portable Blender (Patterson-Kelly LB-16P) is used for blending very fragile,
heat or shear sensitive ingredients. It is shaped like a V and simply splits the blend as it rotates. The
blending takes place in a closed vessel and is completely dust-free.
Materials to be processed are loaded into the “V” shaped blending shell which is set in slow rotation. As
the shell turns, the material falls alternately toward the apex and then toward the legs of the “V”. The
particles of material move in both vertical and horizontal directions so that complete mixing occurs. The
machine achieves uniform blends, with virtually no attrition to sensitive particles, through the
intermeshing of material as the two chambers combine their flows.
The blender is controlled with a control system that uses timers, relay logic, switches, and an IEC non-
reversing motor starter. The operator controls are mounted on the door and are accessible from the front
of the control panel. The controls consist of a blend timer, pushbuttons, selector switches, and status
lights.
This equipment is validated by IQ/OQ. In addition to standard test functions, the following additional
tests are performed:
• Revolutions per minute

• Demonstrate vessel integrity during blending.
• Maximum capacity test
• Physical homogeneity test
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8.5.10. Capsule Filling Machine
The ZANASI 6/12 F capsule filling machine is used for filling two part, hard gelatin capsules. The
machine can fill capsules with products such as: Liquids, Powders, Granular Products, Pellets, and
Tablets. The machine is an alternate movement filling machine with a stage which rotates clockwise with
a stick slip motion about its axis in a clockwise direction transporting the capsules through 8 operative
stations. This machine will primarily be used for filling capsules with solid materials.
Operators load the blended mixture into the encapsulating machine hopper. Empty capsules are placed
into a second hopper. Prior to the batch run, operators adjust the machine and check that the capsules are
the proper and consistent weight. Operators also check the capsules visually to see if they seem to be
splitting or dimpling. Once the equipment is setup properly, the batch is run.
The blended powder and empty capsules flow through the hoppers. The capsules are broken into halves
by the machine. The bottom half of the capsule falls through a funnel into a rotating dosing dish.
Simultaneously the machine measures a precise amount of the blended powder into a tapping station.
Tamping pins push the powder down forming a slug. The slug is then discharged into the open capsule
half. The top halves of the capsules are then pushed down onto the filled bottoms.
The filled capsules are then run through a polishing machine. The capsules are circulated through a
screened drum where any excess dust is removed from the exterior of the capsules by the vacuum system.
The polished capsules are then moved into an inspection machine. The inspection machine removes any
capsules that are too long, split, dimpled, or otherwise imperfect.
The equipment is a fully automatic capsule-filling machine that can fill a large variety of powder
formulations into hard gelatin capsules. Output is rated at ~6,000 capsules per hour for powders and can
fill No. 0, 1, 2, 3, and 4 capsules.
• Component Transport
• Range of Speed
• Fill Weight Repeatability
8.5.11. Glatt Tablet Coater & Mixer
The GPCG 2 LabSystem is a modular constructed fluid-bed system for developing production processes
in the laboratory and for producing clinical samples. The combinability of the components ensures that
the system can be adapted to a broad range of processes.
Depending on the module combination the following processes are possible:
• Drying
• Granulation
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• Coating
• Pelletizing
Batch sizes range from 0.3 kg to 5 kg.

The purpose of the fluid-bed processor is to get powdery or granulated substances floating, with the
fluidizing air stream being able to exchange heat and mass in an efficient manner. The Glatt uses four
fluid bed techniques:
In the drying method the air flow draws the moisture out of the floating product.
• The process air flows through the sieve bottom of the product container into the machine tower.
The air speed should be set so that the produce makes a swirling motion and forms a fluid bed in
the machine tower. In the process the product gives up its moisture to the air stream.
The top spray method is used for coating and granulating particles.
• The process air flows through the sieve bottom of the product container into the machine tower.
The air speed should be set so that the produce makes a swirling motion and forms a fluid bed in
the machine tower.
• In granulation a spray nozzle is used to spray an agglomeration fluid into the fluid bed from
above. The product particles in the fluid bed bind to each other, i.e., they granulate. The velocity
of the particles drops and they fall back into the container in the edge zone of the fluid bed. In the
fluid bed the entire particle surface is exposed to the air stream. This achieves optimal spraying
and optimal drying.
• The goal of granulation is to produce a dust-free and fluid granulate out of a very fine powder. A
granulate is obtained by coating the fine-grain, usually powdery product with a spray medium, so
that powder particles stick to each other and become a granulate. Granulation results in a
controlled agglomeration.
• In particle coating a suspension or dispersion is sprayed. The conditions in the air stream
(including the temperature) must be such that the fluid phase of the suspension is not evaporated
before the drops can wet the product particles in the fluid bed.
The Wurster process, which is also referred to as bottom-spraying, produces a particularly uniform
surface on the coated product. This high surface quality is necessary for example for the controlled
release of active ingredients from drugs.
• The Wurster method allows films to be applied to curved tablets, capsules, pellets and similar
shapes in a closed system. This method is referred to as fluid bed spray coating or film coating.
• The special design of the material container (with an internal riser) and the spray nozzle built into
the sieve bottom ensures the desired particle motion and the desired spray shape of the spray
medium. The process works optimally when the product rises in a rapid eddy motion within the
riser and falls back into the product container outside the riser.
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Rotor process, also known as tangential spraying. The quality of the coating that is applied is comparable
to that achieved by the Wurster process. The advantage of this process is the spherical and very smooth
surface of the coated particles.
• In the rotor method powdery or granulate-like material (with an appropriate bulk density) is
placed in a constant, spiral turning motion. This motion is created by the combined effect of the
air stream with the centrifugal force and gravity. This method ensures optimal drying of each
product particle. At the same time binding agents or catalysts are sprayed in, resulting in an
agglomeration process. A granulate if formed out of powdery material.
• The friction between the rotor disc and container wall affects the shape and density of the
granulate during the process. This method enables granulation, palletizing, layering and coating
processes.
General System Operation

The fan in the exhaust air area draws the process air through the entire system. The process air first flows
through the dehumidifier module where it is brought to the proper moisture. The inlet air handler, brings
the process air to the proper temperature. At the lower section it enters the machine tower and in the
product container produces a fluid bed. Depending on the process type the spray nozzle sprays a liquid
into the fluid bed. The product retaining filter in the machine tower prevents the product dust from
escaping the fluid bed. The after filter in the exhaust air section holds back product dust remnants which
were able to pass through the product retaining filter in the machine tower. This means only clean
process air leaves the system. The three pressure gauges on the working plate show the differential
pressures of inlet air Stage 1, inlet air filter Stage 2, and exhaust air filter.
• Air flow verification

• Moisture removal verification
The end point of a fluid bed drying or granulating process has customarily been determined
by monitoring the temperature of the exhaust gas stream.
•
• Spray application verification
8.5.12. Tablet Filling Machine
The Tablet Filling Machine (IMA, Pressima) is a rotary press which is designed for pressing solid
materials. A maximum tablet diameter of 25mm and a maximum tablet fill dimension of 17mm can be
produced. The output of the tablet press is adjustable between 4,800 and 19,200 tablets per hour.
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Operators load blended materials into a hopper above the machine. The powder then flows through the
hopper to a filling station beneath, and flows from there to a rotating table. The rotating table is fitted with
holes on its outside edge that hold dies with the specified tablet dimensions. The dies are interchangeable,
so the same table can produce whatever shape is required if fitted with new dies. The powder flows from
the filling station to fill the die. When the table rotates, the filled die moves into a punch press. When the
upper and lower halves of the punch meet, several tons of pressure is exerted on the powder. The pressure
compresses the powder into a compact tablet. The punch releases, and the lower punch lifts to eject the
tablet. The speed of the rotation of the table determines how many tablets are made per minute. The
tablets eject onto a vibrating belt which vibrates any loose dust off the tablets. Tablets are formed using
the following method:
• Material Transport
• Range of Speed
• Hardness Adjustments - Adjust tablet hardness by adjusting the wall dimension and fill height.
• Hardness Verification – Run batch and pull samples. Verify hardness variation of samples is
within 5% of target
• Weight Verification – Run batch and pull samples. Verify weight variation of samples is within
5% of target.
• Dimensional Verification – Run batch and pull samples. Verify dimensional variation of samples
is within 5% of target.
• Visual Examination for defects (e.g. color)
• Dissolution
8.6. Laboratory Equipment

8.6.1. Environmental Chambers for Product Storage*
The validation protocol for an environmental chamber should consider temperature, humidity, and CO¬2
concentration as applicable. The range specification should be set from either the user specification or the
manufacturer tolerances. All parameters should be tested initially for 1 week to include typical daily and
weekend variation. Instrument calibration records, for both the test equipment and the chamber control
equipment should be up to date.
o (9) thermocouples
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o Thermocouple Calibration (±0.5C accuracy)

• Humidity: (5) consecutive days
• CO2: (5) consecutive days
8.6.2. Refrigerators and Freezers for Product Storage*
The validation protocol for refrigerator or freezer should consider temperature as applicable. The range
specification should be set from either the user specification or the manufacturer tolerances. All
parameters should be tested initially for 1 week to include typical daily and weekend variation.
Instrument calibration records, for both the test equipment and the chamber control equipment should be
up to date.
o (9) thermocouples
o 24 hours
o Thermocouple Calibration (±0.5C accuracy)
• Humidity:
o as applicable
8.6.3. Sensors
Sensors, such as pressure gauges, flow gauges, particle counters, and voltage meters (pH meters) fall into
a category of instruments which may require an SOP for operation but as a rule do not need validation.
Sensors need to be calibrated, unless they have been tagged as not critical and calibration not required.
8.6.4. In process and Finished Product Test Equipment
Laboratory equipment is used to perform in process and finished product testing. This equipment will be
operated via SOP and have equipment specific IQ / OQ protocols. For instrumentation, the IQ and OQ
documents will often be combined into one document, since it is easier to perform them at the same time.
All of the instruments considered by this master plan are “off the shelf” (OTS) designs from major
laboratory equipment manufacturers. IQ information is largely the same for all laboratory equipment. The
IQ is used to ensure that the instrument has been installed properly. It verifies all of the equipment is
present and received as designed and specified, properly installed, and that the environment is suitable for
the instrument’s operation. It is a simple process that requires document collection and set-up verification.
The following items should be assembled for the instrument IQ.
• tools,
• parts,
• procedures,
• manuals,
• purchase orders,
• certification/calibration records,
• drawings – Produce a unit connection drawing with identification of cables.
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• Verify the software versions

• Make backup copies if possible.
The following instrumentation will be covered under this category:
8.6.4.1. HPLC
HPLCs are system integration devices, being composed of several modules and assembled into a working
unit (the HPLC) according to which specific modules are linked. In the OQ section of the equipment
qualification, the following system tests should be considered. These need not be performed as written
herein, since the specific system must be addressed and it may require substantially different
implementation.
• Temperature accuracy and stability of column heater/cooler
• Detector wavelength accuracy
• Detector linearity
• Injection volume linearity and precision
• Detector noise and drift
• Gradient composition accuracy and linearity
• High and low pressure cut off accuracy
• Sample to sample carryover
8.6.4.2. Mass Spectrophotometer
Finnigan LCQ™ Series instruments are members of the Finnigan™ family of MS detectors. The Finnigan
LCQ Series MS detector is an advanced analytical instrument that includes a syringe pump, an optional
divert/inject valve, an atmospheric pressure ionization (API) source, a mass spectrometer (MS) detector,
and the Xcalibur® data system. In a typical analysis, a sample can be introduced in any of the following
ways:
• Using the syringe pump (direct infusion)
• Using the inject valve fitted with a loop and an LC (flow injection analysis)
• Using a divert valve and an HPLC fitted with a column (LC/MS)
In analysis by LC/MS, a sample is injected onto an LC column. The sample is then separated into its
various components. The components elute from the LC column and pass into the MS detector where
they are analyzed. Analysis by direct infusion or flow injection provides no chromatographic separation
of components in the sample before it passes into the MS detector. The data from the MS detector are
then stored and processed by the Xcalibur data analysis system.
Installation utility requirements for the MS system are more rigorous than for most laboratory
instrumentation. A typical installation appears as in the figure below.
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In addition to the items depicted, there are also two gas sources (N2 and He) and two vent sources (for
pump exhaust and chemical ionization exhaust). There is also a fluid path that is not shown. All of the
component interconnections as well as the plumbed liquid paths should be clearly shown in a final as built
drawing that is part of the IQ.
8.1.1.1. Polarimeter
8.1.1.2. Differential Scanning Calorimeter
The DSC system consists of an analytical instrument designed to measure heat capacity and changes in
heat capacity as a function of temperature. It includes the Model 4100 Multi-cell DSC from Calorimeter
Sciences Corp. [TA Instruments], a computer display monitor, a computer system with pre-installed
operating system, keyboard, and mouse, a surge protector, a manual, four Hastelloy-C reusable ampoules,
a ampoule storage block, a package of gaskets, tweezers, a rubber ampoule grip and assorted tubing and
insulation for purge gas and accessory cooling bath. There may additionally be a cooling bath, printer, gas
regulator, or second ampoule kit. OQ validation of the system consists of automatic calibration and
confirmation with a water heat of fusion experiment.
8.1.1.3. Capillary Zone Electrophoresis
8.1.1.4. IR Spectrophotometer
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8.1.1.5. Dissolution Testing Devices
8.1.1.6. UV Spec
8.1.2. Laboratory Dishwasher
A Miele Professional, G7883CD dishwasher will be used in the laboratory for cleaning of lab glassware
and reusable materials. The washer is designed to clean, rinse, and dry equipment. The washer consists
of a stainless steel wash chambers, has a direct injection spray nozzles and automatically dispenses of
cleaning agents (if used). The washing cycles are a balance of wash time, water temperature, water
circulation and spray pattern, and specific cleaning agents. Elimination of reside is achieved by using
multiple heated DI water rinses. An electronic controller is used to operate the validated wash cycles.
This equipment is operated via SOP.
In addition to standard IQ and OQ items, the following tests must be performed under a suitable protocol
• Spray Coverage Test – Using a riboflavin paste, materials will be soiled and placed within the lab
dishwasher. An automatic cycle will be run. Following the cycle, the material will be inspected
to verify the soiling has been removed.
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9. Process Validations ............................................................................................................................. 86

9.1. Fill Volume Qualification ............................................................................................................. 87
9.2. Thawing of Frozen Product ......................................................................................................... 87
9.3. Compounding of Product ............................................................................................................ 88
9.4. Filter Sterilization Procedure ...................................................................................................... 88
9.5. Product Sterile Sampling Procedure ........................................................................................... 88
9.6. Media Fills ................................................................................................................................... 88
9.7. Media Fill Policy with respect to Validation ................................................................................ 89
9.7.1. Policy Introduction .............................................................................................................. 89
9.7.2. Initial Performance Qualification ........................................................................................ 89
9.7.3. Periodic Performance Re-Qualification............................................................................... 91
9.7.4. Repeat of Initial Performance Qualification ....................................................................... 92
9.7.5. Media Fill Procedures ......................................................................................................... 92
9.7.6. Media Selection and Growth Support ................................................................................ 93
9.7.7. Incubation and Inspection of Media-Filled Units ................................................................ 94
9.7.8. General Acceptance Criteria ............................................................................................... 94
9.7.9. Contamination with Media ................................................................................................. 95
9.7.10. Data Required from Media Fills .......................................................................................... 96
9.7.11. Media-Fill Runs Exceeding Action Levels ........................................................................... 96
9.8. Visual Inspection of Product (Human inspection) ...................................................................... 97
9.9. Media Fill Procedural Overview .................................................................................................. 99
9.10. Product Lyophilization Validation ......................................................................................... 100
9.11. Support Processes ................................................................................................................. 100
9.11.1. Holding Period for Clean Items Prior to Use ..................................................................... 100
9.11.2. Holding Period for Sterile Items Prior to Use .................................................................... 100
9.11.3. Other Hold Time Studies ................................................................................................... 101
9.11.4. Gowning Procedure .......................................................................................................... 101
9.11.5. Sterile Filter Integrity Testing ............................................................................................ 101
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9.11.6. Washing of Components other than Vials ........................................................................ 101

9.11.7. Sterilization of Loads (Steam or Dry Heat) other than Vials ............................................. 102
9.12. Facility Monitoring ................................................................................................................ 106
9.13. Cross Contamination Control ................................................................................................ 106
9.14. cGMP Procedures and Programs .......................................................................................... 107
9.14.1. Facility Cleaning and Sanitization...................................................................................... 107
9.14.1.1. Equipment Cleaning .................................................................................................. 107
9.14.1.2. Cleaning Process Description .................................................................................... 107
9.14.1.3. References ................................................................................................................ 108
9.14.1.4. Spray Device Coverage Documentation Verification ................................................ 108
9.14.1.5. Equipment Hold Time ............................................................................................... 108
9.14.1.6. Determination of Acceptance Criteria ...................................................................... 108
9.14.1.7. Sampling Method Descripton ................................................................................... 109
9.14.1.8. Visual Inspection ....................................................................................................... 109
9.14.1.9. Visual Plus Analytical Verification ............................................................................ 110
9.14.1.10. Swab Samples ........................................................................................................... 110
9.14.1.11. Controlled Volume Rinse Water Samples ................................................................. 110
9.14.1.12. Routine Production In-Process Monitoring .............................................................. 111
9.14.1.13. Visual Examination .................................................................................................... 111
9.14.1.14. pH .............................................................................................................................. 112
9.14.1.15. Total Organic Carbon ................................................................................................ 112
9.14.1.16. Detergent .................................................................................................................. 113
9.14.1.17. HPLC .......................................................................................................................... 113
9.14.1.18. LAL (Endotoxin) ......................................................................................................... 113
9.14.1.19. Sample Collection Sites ............................................................................................. 113
9.14.1.20. Protocol Development .............................................................................................. 114
9.14.2. Environmental Monitoring Program (non-Viable) ............................................................ 114
9.14.2.1. Room Pressure Control ............................................................................................. 114
9.14.2.2. Room Temperature Control ...................................................................................... 114
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9.14.2.3. Room Humidity Control ........................................................................................... 114

9.14.2.4. Non-Viable Particulate Monitoring........................................................................... 115
9.14.2.5. Viable Particulate Monitoring ................................................................................... 115
9.14.3. Change Control Program .................................................................................................. 115
9.14.4. Training Program .............................................................................................................. 115
9.15. Initial Area Validations ......................................................................................................... 116
9.15.1. Sterile Area Surface Maps ................................................................................................ 116
9.15.1.1. Principle .................................................................................................................... 116
9.15.1.2. Testing....................................................................................................................... 117
9.15.1.3. Test Report ................................................................................................................ 117
9.15.2. Light Level ........................................................................................................................ 118
9.15.3. Smoke Testing .................................................................................................................. 118
9.15.4. Air Change Rates .............................................................................................................. 118
9.15.4.1. Air Velocity Measurements ...................................................................................... 118
9.15.4.2. Rate Calculations ...................................................................................................... 118
9.15.5. HEPA Filter Integrity Testing ........................................................................................... 119
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9. Process Validations
A direct quote from the FDA Guidance Document, “Process Validation: General Principles and Practices”
is as follows. (http://www.fda.gov/cder/guidance/8019dft.htm )
FDA has the authority and responsibility to inspect and evaluate process validation performed
by manufacturers. The CGMP regulations for validating pharmaceutical (drug) manufacturing
require that drug products be produced with a high degree of assurance of meeting all the
attributes they are intended to possess (21 CFR 211.100(a) and 211.110(a)). Effective process
validation contributes significantly to assuring drug quality. The basic principle of quality
assurance is that a drug should be produced that is fit for its intended use; this principle
incorporates the understanding that the following conditions exist:
• Quality, safety, and efficacy are designed or built into the product.
• Quality cannot be adequately assured merely by in-process and finished-product

inspection or testing.
• Each step of a manufacturing process is controlled to assure that the finished product
meets all design characteristics and quality attributes including specifications.
Production processes are validated under a performance qualification protocol. For a filling process, the
ultimate validation is a series of media fills culminating in sterile media filled into vials. It is often useful
to break out the fill process into other activities some of which might not be tested by the media fill
process and some that might be something that can be independently tested and thus aid in future
investigation work. USP <1211> has some general, but useful detail relating to sterility assurance. Far
more information is available in the FDA Guidance document, Sterile Drug Products Produced by
Aseptic Processing (http://www.fda.gov/cder/guidance/5882fnl.pdf ).
A tablet manufacturing process has multiple steps, each requiring some level of validation. While the
rigor of sterility is not applicable, success in manufacturing will require careful attention to critical
process variables. After fluidized bed granulation, a specification may be set for “powder flow”, USP
<1174> based on one of several assessment methods. The specification can be set for 1) angle of repose,
2) compressibility index or Hausner ratio, (3) flow rate through an orifice, or (4) characterization with a
shear cell.
After tablet formation, a few other physical tests are recommended to assure a successful process.
Friability, USP <1216> can be measured for uncoated tablets using a standardized drum apparatus.
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Following a pharmacopeia harmonized protocol, an acceptable weight loss of the tablet sample is less
than 1%.
Having assured that the tablet will not fall apart inadvertently, it is important to assure that it will
disintegrate and dissolve in the patient. Consequently, a test for disintegration, USP <701> and a test for
dissolution, USP <711> may be required.
Additional and exacting criteria for powder, tablet, and capsule manufacturing validation can be found
from the FDA Guidance for Industry, Powder Blends and Finished Dosage Units - Stratified In-Process
Dosage Unit Sampling and Assessment (http://www.fda.gov/cder/guidance/5831dft.htm ).
Finally, instructional information within this section is for guidance only. It is not intended to trump a
carefully prepared protocol and a protocol is required for all process validations.
9.1. Fill Volume Qualification

See Section 8.4.2 about Liquid filling.
9.2. Thawing of Frozen Product

Thawing of product for the purpose of filtration and fill is so common that it should be considered a
process in need of validation. It is important that the product be completely thawed prior to starting work
with it on the fill day. However, in most cases, the product would not have been frozen, if it were
completely stable in liquid form. Consequently, one does not want the product thawed with a large
margin of safety in terms of days sitting as a liquid. Finally, the thawing process is most commonly
carried out in a cold room rather than in a water bath.
Protocol Outline:
Select the shape and mass of product to be thawed. For generic studies, (i.e. no specific product is
available) use water frozen in the following containers. As an alternative or additional generic product,
use saline. As a Product Validation, it is necessary to use actual product and that would be the most
acceptable practice.
• 250 mL Nalgene bottles

o Polypropylene bottles
o Polycarbonate bottles
• 500 mL Nalgene bottles
• Liter Nalgene bottles
1. Place the bottles into a cold room controlled at about 5°C. Carefully record the cold room
temperature. Set the bottles so that they do not touch each other.
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2. Record the thawed or not state of the bottles every X hours. Typically, X will be 6 to 8 hours.
3. A successful validation is one in which all bottles of a specific type are completely thawed.
9.3. Compounding of Product

Compounding of product is not a sterile operation. It is a clean operation, conducted under cGMP.
Compounding may not be specifically validated. If it is, then the validation should be done with a
specific stated goal, such as
o confirmation of weighing
o handling product in conditions of very low light
o compounding to result in <X% oxygen in solution
o compounding to occur within X minutes due to low product stability in aqueous form or at
some necessary pH condition
If aseptic compounding in the clean room (or laminar flow hood) is to be performed, then it should be
validated with 3x performance including 3 complete set-ups on 3 different days. The successfully
compounded aseptic product ( or simulant) should be subjected to growth media to ascertain sterility. Of
course a growth promotion test will also have to occur with the product (simulant).
9.4. Filter Sterilization Procedure

The sterilization procedure should normally be validated as part of the media fill. That is, media sterility
should be accomplished by filter sterilization. For some very complex aseptic formulations, where
components of the formulation are sterile filtered and then aseptically combined, it is reasonable to
validate the sterile filtration and formulation process independently of the media fill. However, whenever
possible, a media fill event should accompany the filter sterilization procedure.
9.5. Product Sterile Sampling Procedure

It is often necessary to sample a compounded product for sterility. The procedure for that sampling
should be validated as a part of the filter sterilization procedure. Follow a sterile sampling SOP and
demonstrate that multiple personnel (all personnel who will ever do the process) can obtain a sterile
sample.
9.6. Media Fills

A media fill is a validation exercise which simulates a fill and demonstrates that product sterility is
achieved and maintained. It may include various manipulations like power failures, component breakage,
and miscellaneous adjustments. Not all manipulations will be included in every media fill, but the overall
collection of media fills must include validation of special events.
Media Fill Protocols should address the following items.

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• An objective describing the operation to be validated.

• A scope outlining the activities to be performed during the media fill including the following:
• Time frame over which the fill is expected to occur.
• Minimum number of technicians.
• Change of technicians, if applicable.
• Lyophilizer loading and unloading procedure if necessary.
• Criteria dictating the minimum number of units to be filled. This is further described within the
Media Fill Policy of 8.5.9.
• Description of the validation as initial or repeat.
• Incubation conditions for the media.
• Preparation instruction for the media and justification if needed.
• Growth promotion testing.
• Criteria for determining successful completion of the media.
Components used in the media fill may have been held (sterilized and held in the sterile storage area) for
a maximum time period, thus simultaneously validating the hold of materials. This hold time validation
may also be performed independently of a media fill.
9.7. Media Fill Policy with respect to Validation

9.7.1. Policy Introduction
Media fills are process simulation tests for aseptic processing. The level of control of aseptic operations
is dependent on and maintained by well-trained personnel, defined procedures and appropriately
designed equipment and facilities. Media filling in conjunction with comprehensive environmental
monitoring is particularly valuable in demonstrating that the aseptic processing of sterile materials is
functioning as intended. The media fill is intended to simulate the aseptic process.
A media fill is a “point in time” representation of the capabilities of an aseptic processing system
including environment, equipment and personnel. It does not automatically ensure that products made on
the same line at other times will have the same level of microbiological quality. However, through control
and validation of all related processes, such as environmental monitoring, qualification of personnel, and
validation of cleaning and sterilization cycles, it is possible to maintain the level of product quality
demonstrated by the media-fill program.
9.7.2. Initial Performance Qualification
Performance qualification shall be conducted for each new aseptic filling line (both a laminar flow hood
and biological safety cabinet if sterile products are filled in each) and for each new product/container
configuration, which has not been represented in a previous performance qualification. The following
criteria will apply:
• Performance qualification shall consist of media-fill studies of a product/container configuration,
which is representative of the actual product to be filled. Representative criteria include:
• The actual product/container configuration that is representative of other filled products on the
filling line.
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• Two products which bracket all others with respect to size, fill, container opening size, line speed,
manipulations, etc.
• Products, which have been deemed to be the worst case with respect to the opportunity for
contamination, e.g. a container that has the largest opening and is conveyed at the slowest line
speed.
• Performance qualification acceptance criteria shall meet the requirements of Table 1.
Table 1 - Initial performance qualification - Media-fill acceptance criteria
•
Minimum Minimum Media-Fill Alert
Production
Number of Number of Total Level and Required Media-Fill Action Level
Batch Size
Media Fills Filled Units Action
(1) One (2) Two contaminated
<500 ≥3, as needed 5,000 1), 2) contaminated unit in units in a single run OR
to achieve total any run= investigate (2) runs with a single
number filled cause AND conduct contaminated unit= Cease
units (1) one additional qualification media fills,
run investigate cause AND
repeat initial qualification
media fills from the start.
≥500 to 4,749 ≥3, as needed 5,000 1) contaminated unit in units in a single run OR
to achieve total any run= investigate (2) runs with single
number filled cause AND conduct contaminated unit= Cease
units (1) one additional qualification media fills,
media fills.
≥ 4,750 3) 3 fills of at least 14,250 contaminated unit in units in a single run OR
4,750 units per any run= investigate (2) runs with single
run cause AND conduct contaminated unit= Cease
(1) one additional qualification media fills,
media fills.
1) Since less than 4,750 units would be filled in any one run, Table 3 cannot be directly applied; however,
on an empirical accumulative basis, no positive units in each of the individual media-fill runs would be
suggestive of a low contamination level.
2) See GUIDANCE regarding considerations for small or infrequent production or clinical batches.
3) The number of units filled in a media-fill run should allow for interventions that are routinely
encountered during production.
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• The number of runs and total filled units are summarized below. Media fills relative to A), B) and
C), below, shall contain a number of units at least equal to the maximum production batch size.
•
• For production batch sizes of less than 500 units, a minimum of three media-fill runs, for a total
of 5,000 units, shall be conducted.
•
• For small, infrequently filled production or clinical batches (< 500 containers, filled less than four
times per year) that are filled on established, qualified manufacturing equipment in a controlled
environment, it shall be acceptable to conduct three media fills preceding the fill.
•
• For production batch sizes between 500 and 4,749 units, a minimum of three media-fill runs, for a
total of 5,000 filled units, shall be conducted.
•
• For production batch sizes of 4,750 units and greater, a minimum of three media-fill runs of >
4,750 each, for a total of > 14,250 filled units, shall be conducted.
•
• GUIDANCE: It may be necessary to fill more than 4,750 units per media-fill run in order to
accommodate process variables and interventions routinely encountered during production.
•
9.7.3. Periodic Performance Re-Qualification
Scheduled media-fill re-qualification shall occur at least every six months for each product/container
configuration and aseptic filling line OR three qualification fills must occur within a six month period
prior to the fill of any product/container configuration.
Risk: Product manufacturing may resume while the media-filled units are being incubated; however,
product release shall not occur until acceptable media-fill data are obtained.
GUIDANCE: Media-fill re-qualification of the process or line prior to the scheduled six-month interval
should be considered in cases of facility and equipment modification changes in personnel, and anomalies
in environmental testing results or end product sterility testing.
Re-qualification acceptance criteria shall meet the requirements of Table 2. The number of runs and total
filled units are summarized as follows:
For small production batch sizes (< 500 containers), three media-fill runs of the maximum batch size shall
be conducted.
Risk: Alternatively, for small, infrequently filled production batches or clinical batches, (< 500 containers
filled less than four times per year), it shall be acceptable to re-qualified the process or line by performing
a single media-fill run, containing a quantity of units at least equal to the production batch, immediately
after and on the same day that the production batch is filled.
GUIDANCE: Any necessary measures should be taken to assure inactivation or elimination of

antimicrobial residuals in filling lines prior to conducting a media fill.
For production batch sizes between 500 and 4,749 units, one media-fill run of at least the maximum batch
size shall be conducted.
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For production batch sizes greater than 4,749 units, one media-fill run of at least 4,750 units shall be
conducted.
GUIDANCE: It might be necessary to fill more than 4,750 units per media-fill run in order to
accommodate process variables and interventions routinely encountered during production.
9.7.4. Repeat of Initial Performance Qualification
When required, repeat of performance qualification shall be conducted using the same procedures,
methods and acceptance criteria as described in previous sections of this policy.
An aseptic process or filling line shall be subject to repeat of performance qualification studies when:
An action level is exceeded, unless an assignable cause for the exceeded action level is identified
Production lines have not been in operation for an extended period of time, e.g. one year
There has been a significant change. Documented change control procedures should identify significant
changes, such as:
• Modifications in equipment directly contacting bulk product or product/containers
• Modifications in equipment or facilities, which can affect air quality or flow
• Major changes in production personnel, e.g. new crews
• Initiation of additional production shifts
9.7.5. Media Fill Procedures
Media fills shall be conducted on separate days, at different times during the normal working period.
A list of permitted and prescribed intervention events that could occur during the aseptic processing shall
be available.
Media fills shall be conducted under processing conditions that include "worst-case" conditions, e.g. the
greatest practical number of permitted interventions, correction of line stoppage, repair or replacement of
filling needles/tubes, replacement of on-line filters, number of personnel involved, etc. The duration of
media fills may need to be adjusted for special processes where planned interrupted filling occurs.
The media-fill run shall be of sufficient duration to cover most manipulations and operations that are
normally performed in actual processing.
GUIDANCE: If multiple sizes of the same container/closure configuration are aseptically filled, selective
sizes may be used for media fills. Containers with the widest diameter openings and lowest line speed
should be included in the media-filling regimen and might be representative of worst-case conditions.
However, there are cases where small containers are representative of worst-case conditions because of
lack of container stability in the line operations and the need for increased manual intervention.
Table 2 - Periodic re-qualification – Media fill acceptance criteria

Production
Batch Size
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Production
Batch Size
(1) One contaminated (2) Two contaminated
<500 ≥3 2) Maximum batch unit in any run= units in a single run OR
size 1) investigate cause (2) runs with single
AND repeat contaminated unit=
performance Cease qualification
qualification media fills, investigate
cause AND repeat initial
qualification per table 1
(1) One contaminated (2) Two contaminated
≥500 to 1 Maximum Batch unit in any run= units in a single run OR
4,749 Size 1) investigate cause (2) runs with single
AND repeat contaminated unit=
performance Cease qualification
qualification media fills, investigate
cause AND repeat initial
qualification media fills.
4,750 Repeat the media fill See Table 3 for
≥ 4,750 3) 1 run IF the alert values maximum action level
in Table 3 are values
exceeded.
1) Since less than 4,750 units would be filled in anyone run. The 95% confidence limit table cannot be
directly applied; however, on an empirical accumulative basis, no positive units in each of the individual
media-fill runs would be suggestive of a low contamination level. Reference Table 3.
2) See GUIDANCE regarding considerations for small or infrequent production or clinical batches.
3) The number of units filled in a media-fill run should allow for interventions that are routinely
encountered during production.
9.7.6. Media Selection and Growth Support
• Verification of growth promotion of media used in specific media-fill runs shall be conducted
following the run.
• The incubation temperature shall be the same as that used for the media-filled units.
• The media selected for media-fill runs shall be capable of growing a wide spectrum of
microorganisms and of supporting microbiological recovery and growth of low numbers of
microorganisms, i.e. 100 colony-forming units (CFU)/unit or less.
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GUIDANCE: Growth-promotion test units should demonstrate growth of the test organisms in
conformance with pharmacopoeia requirements. Such tests should be conducted within the actual media-
filled containers where possible.
9.7.7. Incubation and Inspection of Media-Filled Units
• Leaking or damaged media-fill evaluation units shall be removed, and a record made of such
removal, following processing and prior to incubation of the media.
• Media-filled evaluation units shall be incubated for a minimum of 14 days.
• Incubation temperatures shall be appropriate for the specific growth requirements of
microorganisms that are anticipated in the aseptic filling area.
GUIDANCE: Environmental monitoring data can assist in identifying the optimum incubation
temperatures. Frequently used temperatures ranges for incubation are 20° to 25° and 30° to 35° C, or 28°
to 32° C.
• Media-filled units shall be stored or manipulated to allow contact of the media with all product
contact surfaces in the unit.
•
• After completion of the incubation period, qualified personnel shall visually inspect the media-
filled containers for the presence of microbial growth.
•
• Microorganisms present in contaminated units shall be identified to help determine the likely
source of the contamination.
GUIDANCE: Inspection of the units at an earlier time period (3 to 7 day incubation) can be useful to gain
a preliminary indication of the results.
GUIDANCE: Media-Filled units should be chronologically identified within the batch to help identity
the time at which a contaminated unit was filled.
9.7.8. General Acceptance Criteria
• A contamination rate of 0.1 %, with a 95 % confidence level, shall not be exceeded in a media fill
of not less than 4,750 units.
GUIDANCE: See Initial performance qualification and Media Fill procedures regarding batches of less
than 500 units.
Regardless of the values that appear in Table 3, any contaminated units shall be investigated concerning
the reason and possible origin of the recovered microorganism(s).
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Acceptance criteria tables

• Table 1 shows the acceptance criteria for initial performance qualification of an aseptic
processing line.
• Table 2 shows the acceptance criteria for re-qualification of an aseptic processing line.
• Table 3 shows alert and action levels when a 0.1 % contamination rate is attained for large
numbers of media-filled units, i.e. when one elects to media-fill greater than 4,750 units. The
action levels in Table 3 are based on a 0.1% contamination rate. This value was attained by
dividing the number of media-filled units by the upper 95% confidence limit for the presence of
positives as calculated by the Poisson distribution formula.
9.7.9. Contamination with Media
Contamination of the facility and equipment with media during media-fill runs must not compromise the
quality of the facility and equipment or the product subsequently processed using the same facility and
equipment. Media shall be handled properly and promptly followed by cleaning, disinfection and, where
necessary, Sterilization of the equipment.
GUIDANCE: The validation of cleaning, disinfection and sterilization methods should demonstrate the
removal of media spillage that may have occurred during media-fill runs.
Table 3 - Alert levels and action levels for larger numbers of media-filled units
Number of units in single media Alert Level 1) (number of Action Level 2) (number of
fill test contaminated units) contaminated units)
≤3,000 NA 1
4,750 1 2
6,300 1 3
7,760 1 4
9,160 1 5
10,520 2 6
11,850 2 7
13,150 3 8
14,440 3 9
15,710 4 10
16,970 4 11
NOTE 1 Any recovered microorganisms shall be investigated related to the reason and possible Origin of
the recovered microorganism(s) (see 8.1).
NOTE 2 A sufficient number of units should be media-filled to permit most interventions and worst-case
Conditions that may occur during production conditions.
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1) This alert level is based on selection of a 0.05% contamination rate.
2) ≥0.1 % contamination rate at 95% confidence level.
9.7.10. Data Required from Media Fills
All media-fill runs shall be fully documented, and the following information (where applicable) included
with, or cross-referenced to, the records for each media-fill run:
1. Date and time of media fill

2. Identification of filling room/equipment used
3. Container/closure type and size
4. Volume filled per container
5. Filling speed
6. Filter (manufacturer, lot number, catalogue number)
7. Type of media filled – reference to preparation SOP.
8. Number of units filled
9. Number of units rejected at inspection and reason
10. Number of units incubated
11. Number of units positive
12. Incubation time and temperature for each group of units incubated and whether any group of units is
subjected to two different temperatures during the incubation
13. Procedures used to simulate any steps of a normal production fill, which might include. For example,
mock freeze-drying or substitution of vial headspace gas
14. Microbiological monitoring data obtained during the media-fill set-up and run
15. Environmental monitoring data collected immediately prior to and at the time of the fill
16. List of personnel who participated in the media fill
17. Growth promotion results of the media removed from filled containers
18. Identification of the microorganisms from any positive units, and investigation of any contamination
events observed during media fills
19. Management review
20. Length of time media was stored in holding tank prior to filtration
21. Length of time taken to fill all containers
9.7.11. Media-Fill Runs Exceeding Action Levels
When media-fill action levels are exceeded, an investigation shall be conducted and documented
regarding the cause of the failure.
If action levels are exceeded, there shall be a prompt review of all appropriate records relating to aseptic
production between the current media fill and the last successful one. Results of this review are to be
included in the investigation.
The investigation should include, but not be limited to, consideration of the following:
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1. Microbial environmental monitoring data

2. Particulate monitoring data
3. Personnel monitoring data (finger impressions, etc.)
4. Sterilization cycles for media, commodities and equipment
5. HEPA filter evaluation (airborne particulate levels, smoke-challenge testing, velocity measurements,
etc.)
6. Room air flow patterns and pressures
7. Operator technique and training
8. Unusual events that occurred during the media fill
9. Storage conditions of sterile commodities
10. Identification of contaminants as a clue to the source of the contamination
11. Housekeeping procedures and training
12. Calibration of sterilization equipment
13. Pre- and post-filter integrity test data, and/or filter housing assembly
14. Product and/or process defects, and/or limitation of inspectional processes
15. Documented disqualification of samples for obvious reasons prior to final reading.
Corrective actions
1. Media-fill tests, which exceed action levels, shall require action as described in Tables 1 or 2.
2. Decisions on whether or not to take action against product being held and/or distributed shall be based
upon an evaluation of all the information available.
3. All products that have been produced on a line following the media fill shall be quarantined until a
successful resolution of the media fill has occurred.
GUIDANCE A review of production batches in association with an unsuccessful media fill should
include appropriate environmental monitoring data. The record of sterility test results over this period of
time, possible assignable causes for the current media-fill results, and any other information that would
bear upon the sterility of the product involved.
References:
US-FDA Guideline on Sterile Drug Products Produced by Aseptic Processing, June 1987
ISO 13408-1: 1998 (E), Aseptic Processing of Healthcare Products- Part 1: General Requirements, 1998
ISO 13408-2, Aseptic Processing of Health Care Products- Part 2: Filtration, Draft
9.8. Visual Inspection of Product (Human inspection)

Visual inspection is not a test but is rather a process for the reduction of visible contaminant within
product vials. Consequently, every vial is subjected to examination and any found to contain particulate,
or otherwise fail to meet inspection criteria, are culled. It is not a regulatory requirement to place a
pass/fail limitation on the visual inspection process, though a few manufacturers have imposed action
limits when the numbers of culled units in or not in a single category approach some level of concern.
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Recognize that the inspection is not infallible. Lots that are 100% re-inspected by the same personnel
(from when those personnel were fresh) should contain less culled units than they did on the first
inspection but they will continue to show culled units because inspectors are not very close to 100%
effective.
o Define the inspection process exactly. Although vials are inspected for all of the following
criteria, the particle inspection is often done to the detriment of the other processes.
In the process definition, consider the following.
• Particle motion
• Vial motion (swirl/invert)
• Volume of container inspected
• Type and intensity of lighting
• Background-particle contrast
• Time of inspection
• Background illumination
• Employee comfort (time between breaks, chair height, etc.)
• Use of magnification
o Some manufacturers define a separation of the particle inspection from the remainder of the
visual inspection and thereby obtain improved compliance.
Vials are visually inspected for

• High fill
• Low fill
• Dark Particles
• Light Particles
• Container defect
• Stopper defect
• Seal defect
• Missing stopper
• Empty vial
• Broken vial
• Solution color
• Appearance of cake (lyophilized products only).
1. Check the inspectors for visual acuity. This can be done by an optometrist or in-house by use of vision
cards, but it must be done on a routine basis like a calibration.
2. Prepare a set of test vials that have known defects from the classes listed here. Require visual inspectors
to pass (95%) a test at each calibration period.
3. Collect statistics on all lots inspected. Re-inspect some lots 100% to determine the group effectiveness of
a single visual inspection process. If possible, operators should not know that they are re-inspecting the
same vials.
Use a statistical sampling of “inspection completed” vials to asses the inspection process. This “2nd
inspection” should omit the particle inspection because the group of 2nd inspectors may not have the same
training and equipment (inspection stations) as the qualified inspectors.
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9.9. Media Fill Procedural Overview
• A process simulation using microbiological growth media shall be performed by:
• Sterile filtration of non-sterile Trypticase Soy Broth (TSB), or Soy Casein Digest (SCD), pre-
formulated at 30g/Liter WFI, into the pre-sterilized receiving vessel.
• Holding the TSB (or SCD) in the product tank, while mixing, and sterile filtering with Sterile Air
or peristaltic pump, for a time not less than the maximum time to be used during routine
manufacture.
• Holding the TSB (or SCD) in the Receiving Vessel for a time not less than the maximum time to
be used during routine manufacture.
• Sampling TSB (or SCD) in the Receiving Vessel.
• Transferring the TSB (or SCD) from the Receiving Vessel to the filler pumps.
• Filling the TSB (or SCD) in xxx ml glass vials, using a fill time not less than the maximum
expected during the routine production.
• Stoppering with sterile lyophilization stoppers or serum stoppers if lyo simulation is not planned.
• Transferring TSB (or SCD) filled and stoppered glass vials into the presterilized lyophilization
trays.
• Transferring the filled lyo trays to the sterilized lyo chamber and loading the lyo with vials of
product. The total transfer time must equal the maximum transfer time expected during routine
production.
• Simulating a lyophilization cycle by evacuating the chamber to 10 PSIA, holding, then re-
pressurizing with sterile filtered air.
• Using the lyophilizer to completely stopper the vial.
• Unloading the lyophilizer, loading trays, and transferring the filled trays to the capper.
• Capping the vials with the specified aluminum seal.
• Incubating the sealed vials at 20-25ºC for (7) days, followed by incubation at 30-35ºC for (7)
days.
• Inspection of the TSB (or SCD) for growth, followed by performance of current USP specified
Growth Promotion testing.
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• There shall be no growth observed in the incubated (simulation) media, and the Growth
Promotion media shall meet the USP acceptance criteria.
•
Three consecutive, successful media simulations are required for process validation.
9.10. Product Lyophilization Validation

Performance validation of product in the lyophilizer is determined by final product parameters. Such
parameters usually include assay, moisture content, reconstitution time, cake appearance, product on
stopper, and sometimes head space conditions (vacuum, O2, etc.). Machine performance may affect these
parameters. Typical validation is to look at varying the cycle pressures, times, and temperatures to the
extremes thought to be tolerable and then forcing an operation to within those parameters.
Nearly all phase I/II lyophilized products are both filled and lyophilized without the benefit of specific
product performance validation.
9.11. Support Processes

The processes listed in this sub section require specific validation.
9.11.1. Holding Period for Clean Items Prior to Use
Vials that have been washed and prepared for sterilization, but not sterilized should have a validated
holding period prior to sterilization, or else they must be re-washed. Other items that have been washed
and wrapped for sterilization or depyrogenation should also have a hold expiry period.
The only concern related to trayed (with lid applied) vials held for oven depyrogenation is that the grade
C air or perhaps an uncontrolled insect might add contamination to the lot. The less time that the vials are
held, the more likely such contamination might occur. Validate a hold time for prepared items of 1 week
(7 days). Do this with vials by use of the USP <788> SVP (small volume parenteral) particulate test.
Show that after 1 week, sitting in the grade C prep room, or in an alternative holding room (a grade C
storage room), that the vials will continue to pass the test. Statistical sampling should be addressed.
9.11.2. Holding Period for Sterile Items Prior to Use
Materials and especially components that have been sterilized will remain in Sterile Storage until their
use. Validate by sterility testing that they these items will remain sterile for the maximum period that
they will ever be permitted by SOP to remain in sterile storage. For glass and metal items, that validated
period should not exceed one month. After 1 month, even validated items should be either discarded or
re-sterilized.
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9.11.3. Other Hold Time Studies
Liquid products that must be held for any significant (compared to the room temperature stability data of
the product) period of time after sterilization must undergo a hold time study to assure product stability
after the hold.
Liquid products that may be compounded and held prior to sterilization must undergo a hold time study
which includes bioburden to assure that the microbial level is not adversely affected.
In some instances, it may be necessary to perform a hold time study on partially stoppered lyophilizer
vials, although such efforts may more readily be achieved through media fills. In general, partially
stoppered lyo vials will be placed into the lyo maintained at about 2°C and held for no more than 8 hours.
9.11.4. Gowning Procedure
The gowning procedure must result in a completely gowned person that when tested by RODAC
application to various body locations results in biological plates showing no growth. This validation
should be performed in triplicate on team members going in for fills in order to qualify them for filling.
Once qualified, each team member should undergo re-testing at least once per week, or if a person enters
the clean room less frequently, then retesting on each entry. That method maintains the personnel in a
continuous state of validation.
Finding growth on a person who has already been qualified by passing the test in triplicate does not
immediately disqualify the person. Finding growth on three out of five consecutive entrees (grouped any
way) will result in a need to stop that individual from assisting in the fill room until they can be re-
qualified.
9.11.5. Sterile Filter Integrity Testing
When using a machine sterile filter integrity tester, such as is sold by Pall, Millipore, Sartorius, or others,
then no process validation is required beyond the equipment IQ and OQ. If, however, one chooses to
perform a product bubble point determination which employs the manual reading of a gauge, then it is
necessary to draft an SOP for the procedure and to validate the performance with multiple employees
using a selection of filters that are known to both pass and fail. The bubble point manual procedure is
sufficiently qualitative that process validation is required.
9.11.6. Washing of Components other than Vials
Cleaning validation for utensils, tanks, and other steel, or glass objects is not substantially different from
the cleaning of vials. One must state in the protocol what SOP is being validated and what that SOP is
expected to accomplish. If the cleaning is for the removal of endotoxin, particulate, or chemical
contamination, then the procedure is challenged with the simulant endotoxin, particulate or chemical
(usually sodium chloride), and a test is performed post cleaning to assure that the objective has been
accomplished.
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9.11.7. Sterilization of Loads (Steam or Dry Heat) other than Vials
IQ and OQ for the Steam Autoclave or for the Depyrogenation Oven have been addressed previously. It
is the purpose of this section to address Performance Qualification. All items subjected to heat (dry or
steam) sterilization must be validated. Maximum load configurations (by weight or bulk) may be
developed but every item that is subjected to sterilization must be included in a validation load
somewhere. It may be possible to show that the exact placement of items within the chamber is
unimportant but doing so may take more validation work than just specifying the exact location of items
within the equipment. When performing triplicate or even quintuplet load validations, rearrangement of
similar items within the load (by protocol design) will permit greater flexibility under operating
conditions.
Steam: (PQ) Loaded chamber steam penetration runs are conducted on every load. This is a very time
consuming process. The above paragraph notwithstanding, it may be necessary to determine which load
items are the most difficult to sterilize and which locations within the items presents the worst-case
conditions.
There are two commonly used methods for determining the worst-case items/locations, thermocouples,
and steam integrators. Steam integrators are commercially available strips that provide a quantitative
indication of the exposure to steam. The amount of steam exposure can be determined by measuring the
movement of a chemical indicator on the integrator strip. Steam integrators are slightly superior to
thermocouples because thermocouples do not take into account the presence or absence or air but rather
look only at temperature. The integrators see only steam and not temperature. [For example, STERIS –
“Verify Steam Integrator Strips”.]
Determining which load items are the most difficult to sterilize and which locations within the items
presents the worst-case conditions can be a daunting task. With a large load containing a wide variety of
different types of items, the number of possible test locations seems to approach infinity. It also can be
difficult to get the thermocouple and or steam integrator into the item without adversely affecting the
item’s ability to be sterilized and/or ruining the item (a concern with expensive items).
One must evaluate an item on a case-by-case basis and determine how best to challenge the item. Often
the item must be sealed somehow to return the item to a state that represents equivalency with respect to
steam penetration No attempt will be made to provide an exhaustive commentary here, but rather
provide basic techniques for answering questions that arise.
The center of a hose is the most difficult point to sterilize. To get a thermocouple or steam integrator into
the center of a hose, either slit and reseal the hose, or use a connector to join two hoses. Of course, insert
the device prior to joining and sealing. The connector technique is particularly useful for small diameter
tubing that would exclude the thermocouple or integrator due to their size. Just put them into the
connector.
The worst-case location within a bottle, flask or cylinder (small tank) is in the center, near, but not at the
bottom.
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How can you minimize the number of runs required to challenge a load? Using steam integrators can
help minimize the number of runs required to challenge a load. There are a limited number of
thermocouples available, but as many integrators as desired can be place in the load.
Fixed vs. Flexible load configurations:

Example load: three flasks, four graduated cylinders, 24 plastic bottles with vent filters.
Fixed Load / Fixed Position:

In this situation, all of the load items are placed in the autoclave, each time in the same position for each
item, and a diagram of the load configuration is available in the procedures so that the operators can
reproduce the load for every processing run. This situation will require the least validation runs, but
offers no flexibility in load configuration.
Fixed Load / Variable Position:

In this situation, all of the load items re placed in the autoclave, but the location of the item in the
autoclave can vary and only a list of the load items is required for the procedures. The validation runs
must demonstrate positional equivalency by rotating the items from location to location during the test
runs. It may be possible to accomplish this with the same number of validation runs as above and offers
the operators some flexibility in loading the autoclave. This can be an advantage especially for large
loads containing numerous items.
Variable Load / Variable Position:

In this situation, any or all of the load items (i.e. any combination of from 0 to 3 flasks, form 0 to 4
cylinders, form 0 to 24 bottles) can be placed in the autoclave in any position in the autoclave and only a
maximum load list is required for the procedures. The validation runs must demonstrate positional
equivalency by rotating the items from location to location during the test runs. The validation runs also
must demonstrate that the cycle is adequate for both a maximum load and minimum load configuration.
The minimum load tests are done with only one item in the autoclave, that item being the load item
demonstrated as being the most difficult to sterilize. This method will require the greatest number of
validation runs, but offers the operators a great deal of flexibility in loading the autoclave. This can be a
significant advantage in many situations.
Biological Challenge Tests

After determining the worst-case items and worst-case locations within items, the items are then
challenged with biological indicators (spore strips and/or vials for placement within liquids). A
thermocouple should be placed along with each indicator, as the temperature data will be required to
extrapolate the cycle to achieve the SAL of 10-6.
Tests are conducted until a cycle time results in three consecutive runs where the biological indicators
show no growth. NIH/PHAR does not normally consider it important to find the shortest possible cycle,
so attempt to predict a cycle time that might pass and add a minute or so to it. Some firms use a half
cycle method. The half cycle is the shortest achievable exposure time (only count product TC
temperature greater than 121°C) that results in F0 > 12. Then the full cycle is double the half cycle. This
could be referred to as double overkill.
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Once one has achieved three consecutive runs resulting in no growth and therefore demonstrating a 6-log
reduction (assuming you were using indicators of 106 spores/strip), the following equations show how to
extrapolate the full cycle required to achieve the SAL of 10-6.
F0
La 12⋅ − F0 EQ#1
R
12 = used to extrapolate a 12 log reduction

La = the additional lethality (F0) required
F0 = the minimum accumulated F0 value from the biological challenge runs at the end of the cycle
R = the log reduction demonstrated (i.e. log[spore population])
( T − 121.1)
10
Fi 10 EQ#2
Fi = the instantaneous F0 value

T = the minimum temperature expected during the additional lethality period (Note: this temperature
should be taken as the temperature achieved at the end of the dwell period at the challenge location where
the minimum accumulated F0 value resulted)
La
Ta EQ#3
Fi
Ta = the additional time required
C Ta + D EQ#4
C = total dwell period time required

D = the dwell period time which resulted in the demonstrated reduction
Example Calculation:
The biological challenge runs were performed using spore strips that were enumerated at 1.21 x 106
spores/strip. Therefore R = log(1,210,000) = 6.08
The minimum accumulated F0 value (at the end of the cycle) from the biological challenge runs was 30.2
minutes. Therefore F0 = 30.2 minutes.
The additional lethality (F0 required by Equation 1 is 12*30.2/6.08 – 30.2 = 29.4 minutes.
The temperature in the coldest item at the end of the dwell period was 119.4°C.
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119.4− 121.1
10
Fi := 10
= 0.676
Ta = La/Fi = 29.4/0.676 = 43.5 min
The biological challenge runs were conducted with a dwell period of 45 minutes. Therefore D = 45
minutes.
C = 43.5 + 45 = 88.5 minutes (note this number should be rounded up).
Thus, the dwell period should be 89 minutes to achieve a 12 log reduction.
Additional information:
1. Be sure that items are wrapped so that they can withstand the vacuum cycle of the autoclave. Big
cans will collapse if wrapped air tight.
2. Rotate thermocouples from run to run. This avoids misinterpreting thermocouples that read slightly
lower temperatures (i.e. cold TC’s) as cold spots or cold items.
3. Label the TC’s by number with a small strip of tape. Avoid mixing up where the TC have been
placed.
4. If you are performing a large number of test runs, compromise between post-calibration verification
of thermocouples after every run and at the end of the entire testing period.
5. Be cautious with the acceptance criteria you employ for post-calibration of TCs. Usually, permit 20%
of the TC’s in any run to fail and still count the run. Perhaps require an additional run (still with a
20% TC failure criteria) to make up for the lost TC’s of all runs collectively.
6. Documentation must include:
• Load diagram with all items and all TC numbers and date/time and data-set
• Integrators and BI’s that were present and their location
• Printout of the data – including file names and locations of backup data set.
• The time within the dataset that the dwell begins and ends. Get it on the spot. It is much easier to
locate the begin end time when you are actually doing the work than it is later.
• A TC should always be placed next to the controlling TC of the equipment. Usually that is in the
drain of an autoclave.
Comments:
• Non-liquid loads require cycles with vacuum.

• Some regulatory bodies are extremely concerned that all points within the load achieve sterilization
temperature when starting the dwell period. To achieve the goal, it may be needed to draw additional
vacuums, rearrange the load, or modify the way items are wrapped to get more efficient steam
penetration.
• Biological Indicators must be used as a part of the validation. Temperature alone will not suffice for
regulatory approval.
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• If you must place a small quantity of water within load items to assist with sterilization, you must
have appropriate procedural controls in place to ensure ongoing consistency with the amount of water
present during the validation runs and all subsequent processing runs.
Dry Heat
Dry Heat Sterilization validation could be performed similarly as autoclave validation, except that in
place of F0, a variable called FH will be used. In practice, it is acceptable to claim sterility when
depyrogenation is achieved. Consequently, it is recommended to include endotoxin indicators at all
locations where a thermocouple will be placed and subsequently demonstrate that the endotoxin has been
reduced to an acceptable level. Common criteria for a successful Dry heat sterilization event are shown
below.
• There will be at least a 3 log reduction in LAL endotoxin at all seeded locations.
• The temperature at all probed locations shall reach a minimum of 235°C [250°C set point less
15°C maximum acceptable lower range.] . [USP 30 <1211> Sterilization and Sterility
Assurance, recommends “A typical acceptable range in temperature in the empty chamber is +
15°C when the unit is operating at not less than 250°C”.]
• The oven shall faithfully execute the cycle for time and temperature as demonstrated from the
time/temperature profile of at least one valid probe located close to the oven controller probe.
9.12. Facility Monitoring

The primary function of facility monitoring is to provide physical and environmental data which together
may demonstrate that the manufacturing areas are being operated in a state of compliance.
The type of data that should be collected consists of the following.

• Equipment alarms
• Manufacturing Room Differential Pressures
• Laminar Flow Hood and Biological Safety Cabinet Differential Pressures.
• Room Temperature
• Room Humidity
• Non-Viable particulate counts
• Viable particulate counts
The equipment and methods for obtaining and recording these data should be calibrated and validated.
Automated SCADA systems can collect these data when suitable sensors and transmitters are connected.
Sensors should be calibrated traceable to NIST. SCADA systems must be shown to reliably record data
and are subject to Part 11 validation. [See: Guidance for Industry Part 11, Electronic Records; Electronic
Signatures - Scope and Application] [ http://www.fda.gov/cder/guidance/5667fnl.htm ]
9.13. Cross Contamination Control

The HVAC system consists of two air zones to minimize cross contamination during operations. The
HVAC system will provide the necessary flow, pressurization, temperature, and humidity to the facility.
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Offices and non-GMP areas are on an independent air system. The GMP operation is controlled by two
HVAC air handling units, which are combined for output, but use single pass air. As a consequence of
the single pass air, there is no opportunity for comingling of air from separated parts of the operation
within a common return, since there is no return.
9.14. cGMP Procedures and Programs

9.14.1. Facility Cleaning and Sanitization
Facilities cleaning is the general cleaning of the facilities, rooms, floors, and outside of equipment. Such
cleaning will be performed manually using various surfactants and sanitizers, to ensure that the
manufacturing facilities conform to the requirements of their respective environmental classifications.
The facilities cleaning regimen has two primary targets:
1. Remove residual product and / or non-viable particulate matter on facility and external equipment
surfaces, to prevent possibility of ingress into the manufacturing process.
2. Remove and / or destroy viable microorganisms that represent the normal environmental flora.
SOPs will be generated to codify the following initial cleaning program.
1. For Rooms Currently in Use

a. Mop floors daily with disinfectant solution.
b. Spray or wipe walls and enclosed, sealed equipment exteriors weekly with disinfectant
solution.
2. For Rooms Unused since Previous Cleaning
a. Mop floors monthly with disinfectant solutions
b. Spray or wipe walls and enclosed, sealed equipment exteriors monthly with disinfectant
solution.
3. All rooms
a. Wipe down walls, ceilings, equipment, and piping every size months.
9.14.1.1. Equipment Cleaning
Equipment cleaning processes are designed to remove residual product, non-viable particulates, viable
particulates, and bacterial pyrogens, from direct and indirect product contact surfaces. Specific cleaning
SOP’s may be appropriate in the case of specialized equipment. Usually, equipment cleaning will be
covered in an Equipment Operations SOP.
9.14.1.2. Cleaning Process Description
Prior to cleaning, the lines and fittings are completely disassembled, and the silicone parts are discarded.
The stainless steel parts are disassembled to component level and manually cleaned using RODI. No
detergents or surfactants are used. Parts are inspected at the wash station, and rewashed if required.
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Following visual inspection, dedicated equipment will be staged in the Storage Room. Non-dedicated
equipment will be forwarded for additional cleaning processes prior to use. Clean components are rinsed
with WFI immediately prior to wrapping and sterilization.
9.14.1.3. References
• Fourman G., Mullen M., Determining Cleaning Validation Acceptance Limits for Pharmaceutical
Manufacturing Operations, Pharmaceutical Technology, April 1993
• APHA Standard Methods for the Examination of Waste Water, 10th ed., p. 5-42, method 5540 C
(1995).
• Guidance For Industry, QB2 Validation of Analytical Procedures: Methodology, ICH November
1996.
9.14.1.4. Spray Device Coverage Documentation Verification
For equipment employing internal or external spray assemblies, spray device coverage studies will be
performed by covering surfaces with a fluorescing material, such as Riboflavin, and verifying that the
material is completely removed by the spray devices (based on visual examination with a UV light
source) using only water, without detergent. Spray Device coverage verification will be documented as
part of each equipment-specific cleaning validation protocol.
9.14.1.5. Equipment Hold Time
The cleaning validation performed will determine the allowable equipment hold times relative to:
• Maximum time between use and cleaning.
• Maximum time between cleaning and use (time before re-cleaning is required).
9.14.1.6. Determination of Acceptance Criteria
The objective of the cleaning validation studies is to verify that residuals do not compromise the safety of
subsequent batches of product. The setting of acceptance criteria establishes what level of residue is
acceptable upon completion of cleaning procedure. Acceptance criteria are determined in total amount of
allowable carryover per unit surface area of equipment. Acceptance criteria will be determined prior to
execution of actual qualification studies.
Chemical residual testing will be performed, where required, on the primary product constituent.
Excipients and vehicles that are Generally Recognized as Safe (GRAS) will not be tested. Excipients and
vehicles that are not GRAS will be part of the residual testing process, and limits will be determined using
the same methodology as used for residual product limits, unless:
1. The constituent being tested for, and each excipient, are freely soluble (as defined in compendial
literature such as USP, Merck Index, etc.) in the vehicle(s) used, AND;
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2. The LD50 for each excipient, or vehicle constituent, is not less than two times the LD50 of the
most toxic constituent being tested for.
For non-GRAS excipients and vehicles that do meet both criteria, removal of the constituents being tested
for, will be considered sufficient to demonstrate removal of the excipients and vehicles to the desired
residual level. The 2X-LD50 specification ensures an additional safety factor for each of the non-GRAS
constituents.
Dedicated Equipment – Direct Contact Equipment, Bulk and Final Container

Product Residuals: Dedicated equipment, by definition, presents no risk of cross-contamination by other
product residuals. Since there is no risk for subsequent processes, the product excipient, and vehicle
residual limits for Dedicated Equipment will be Visually Clean.
Bacterial Endotoxin Residuals – Sterile Product Manufacturing Equipment Only: For sterile injectable
products (or for any product with in-process or final product endotoxin specifications), bacterial
endotoxins are of concern. The cleaning methodology used must render the system capable of producing
product meeting the bacterial endotoxin limits. Endotoxin residual limits will be calculated based on the
following assumptions:
1. All residuals are evenly distributed on the surfaces of the manufacturing process stream.
2. All residuals will be evenly distributed in the subsequent batch.
3. Residual limits will be calculated based on product specifications, where applicable, or calculated
to meet WFI endotoxin limits where product limits are not established.
9.14.1.7. Sampling Method Descripton
Several types of sampling methods will be considered for use. The applicable sampling method(s) will be
determined based upon the calculated acceptance limit, limit of detection, equipment configuration,
equipment size (surface area), residue characteristics and physical safety & toxicity. Typically, a
combination of methods is used when validating large scale operations, or facilities with a wide range of
product types. The methods to be considered are presented below, with advantages and disadvantages
detailed to aid in determination of which is / are most appropriate.
9.14.1.8. Visual Inspection
Visual inspection will be used whenever possible in the determination of equipment cleanliness.
Residues remaining after cleaning procedures are often visible at relatively low concentrations.
Individual inspectors must be qualified under worst-case visual conditions.
Advantages:
• Results are immediate.
• Method is applicable to all residues.
Disadvantages:
• Test is not quantitative or selective.
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• All required equipment may not be inspectable.

• The visual examination is subjective, which may add variations between inspections.
9.14.1.9. Visual Plus Analytical Verification
An acceptable visual examination of the equipment may be followed by direct surface sampling with
swabs, or via evaluation of a controlled volume of rinse water. The addition of a quantitative, or semi-
quantitative, method provides assurance that a buildup of visibly undetectable residue does not occur.
Use of this combination method is indicated when the visual examination is used to confirm a quantitative
limit (e.g., a fluorescing compound may be visible to a level of 1 mg/cm3, with an acceptance limit of 5
mg/cm3. In such a case, the visual inspection would be an acceptable limit test for determination of an
“analytical” limit). When used to verify an “analytical” (i.e., calculated) limit, visual inspection will be
used only when it can be demonstrated that the calculated limit is at least four times (4x) the visual
detection limit.
Advantages:
• Results are immediate for gross contamination
• Method is applicable to all residues.
Disadvantages:
• Results for the quantitative test may not be available quickly.
• All required equipment may not be inspectable.
The visual examination is subjective, which may add variation between inspecti
9.14.1.10. Swab Samples
Swab sampling is the preferred method of sampling when more than a visual inspection is required, and
where the equipment is readily accessible. Swabbing allows control of the surface area sampled and
recovery solution volume, making quantitation of residue easier than when using rinse water methods.
Advantages:
• Hard to clean areas that are reasonably accessible can be evaluated.
• Allows for recovery of dried or are insoluble residue by mechanical action.
• Provides the highest level of contaminate recovery.
Disadvantages:
• Swabs may be method-specific, and swab interference studies must be conducted.
• Some equipment areas may not be accessible, or may require disassembly.
• Physical entry into equipment may be required.
9.14.1.11. Controlled Volume Rinse Water Samples

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Controlled volume rinse water samples will be used only when the equipment is not easily accessible to
swab sampling and more than a visual inspection is required. The analytical method of detection must be
considered when determining the quantity of rinse water to use in sample collection.
It is important to recognize that a direct measurement of the residue or contaminant must be made for the
rinse water when used to validate the cleaning process. It is not acceptable to simply test rinse water for
compendial water quality requirements.
Advantages:
• Samples are easy to collect.
• Large surface areas may be sampled.
• Physically inaccessible systems may be sampled.
• Standard water testing methodology can often times be used.
Disadvantages:
• Insoluble residues may not be detected.
• Insoluble residues adhered to equipment surfaces may be left behind, and not removed by the
rinse. Such residues would evade detection by this method.
• Samples are harder to quantitate.
• Large dilution factors in some situations may decrease method sensitivity to below the calculated
acceptance limit.
9.14.1.12. Routine Production In-Process Monitoring
Indirect testing, such as in-line conductivity testing, may be of value for routing monitoring once a
cleaning process has been validated. This method is particularly useful for reactors, centrifuges, and
piping between large equipment that can only be sampled using rinse solutions samples.
All indirect test methods must correlate with the condition of the equipment. To confirm correlation, it
must be documented during validation testing that testing the solid equipment, using the desired
monitoring method on the equipment prior to cleaning, give a “not acceptable” result, while testing of the
cleaned equipment results in “acceptable” results.
9.14.1.13. Visual Examination

All pharmaceutical manufacturers are currently required to perform visual inspection of manufacturing
equipment per CFR 21..67(b)(6). Current literature suggests that most solid residues can be seen at levels
of 1 to 4 µg/cm3. In order to use visual examination, the surfaces to be inspected must be readily
observable.
Advantages of visual examination include immediate results and applicability to all solid residues. Visual
examination in neither quantitative, nor selective, and should thus be used only as a limit test.
Visual examination is also subjective; and variance in observations between inspectors may result. For
this reason, when visual examination is used to verify a quantitative limit, inspectors must be qualified.
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Such qualification involves verification, through identification of spiked sample coupons, that each
individual is capable of discerning contamination when present at the limit of quantity. When visual
examination is used for verification of “presence / absence”, as in the case of examination of dedicated
equipment, the individual need not be formally qualified, but should be tested to ensure that they have at
least normal visual acuity.
9.14.1.14. pH
Essentially utilized as a limit test for acids and bases, application consists of testing rinse samples for
deviation from published pH specifications for the appropriate grade of water used to perform sampling.
The advantages of pH test methods include its high speed and low cost. The methodology is adaptable
for on-line analysis, and is applicable for detection of residual acids and bases. This essentially limits the
technique’s usefulness to detection of residual cleaning agents. Lack of selectivity, limited sensitivity and
low precision are disadvantages of the technique.
As applied to cleaning validation, pH should only be considered a limit test. In addition, application of
the test is problematic for a number of reasons. Interpretation of results consists of comparing
measurements obtained from cleaning validation studies to published pH specifications for the
appropriate grade of water used to perform rinse sampling and / or swab extraction. As such, results
obtained through cleaning validation studies can vary from day to day due to the pH of the water used to
perform sampling.
9.14.1.15. Total Organic Carbon
Total organic carbon is a simple, quantitative approach to monitoring organic contaminants in water. The
procedure is non-selective and appropriate for determination of aqueous soluble carbonaceous residues.
Fundamentally, there are two components of TOC analysis; oxidizing organic materials, and measuring
the products of oxidation. Several techniques are available for oxidizing organic matter in water, such as
UV, UV/persulfate or high temperature. In general, all of the commercially available techniques are
capable of oxidizing the low levels of organic compounds in purified water. It is important to not that
high molecular weight proteinaceous residues may be difficult to completely oxidize, particularly with
TOC analyzers utilizing UV oxidation.
Advantages of methods employing TOC include high sensitivity, broad spectrum, rapid analysis, and
minimal sample preparation. The technique is non-selective and is limited to analytes that are soluble in
water, acid or basic solutions, and solutions of low carbon surfactant. The technique is appropriate for all
carbon containing residues soluble in aqueous solutions, which may include proteins, denatured proteins,
process additives, and cleaning agents. A supplementary technique for the determination of residual
cleaning agents may be required if TOC is chosen.
A disadvantage of the method is the ease with which the sample can become contaminated. Most
environmental contaminates are carbon containing, providing a significant opportunity for introduction of
a confounding variable. Additionally, adsorption of CO2 from the atmosphere can interfere with assays
relying on extremely low limits of detection, requiring additional care in sample acquisition and storage
methods.
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9.14.1.16. Detergent
A number of colorimetric procedures are available for the analysis of detergents. Anionic surfactants in
water may be analyzed by reaction with methylene blue to form a blue colored salt. The salt is extracted
using an immiscible organic solvent such as chloroform. Absorbance is then measure using UV
spectroscopy; detergent concentration is calculated by comparison to a standard. Materials other than
surfactants that are known to react with methylene blue are organically bound sulfates, sulfonates,
carboxylates, phosphates, phenols, cyanates, thiocyanates and some inorganic ions such as nitrates and
chlorides. Naturally occurring organic materials that react with methylene blue are relatively rare.
Advantages of quantitative techniques such as this include moderate selectivity, high sensitivity, and
relative simplicity. The procedure is relatively time consuming and produces solvent waste. It is
important to note that interference due to residues other than anionic surfactants are possible, and during
method validation, studies to determine if interference is present are necessary.
A similar method exists for the analysis of nonionic detergents, utilizing cobalt thiocyanate as the
complexing agent.
9.14.1.17. HPLC
High Pressure Liquid Chromatography is a highly specific assay method. Specificity and low limit of
detection are advantages of HPLC, making it a good choice for analyzing low level residuals. The HPLC
method, using organic mobile phases, is also better suited to quantitate analytes that are not soluble in
water (analytes for which methods such as TOC may be more easily employed).
HPLC methods have the disadvantages of being more time consuming to perform and validate, and
require costly hardware. Additionally, different columns may be required for each specific analyte, and
column-to-column variability must be considered when validating low-level HPLC methods.
9.14.1.18. LAL (Endotoxin)
Limulus Amebocyte Lysate (LAL) testing is a simple, quantitative approach to monitoring the presence
of endotoxin in the cleaning process of equipment and systems. It is important to include Endotoxin
testing during the cleaning validation in order to evaluate the level of pyrogens, on product contact
surfaces, where the sterilization methods for the equipment do not provide the depyrogenation. Testing
for Endotoxin is also important when validating storage conditions and storage times for cleaned
equipment, since Endotoxin levels can be indicative of growth of gram-negative organisms.
9.14.1.19. Sample Collection Sites
Appropriate sites for sample collection will be identified based on worst-case methodology.
Consideration for sampling sites should include areas where process fluids may have dried during routing
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manufacturing and steam-in-place procedures. Worst-case sampling sites should be based on physical
inspection of equipment and P&IDs to determine worst-case sites.
Sample sites will be delineated and approved in the individual process protocols. Additional sample sites
may be added upon further inspection of the equipment during the validation studies.
The number of samples to be taken and subsequently analyzed will be largely determined by the number
of sites sampled. A minimum of four samples per piece of equipment should be considered when using
swab sampling techniques.
9.14.1.20. Protocol Development
Using the methods described, develop a cleaning validation protocol for each piece, or group, of
equipment that contacts, directly or indirectly, the manufacturing process. At a minimum, the validation
protocols will address:
1. Purpose
2. Scope
3. Process Description
4. Analytical Method Determination
5. Acceptance Criteria – Residual Limits Calculation
6. Sampling Methodology and Procedures
7. Data Analysis Methodology
8. Summary Report Requirements
9.14.2. Environmental Monitoring Program (non-Viable)
9.14.2.1. Room Pressure Control
Control of room pressurization is done by a Siemens control package that is integral to the HVAC air
handlers. The GMP manufacturing space has positive, negative and neutral areas for differential pressure.
All pressure differentials are nominally 0.05 inches of water. A drawing showing the pressurization
scheme is available. (NIH Drawing M6_04, First Floor Pressurization Plan). Pressure is monitored and
recorded by a Rees SCADA system.
9.14.2.2. Room Temperature Control
Temperature is controlled by the same Siemens system that controls pressure. Temperature is held at 65
+ 3°C in the sterile areas. Temperature is monitored and recorded by a Rees SCADA system.
9.14.2.3. Room Humidity Control
Humidity is controlled by the same Siemens system that controls pressure. Humidity is regulated
between 30 and 60% relative humidity. Humidity is monitored and recorded by a Rees SCADA system.
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9.14.2.4. Non-Viable Particulate Monitoring
NIH personnel will use a portable particle monitor for routine monitoring of classified air quality areas.
Data compilation will be in log books maintained by a Quality group.
Continuous non-viable particulate monitoring is recommended for the laminar flow hood and biological
safety cabinet in which sterile filling activity will occur. Such equipment should be integrated to the Rees
system for monitoring and recording.
9.14.2.5. Viable Particulate Monitoring
Viable particulate monitoring will be performed by NIH personnel as part of an integrated monitoring
program. Additional information is contained in a separate section of this document.
9.14.3. Change Control Program
A formal change control program assures that changes in facilities, equipment, and processes are
reviewed to determine if validation, qualification, re-validation or re-qualification are necessary. The
SOP, which defines the program, defines the required re-qualification actions, as well as the action of
notifying all departments that the change could impact. In addition, it is a responsibility of the system to
track and define the pending actions required to close out the change request. The program administrator
ensures that the associated instrument, equipment, or area is not used prior to completion of the changes,
post change testing or validation, and regulatory release including completion of all forms and any
necessary retraining.
Under the program, change is defined as the purchase of new equipment for a new application or the
purchase of a new equipment design for an existing application. Other examples include:
• Any reassignment of equipment or processes to a new or different application.

• Any deviation from current design by providing a new function or by the deletion of any existing
functions.
• Replacement of existing parts of equipment with non-equivalent parts.
• Change to any equipment, utilities or facilities where the nature of this change could compromise
the integrity of the environment if the area has environmental restrictions.
• Construction, renovation or repair of facilities.
“Like for like” change typically involves the purchase of new instrumentation or equipment to replace an
existing part that is an exactly the same and for the same exact use. This requires no additional re-
validation or equivalency studies before implementation though one must follow the same Change
Control Program for documentation purposes. A notation on the Change Control Request (CCR) that this
is a like for like change is entered and no other follow up is required. Approvals are still required before
the change takes place, and after the part is replaced. No other support documentation is required.
Major equipment or systems, if changed or altered, may require revalidation or re-qualification.
9.14.4. Training Program

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All personnel (including those concerned with cleaning and maintenance) employed in such areas should
receive regular training in disciplines relevant to the correct manufacture of sterile products. This training
should include reference to hygiene and to the basic elements of microbiology. When outside staff who
have not received such training (e.g. building or maintenance contractors) need to be brought in,
particular care should be taken over their instruction and supervision.
9.15. Initial Area Validations
9.15.1. Sterile Area Surface Maps
Sterile area surface maps are plan drawings of a sterile area (e.g. laminar flow hood) that is to be tested in
multiple locations for viable or non-viable particulates.
Definitions
Particle
Solid or liquid object which, for purposes of classification of air cleanliness, falls within a cumulative
distribution that is based upon a threshold (lower limit) size in the range from 0.1 µm to 5 µm.
Particle Size
Diameter of a sphere that produces a response, by a given particle-sizing instrument, that is equivalent to
the response produced by the particle measured. Note: For discrete-particle-counting, light-scattering
instruments, the equivalent optical diameter is used.
Particle Concentration
Number of individual particles per unit volume of air.
As-Built
Condition where the installation is complete with all services connected and functioning but with no
production equipment, materials, or personnel present.
At-Rest
Condition where the installation is complete with equipment installed and operating in a manner agreed
upon by the customer and supplier, but with no personnel present.
Operational
Condition where the installation is functioning in the specified manner, with the specified number of
personnel present and working in the manner agreed upon.
9.15.1.1. Principle
Compliance with the air cleanliness (ISO class) requirements specified by the customer is verified by
performing specified testing procedures and by providing specified documentation of the results and
conditions of testing, as agreed upon by the customer and the supplier.
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9.15.1.2. Testing
The reference test method for demonstrating compliance is given in annex B. An alternative method
having comparable accuracy may be specified, although if no method is specified or agreed upon , the
reference method shall be used.
Tests performed to demonstrate compliance shall be conducted using calibrated instruments.
Airborne particle concentration limits

Upon completion of testing, average particle concentrations and the 95% upper confidence limit shall be
calculated using the equations shown in annex C.
Average particle concentration(s), calculated in accordance with equation (C.1), shall not exceed the
concentration limit(s) determined by use of equation (1) in 3.2, as specified for the considered size(s).
In addition, for situations in which the number of sampling locations involved is at least two but not more
than nine, the calculation of 95% upper confidence limits in accordance with C.3 shall not exceed the
concentration limits established above.
Particle concentrations used for determination of conformance to classification limits shall be measured
by the same method for all considered particle sizes.
9.15.1.3. Test Report
The results from testing each cleanroom or clean zone shall be recorded and submitted as a
comprehensive report, along with a statement of compliance or noncompliance with the specified
designation of airborne particulate cleanliness classification.
The test report shall include the following:
a) the name and address of the testing organization, and the date on which the test was
performed;
b) the number and year of publication of this part of ISO 14644, i.e., ISO 14644-1:date of
current issue;
c) a clear identification of the physical location of the cleanroom or clean zone tested
(including reference to adjacent areas if necessary), and specific designations for
coordinates of all sampling locations;
d) the specified designation criteria for the cleanroom or clean zone, including the ISO
classification, the relevant occupancy state(s), and the considered particle size(s);
e) details of the test method used, with any special conditions relating to the test or
departures from the test method, and identification of the test instrument and its current
calibration certificate;
f) the test results, including particle concentration data for all sampling location coordinates
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9.15.2. Light Level
The CFR part 211 requirement for lighting in a manufacturing area is “Adequate Lighting will be
provided in all areas”. The facility is designed to have between 80 and 100 ft candles of light provided by
fluorescent lamps placed either in the ceiling grid or between grids (grade A). Light level testing by
photometer reading will be documented at start up. Unless an engineering change is put into place, or
there is an expressed concern over the light level, it will not be routinely tested.
9.15.3. Smoke Testing
Smoke testing will be conducted in the Grade A Fill Room to demonstrated suitable laminarity in the
areas of filtration, filling, lyo loading, vial loading, and vial unloading. Smoke will be conducted in
dynamic and static states. Testing will be conducted after each significant change in the placement of
equipment or wall configuration, to be indicated in the associated change control document.
9.15.4. Air Change Rates
Air change rates will be calculated based on face velocity results. Air changes rates will be calculated
every time face velocity testing is performed. Rates will be compared to original conditions and reported.
Classification
Standard # / hr
As Measured, assumes full HEPA
Grade A coverage
Grade B 200
Grade C 65
Grade D 22
A drawing showing the design air change rates for all areas is available. (NIH Drawing M6_04, First
Floor Pressurization Plan). Air change rates as high as 150 changes/hr are used for Grade C in front of
the laminar flow hood and biological safety cabinet that are used for filling.
9.15.4.1. Air Velocity Measurements
A specific SOP will exist for Air Velocity Testing even if the Testing is conducted by an outside
contractor. That SOP may be written and approved after consultation with the contractor.
Several methods exist for performing an air velocity test.
Since the rooms are independently monitored for velocity by a continuous system, the need for checking
velocity at all filters is only once every two years or if the environmental program shows an OOS, then it
may be necessary as part of an investigation to perform additional velocity testing.
9.15.4.2. Rate Calculations

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Air change rate is a simple calculation that can be performed by a subcontractor or NIH/PHAR. The
data required are
• Linear flow of air from each filter going into the room = Lf (in ft/min)
• Filter area for each filter = Af (in ft2)
• Volume of the room in cubic feet = V (in ft3)
An example is provided.
 90 
 87 
 
Lf :=  82.5  <= ft/min measured at each of 5 filters
 102 
 
 84 
A f := 7.5 <= Acutal filter area in ft 2
V := 2484 <= Room volume in ft 3
60⋅ ∑(Lf⋅Af)
= 80.707 <= Room changes / hr
V
9.15.5. HEPA Filter Integrity Testing
A specific SOP will exist for HEPA Filter Integrity Testing even if the Testing is conducted by an outside
contractor. That SOP may be written and approved after consultation with the contractor.
Several methods exist for performing a particle challenge and test. All of the methods depend on
releasing a particle challenge upstream of the filters and then detecting them in the room.
Since the rooms are not independently monitored for particles by a continuous particle monitoring system
(PMS), the need for checking all filters is once every 6 months for Grade A [ISO 5]. [Reference ISO
14644-2 see below]. If the PMS shows an OOS, then it may be necessary as part of an investigation to
perform HEPA filter integrity re-certification.
The following is excerpted from ISO 14644-2
4.2 Testing for continued compliance

4.2.1 The reference test method and the maximum time intervals between such tests to prove continued
compliance with the designated ISO class are given in Table 1.
Table 1 — Schedule of testing to demonstrate compliance

with particle concentration limits
Classification Maximum time interval Test method
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< ISO Class 5 6 month Annex B in ISO 14644-1:1999

> ISO Class 5 12 month Annex B in ISO 14644-1:1999
NOTE Particle count tests will normally be performed in the operational state, but may also be
performed in the at-rest state in accordance with the designated ISO classification.
.
4.2.2 Where the application requires them, tests as given in Table 2 shall be carried out to demonstrate
compliance. The requirement for each of these tests shall be determined by agreement between the
customer and supplier.
Table 2 – Schedule of additional tests for all classes

Test parameter Maximum time interval Test procedure
a
Airflow volume or airflow velocity 12 months ISO 14644-3: -, clause B.4
b
Air pressure difference 12 months ISO 14644-3: -, clause B.5
a
NOTE These tests may normally be performed in either the operational or at-rest state in accordance
with the designated ISO classification.
b
This test will not apply to clean zones which are not totally enclosed.
4.2.4 Where the installation is equipped with instrumentation for continuous or frequent monitoring of the
airborne particle concentration, and air pressure difference, where applicable, the maximum time
interval as stated in Table 1 may be extended, provided that the results of continuous or frequent
monitoring remain within the specified limit(s).
4.2.5 In those installations that require additional tests, and where the installation is equipped with
instrumentation for continuous or frequent monitoring of the test parameter applicable, the
maximum time interval(s) as stated in Table 2 may be extended, provided that the results of
continuous or frequent monitoring remain within the specified limit(s).
HEPA filters should have a specified life span. Initially, the expiry can be that given by the manufacturer.
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10. Environmental Monitoring Program I – Physical Tests 123

10.1. Equipment ............................................................................................................................. 123
10.1.1. Sample locations and Volumes (non-viable particles) ...................................................... 124
10.1.1.1. Sampling Rational ..................................................................................................... 125
10.1.1.2. Frequency Rational ................................................................................................... 126
10.1.1.3. Sampling in Clean Air Devices ................................................................................... 128
10.1.2. Method of Sampling.......................................................................................................... 128
10.1.3. Trending and Reading of Particle Data ............................................................................. 128
10.1.4. Laminar Flow Hood ........................................................................................................... 129
10.1.5. Interpretation of Results ................................................................................................... 129
10.1.6. Actions............................................................................................................................... 130
10.2. Pressure Differentials ............................................................................................................ 130
10.2.1. Frequency of Sampling...................................................................................................... 131
10.2.2. Method of Sampling.......................................................................................................... 133
10.2.3. Results (Pressure Differential) .......................................................................................... 133
10.2.4. Action (Pressure Differential)............................................................................................ 133
10.3. Air Velocity ............................................................................................................................ 133
10.3.1. Introduction ...................................................................................................................... 134
10.3.2. Equipment [Air Velocity] ................................................................................................... 134
10.3.3. Sample Locations [Air Velocity] ........................................................................................ 134
10.3.4. Frequency of Sampling [Air Velocity] ................................................................................ 134
10.3.5. Method of Sampling [Air Velocity] .................................................................................... 134
10.3.6. Results [Air Velocity] ......................................................................................................... 135
10.3.7. Action [Air Velocity] .......................................................................................................... 135
10.4. Air Change Rate..................................................................................................................... 135
10.4.1. Introduction ...................................................................................................................... 135
10.4.2. Equipment [Air Changes] .................................................................................................. 136
10.4.3. Sample Locations [Air Changes] ........................................................................................ 136
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10.4.4. Frequency of Sampling [Air Changes] ............................................................................... 136

10.4.5. Method of Sampling [Air Changes] ................................................................................... 136
10.4.5.1. Using an anemometer............................................................................................... 136
10.4.5.2. Using a flow measuring hood.................................................................................... 137
10.4.6. Results [Air Changes] ........................................................................................................ 137
10.4.7. Action [Air Changes].......................................................................................................... 137
10.5. Filter Integrity Testing (HEPA filters)..................................................................................... 137
10.5.1. Introduction ...................................................................................................................... 137
10.5.2. Equipment [Filter Integrity] .............................................................................................. 138
10.5.3. Sample Locations [Filter Integrity] .................................................................................... 138
10.5.4. Frequency of Testing [Filter Integrity] .............................................................................. 138
10.5.5. Method of Testing [Filter Integrity] .................................................................................. 138
10.5.6. Results [Filter Integrity]..................................................................................................... 139
10.5.7. Action [Filter Integrity] ...................................................................................................... 139
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10. Environmental Monitoring Program I – Physical Tests
The purpose of an environmental monitoring program is to test and certify on a continued basis that a
particular space is appropriate for conducting the activities for which the space is designed. Specifically,
the program outlines a set of tests and specifications that assure the continued maintenance of the
designated space as being suitable to conduct those activities. Other tests, which might include with
more frequent sampling, or simply different testing may be considered for validation and fall within the
boundaries of a validation protocol deemed separate from this environmental monitoring program.
The monitoring program is composed of two parts, physical parameter testing and microbiological
methods. The performance of equipment and facilities can be measured and generally accepted
through the testing of physical parameters. Such tests include airborne particulate contamination,
pressure differentials, temperature, humidity, and air flow rate and turnover. Microbiological
measurements are considerably less precise, not uniformly distributed within the space, and poorly
reproducible. Still, microbiological environmental sampling and testing is necessary if one is to produce
aseptic products. It is true that statistical sampling for sterility is impossible. Consequently, control of
the production environment is paramount in making the argument that there is a very low probability
for a non-sterile unit.
This document contains an outline of the general procedures to be followed to assure the suitability of
a parenteral operation for manufacturing sterile products.
10.1. Equipment
An automated particle counting device is used to obtain counts by drawing air through a counter. A
common technology uses a light scattering counter. The generated signal is electronically processed to
display particle counts at different size ranges. The sampling rate can vary but each sample will be
calculated to express particles per one m3. The minimum sample volume will be 1 ft3, with a greater
volume giving more precise results. Particles will be counted at two sizes as specified by the standard.
Pharmaceutical standards quote limits for the total number of particles equal to or greater than 0.5µm
and 5µm, and these are the particle sizes that are measured. Counts of other particle sizes may be
useful for investigation in the event of problems occurring.
Since counts must be collected from all classified rooms, it is most convenient to automate the
collection process and centralize the tabulated results in a single location. This is most readily
accomplished with a facility monitoring system.
However, portable counting systems that travel from location to location can be used. The system then
requires personnel to both move and operate the equipment and to collect, collate, and store the data.
The effort needed to operate a portable counting system with human labor is considerable.
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10.1.1. Sample locations and Volumes (non-viable particles)
The area operational condition is the most important measurement as it reflects the actual
contamination when the area is working and can be expected to yield the highest particle counts. It is
necessary to take sufficient samples within the clean area to have confidence that the HEPA filtration
and air flow is performing within the limits set by the standards. A statistical technique for sampling may
be employed. Using this technique the number of sampling locations reflects the size of the room or
area and its cleanliness [e.g. the larger and cleaner the room the more sampling locations that must be
taken]. A common method for selection of the number of sampling locations can be determined using
the formula:
N.L Area
Where NL is the minimum number of sampling locations (rounded up to the next whole number) and
‘Area’ represents the floor or base cabinet area in square meters. However, it is most important that
samples be collected from the most critical zones close to open product or vials or stoppers that might
be adversely impacted by the contaminating particles.
The sample locations selected should be rationally distributed within the area under test approximately
at the working level, unless scientific rational dictates otherwise. A site plan has been prepared
indicating sampling locations for each area. The minimum volume to be sampled is 27L (approx 1ft³).
The sampling rate and the volume of air sampled will be recorded. Where the sampling rate is 1 ft³ per
min, 1-minute samples should be taken and this should be repeated at least five times per sample
location. It is common to take more samples at each location to allow equipment to electronically
equilibrate following start up.
.
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Room Size Size Air Changes Number of Sample

Area Name Num AC/hr
Feet2 Sites
Laminar Flow 1C166A7A2 40.00 1.928 2
Bio Safety 1C166A7A2 20.27 1.372 2
Fill Room 1C166A7A2 193.68 4.242 150 5
Air Lock 1C166A7A1 32.50 1.738 60 1
Air Lock 1C166A5A1 74.14 2.625 60 3
Clean Prep 1C166A5A 243.13 4.753 60 5
Air Lock 1C166A5 46.66 2.082 25 1
Weigh Rm 1C166A3 52.09 2.200 21 2
Weigh Rm 1C166A4 48.73 2.128 21 2
Liq. Form. 1C166A7F 103.80 3.100 14 3
Wash Rm 2 1C166A7G 96.67 2.997 13 3
Wash Rm 1 1C166A7A 78.21 2.696 11 3
Test Lab 1C166A7B 172.99 4.01 50 4
Large Batch 1C166A7C 132.71 3.51 14 4
Blending 1C166A7D 104.45 3.12 14 3
Corridor 1C166AB 271.70 5.02 40 5
Tablet 1C166A7E 149.69 3.73 13 4
Fin. Packag. 1C166A9 204.78 4.36 7 4
Inactive Equi. 1C166A11 88.39 2.87 14 3
Ctrl Temp Sto 1C166A2 127.50 3.44 29 3
Corridor 1C166A & A1 734.94 8.26 6 or 7 8
10.1.1.1. Sampling Rational
The number of sampling sites in an area has been chosen as the integer area number in meters, usually
rounded up to the next highest integer. Exceptions to that rule are as follows. The air lock entry to the
clean prep room [1C166A5] has two inward opening doors. Due to the extremely simple traffic pattern,
a straight walk through, and relatively low air changes per hour, 25, only one sample site was selected in
the middle of the room. Similarly, air lock 1C166A7A1, between the fill room and wash room #1, is so
small at 32 square feet that only one sample site was justified. The two weigh rooms would have
required three sample sites each according to the rule, but were reduced to two because most of the
room is taken up with a bench which effectively reduces the overall floor space.
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10.1.1.2. Frequency Rational
Product is exposed and at risk while it is in the laminar flow hood or bio safety cabinet and being filled.
Consequently, particle monitoring should be continuous during the filling events. Otherwise, the fill
room, which has air change rates suitable to a very good grade C room, should be sampled at all
locations, twice per week. Other similar rooms are listed likewise. As the perceived risk decreases, such
as in weigh rooms, the frequency is decreased to 1 time per week. Other areas, where air is filtered but
particles are of less concern are monitored but at either once every two weeks or one time per month.
During sanitization activities, it is best to not sample, since micro particulate dispersions are created by
the liquid sanitizers and these will be meaningless counts. Note: It is preferable that sampling takes
place with the facility in the operational condition i.e. personnel present and normal operations being
carried out or in a condition specified.
Room Air Frequency
Number of
Area Name Num Changes
Sample Sites
AC/hr
Laminar Flow 1C166A7A2 2 Every Fill
Bio Safety 1C166A7A2 2 Every Fill
Fill Room 1C166A7A2 150 5 2/week
Air Lock 1C166A7A1 60 1 2/week
Air Lock 1C166A5A1 60 3 2/week
Clean Prep 1C166A5A 60 5 2/week
Air Lock 1C166A5 25 2 2/week
Weigh Rm 1C166A3 21 2 1/week
Weigh Rm 1C166A4 21 2 1/week
Liq. Form. 1C166A7F 14 3 1/week
Wash Rm 2 1C166A7G 13 3 1/(2 week)
Wash Rm 1 1C166A7A 11 3 1/(2 week)
Test Lab 1C166A7B 50 4 1/(2 week)
Large Batch 1C166A7C 14 4 1/(2 week)
Blending 1C166A7D 14 3 1/(2 week)
Corridor 1C166AB 40 5 1/(2 week)
Tablet 1C166A7E 13 4 1/(2 week)
Fin. Packag. 1C166A9 7 4 1/month
Inactive Equi. 1C166A11 14 3 1/month
Ctrl Temp Sto 1C166A2 29 3 1/month
Corridor 1C166A & A1 6 or 7 8 1/month
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Figure: Shows the layout of Particulate control with recommended sites (red circles) for non-viable
particle sampling.
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10.1.1.3. Sampling in Clean Air Devices
Measurements must be made in clean air devices where the contamination level is critical. It should be
stressed that the sampling procedure must not interfere with work zone air quality or airflows when
monitoring in the operational state. PII has a HEPA zone within the Grade A area and over the filling
equipment. The background to this zone is the filling room and it too is Grade A. Nevertheless, the
Filling Equipment can be treated as a clean air device and measurements at critical locations will be
obtained. These measurements will only be meaningful when the HEPA’s for the device are
operational.
10.1.2. Method of Sampling
Automated remote particle counters can provide a continuous particle monitoring system for all counter
sites on a schedule. It is recommended that data be collected every hour, or as often as a particle
counter can obtain a sample and report the results. Automated counters are recommended for the
most critical sites, such as laminar flow hoods and bio safety cabinets. Automated counters should be
considered for the fill room as well.
The results are only useful if they can be directly correlated to activities occurring within the rooms and
at the locations of the counters. A room entry log for the fill room is critical. Record all operators who
enter the room and the time of entry. Further, batch records must indicate which workers are
performing critical functions. Card entry readers may provide for the needed logs.
Probes will have been pre-positioned to point toward the critical operations and should be at
approximately working level.
10.1.3. Trending and Reading of Particle Data
On a regular schedule, and also prior to the release of any product, data must be tabulated and checked
against a specification. It is recommended that a supervisor also check the room counts prior to the
beginning of any operation, and frequently though the progress of a fill. Increase in particulate levels is
often an early indication of problem, and if noticed immediately, loss of product may be avoidable.
If automatic counters are in place, it is recommended that an audible alarm be located in the fill room
and made to alarm when any probe in the fill room exceeds the specification.
Monthly, a trend report should be produced for all rooms at rest and for all rooms during periods of
activity. A person review and report should be written and submitted to management. Remember that
data analysis should disregard startup data for the counters and should average 5 consecutive samples
(1 ft3 samples) for the purpose of comparing to the specification. The two groups of interest are 0.5µm
and 5.0µm counts.
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While the most critical results will come from fills, it is also important to control particulates throughout
the GMP facility to assure that particles don’t get tracked into critical areas. Trending reports from
these lesser grades will be a first warning signal for problems in the cleanest areas.
10.1.4. Laminar Flow Hood
The laminar flow hood is essentially a “clean air device”. These HEPA’s must be switched on prior to the
beginning of operations and allowed to stabilize. At the same time, or shortly after stabilization, the
probes which are located inside of the “clean air device” can be activated. At other times, the probes
inside the filling machine enclosure need not actively collect data, since they are not exposed to HEPA
filtered air. As such, if the probes are left on, they will be suctioning air through the walls of the clean
air device and may likely find particles that would not otherwise be encountered and that should be
considered artifacts.
10.1.5. Interpretation of Results
Current clean rooms standards state that a room will be accepted as having passed the test if the
particle concentration at each of the locations falls below the class limit. Therefore, if the mean number
of particles per cubic meter for each particle size under consideration at each sampling position is equal
to or less than the appropriate number given in the table below, the controlled space (clean room or
clean air device), under the conditions specified, is deemed to have a satisfactory level of environmental
cleanliness. Be clear that the results tabulated are correct for 1 m3 of air! This is especially important
since most equipment collects data for 1 ft3 of air. The conversion should be performed by the
equipment and reported out correctly. Refer to the specific equipment SOP if in doubt.
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Grade Location example Maximum permitted number of particles/m3 equal to

or above
At rest (c) Operational
0.5µm 5µm 0.5µm 5µm
A Fill Hood / Cabinet 3500 1(d) 3500 1(d)
B Not applicable 3500 1(d) 350,000 2,000
C Fill room 350,000 2,000 3,500,000 20,000
D Inspection 3,500,000 20,000 NS 200,000
(a) Not applicable
(b) Not applicable
(c) The conditions given for the “at rest” state should be achieved after a short “clean up” period of 15-
20 minutes (guidance value) in an unmanned state after completion of operations.
(d) These areas are expected to be completely free from particles of size greater than or equal to 5µm.
As it is impossible to demonstrate the absence of particles with any statistical significance the limits are
set to 1 particle per m3. During area qualification it should be shown that the areas can be maintained
within the defined limits.
10.1.6. Actions
It can be appreciated that the airborne contamination level of a given room or area is dependent on the
particle generating activities in the room. If the room is at rest, very low particle concentrations can be
achieved closely reflecting the quality of the air supplied to the room and hence the efficiency of the
filter system. If the room is operational there will be a greater particle concentration, which is wholly
dependent on the number of staff and the activities they are performing. It is expected that a drop in
classification will occur as a result of those activities. This information should be available when
interpreting the data generated during the monitoring as it may allow problems to be pinpointed. It is
difficult to replicate conditions when monitoring environments in the operational state and this should
be considered when interpreting data and deciding whether or not an investigation and corrective
action are necessary. Where limits are exceeded for clean air devices, an investigation into the problem
should always be carried out. Any corrective action taken as a result of investigations should be
recorded. Retesting may be carried out immediately in clean rooms, where there is a known reason for
the failure e.g. due to a certain activity or task being carried out, and if the results are acceptable the
facility will be considered to have passed.
Specific Alert Limits and Action Limits will are left to SOPs.
10.2. Pressure Differentials

In a controlled environment installation it is important to ascertain that the correct degree of
overpressure can be maintained relative to the adjacent areas of lower classification in order that
contamination is not drawn into the controlled environment from its surroundings. It is necessary to
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ensure that air moves from clean areas to less clean areas and not vice versa. Measurement of room
pressure is an indirect means of determining this as it may be assumed that air will flow from an area of
high pressure to one with lower pressure. The highest quality clean rooms should therefore have a
higher pressure than adjacent clean rooms. The pressure differentials between clean rooms are usually
monitored on a continuous basis by either gauges (electronic/magnahelic) or incline manometers.
A Rees Scientific monitoring system is installed to monitor differential pressures at the locations shown
in the figure below. In addition to the pressure transmitting sensors, there are sight gauges on the
doors at critical locations which include all of the ones shown in the figure.
10.2.1. Frequency of Sampling
Samples will be obtained continuously. The actual sample frequency will depend upon the facility
monitoring system and its data collection and data storage capability.
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10.2.2. Method of Sampling
For an “in specification” test the following conditions should be arranged at least one time per day.
• The test must be carried out with the installation unmanned and operating at its designed flow
rate (for both supply and extract) and in a balanced condition. To check the pressure
differentials the air supply system must be running at design or as per any modification, and
therefore supplying and extracting the correct volumes of air.
• All doors in the facility/suite must be closed.
Note: The probe device must be installed well clear of the door such that it is free from obstruction and
so that no pressure is registered from air movement along the floor near the door.
Following the procedure for operating the equipment, record the reading.
10.2.3. Results (Pressure Differential)
If the pressure difference is equal to or greater than the appropriate minimum pressure drop, the
controlled environment installation shall be deemed to have maintained a satisfactory degree of
pressure difference. The pressure difference between clean rooms and adjacent areas shall be equal to
or greater than the values specified below:
• 10 Pa [0.04 inches of water] between classified area and adjacent area of lower
classification
The building pressure design specification is for > 0.05 inches of water between all pressure change
areas. Some areas are indicated as neutral and thus have no differential pressure.
10.2.4. Action (Pressure Differential)
Where the pressure differential does not meet the requirements an investigation into the problem
should be carried out. Low pressure differentials may be addressed by replacing blocked (or partially
blocked) HEPA filters, increasing fan speeds, increasing airflows to specific areas by altering damper
positions or by rebalancing air supplies. Major changes to the air supply system e.g. alterations of room
layouts, addition of extra HEPA filters, must only be undertaken by authorized personnel. Any corrective
action taken as a result of the investigation should be recorded.
10.3. Air Velocity

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10.3.1. Introduction
All critical operations in aseptic units must be carried out in clean air devices, which are designed,
constructed and operated in such a manner as to provide localized clean conditions where the
particulate and microbiological contamination is reduced to a pre-defined level. The operational
requirements of different devices requires that specific air velocities are delivered through HEPA filters
in order to ensure the localized environment is fit for its intended purpose. Airflow velocities are
determined for clean air devices supplying unidirectional airflow to the work zone. Clean air devices with
turbulent or non unidirectional air supply to the work zone e.g. turbulent flow isolators, have the air
supply volume or air change rate determined rather than the airflow velocity.
10.3.2. Equipment [Air Velocity]
The equipment required for measuring the airflow velocity is a thermal anemometer, vane anemometer
or equivalent. The test defined here is an independent check. It is likely that the test, as defined, will
not be carried out in-house and will usually be carried out by an external service contractor.
10.3.3. Sample Locations [Air Velocity]
Measurements are to be made twice a year (minimum) to hoods and HEPA filters in Grade A and B
areas. Other HEPA filters are to be checked for integrity, but not necessarily air velocity for the purpose
of this specification.
The work zone for which airflow velocities are measured is characterized by airflow at right angles to the
supply HEPA filter, often referred to as the entrance plane of the airflow. Sample locations for
measuring the airflow should be such that the work zone entrance plane is divided up into a grid of
equal areas. Alternatively, some contractors measure all of the air coming out of each HEPA filter and
sum the result. Velocity readings are to be collected 6 inches from the filter face.
There is also a need to collect some velocity data in the vicinity of the work surface.
10.3.4. Frequency of Sampling [Air Velocity]
Testing should be performed every six months.
10.3.5. Method of Sampling [Air Velocity]
Because the sampling method is dependent on the style of equipment used by a contractor, it is best to
follow the contractor’s operating procedure. A copy of his procedure will be required before any testing
is commenced.
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10.3.6. Results [Air Velocity]
The mean of the air velocities obtained at each sample position should be determined and be within the
limits specified in the table below.
Clean Air Device Air Flow Limits (m/s) Limits (ft/min)

Unidirectional Air Flow Horizontal Air Flow 0.45 + 0.1 90 + 20
Cabinet Vertical Air Flow 0.30 + 0.05 60 + 10
Safety Cabinet Vertical Air Flow 0.25 – 0.50 50 - 100
Inward Air Flow Not less than 0.4
Unidirectional Air Flow Vertical Air Flow 0.30 – 0.60 60 - 118
Isolator
Clean Rooms (Grades Vertical Air Flow .45 + 0.1 90 + 20
A/B)
The air velocities measured for pass through hatches should be used to determine air change rates in
accordance with the design specification for the device under test. Likewise the air velocities measured
in the critical work zone of the isolator can used to determine the air change rate for the main work area
in accordance with the device design specification.
All determined mean values should be within limits. Air velocities exceeding the stated value may cause
excessive air movement and affect work zone protection. Air velocities below the limit may be
insufficient to maintain critical work zone protection.
10.3.7. Action [Air Velocity]
Where the values obtained are outside of limits, an investigation into the problem should be carried out.
Excessive variation across the measured values may be caused by poor device design or uneven
blockage of the filter. Alteration of fan speed or replacement of the HEPA filter may solve the problem.
Major changes to the air supply system must only be undertaken by authorized personnel. Any
corrective action taken as a result of the investigation should be recorded.
10.4. Air Change Rate
Conventional clean rooms operate on the principle that the air supplied to the room is of sufficient
quantity to dilute or remove the contamination generated within the room. Measurement of the air
supply volume and determination of the air change rate (ACR) is a measure of the frequency of air
turnover in the clean room. This gives some idea as to how quickly contamination may be removed from
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the clean room provided there is acceptable mixing of air in the room. The ACR can be determined by
measuring the mean air velocity at the supply HEPAs and calculating the air change rate based on the
mean air supply volume or by using a flow measuring hood (bolometer), which collects all of the air from
the supply and gives an air supply volume. Whichever method is used to determine the ACR it should be
consistent, as the two techniques will give different readings. Always compare like with like. Using a flow
measuring hood will give a more accurate air change rate reading, while an anemometer will allow a
gross measurement that can be used for trend analysis.
10.4.2. Equipment [Air Changes]
The equipment required for measuring the air velocity is a thermal anemometer, vane anemometer or
equivalent. A flow measuring hood with appropriate measuring device can be used.
10.4.3. Sample Locations [Air Changes]
Where an anemometer is used to determine the mean air velocity sufficient measurements should be
made across the HEPA filter or grille face. It is suggested that at least four positions are tested across the
filter or grille face to obtain the mean supply air velocity. Where a flow measuring hood is used the flow
hood opening should completely cover the filter or diffuser grille and three readings taken to obtain an
average of the air supply volume from the filter or grille. It should be noted that when using a flow
measuring hood, cross-reference to air velocity readings in the duct is required to calculate the
correction factor for the equipment.
10.4.4. Frequency of Sampling [Air Changes]
This measurement should be obtained every six months.
10.4.5. Method of Sampling [Air Changes]
10.4.5.1. Using an anemometer
1. Support the anemometer such that the airflow is unobstructed.
2. Position the sample head of the anemometer such that it is against the HEPA and approximately 10cm
from the HEPA filter, where installed. If the filter is set back behind a grille it is acceptable to take the
readings on or at the surface provided the vanes or eyelashes of the grille are not moved. The effect of
the non-uniform velocity across the supply area can be minimized by taking more readings per unit area.
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3. Repeat for each supply filter or grille.
4. Calculate the mean air velocity (m/s or ft/min) at each HEPA filter/grille and then calculate the air
supply volume (m³/s or cfm) for each HEPA/grille by multiplying the mean air velocity by the HEPA
filter/grille area (m² or ft2).
10.4.5.2. Using a flow measuring hood
1. Place the flow measuring hood opening completely over the filter or grille, seating the face of the
hood against a flat surface to prevent air bypass and inaccurate readings.
2. Measure and record the mean air supply volume (m³/s) by taking three readings at each supply HEPA
or grille.
10.4.6. Results [Air Changes]
The ACR (per hour) can be calculated using the following formula:
m ft
Air_Volume⋅ ⋅ 3600 Air_Volume⋅ ⋅ 3600
s min
ACR ACR
3 3
Room_Volume⋅ m Room_Volume⋅ ft
10.4.7. Action [Air Changes]
Where a facility has been designed to achieve a specific ACR this should be determined. The supply
volume will drop as HEPA filters block and as a result the ACR will fall. Where the ACR does not meet
design requirements an investigation into the problem should be carried out. Any corrective action
taken as a result of the investigation should be recorded. The corrective action may require filters to be
changed and the air supply system to be re-balanced to achieve the desired ACR. Major changes to the
air supply system must only be undertaken by authorized personnel.
10.5. Filter Integrity Testing (HEPA filters)

Filter integrity testing (sometimes referred to as DOP – dispersed oil particulate – testing) is carried out
to ensure that filters comply with the standard required for efficiency. The following is a brief overview
of the test used to determine the filter integrity. It is described here in order to allow an interpretation
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of test results. It is likely that this test will not be carried out in-house and will usually be carried out by
an external service contractor.
Polystyrene Latex Spheres (PSL) are most commonly used as a challenge medium. The use of PSL
provides a consistently controllable and repeatable means for integrity testing of clean room filters,
their installation and method of seal. Employing PSL as a challenge allows for either a poly-dispersed or
mono-dispersed upstream challenge. When a mono-dispersed PSL challenge is utilized, actual filter and
system efficiencies can be verified during testing although there are no regulatory requirements in the
pharmaceutical industry for that level of determination.
10.5.2. Equipment [Filter Integrity]
Equipment used should be calibrated and traceable to national standards. Where the test is performed
by an external service contractor a valid certificate of calibration for the test equipment used should be
supplied with the test report.
PSL is used as an aerosol challenge to test filter integrity at the filter manufacturer and in situ in clean
room installation. The PSL is specified where oils or oil vapor / out gassing is considered an airborne
molecular contaminants. The solution contains no cations and is safe to use in all class clean rooms.
The PSL stock solution is diluted into several gallons of WFI and then put into an aerosol generaor
directed upstream of the filters.
10.5.3. Sample Locations [Filter Integrity]
All HEPA filters should be tested.
10.5.4. Frequency of Testing [Filter Integrity]
Testing should occur every six months.
10.5.5. Method of Testing [Filter Integrity]
The smoke challenge is introduced as close as possible upstream to the filter under test at a
concentration of between 50 and 100 microgram/L as validated by the contractor.
The downstream side of the HEPA filter is scanned using a photometer. The photometer operates on the
light scattering principle and any “smoke” passing through the HEPA filter under test will enter the
detection chamber of the photometer where the “smoke” concentration is measured. The
concentration detected will be presented as a reading on the photometer. In order to calculate the
efficiency of the filter, subtract the reading from 100%.
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10.5.6. Results [Filter Integrity]
The efficiency of the filter must be > 99.97% (rating).
10.5.7. Action [Filter Integrity]
If the calculated filter efficiency is less than the limits above then action should be taken to replace the
leaking filter. Where the leaking filter is in a clean air device this may necessitate taking the device out of
use. Sealing leaks detected in filters is not advocated, but in the case of isolator hatch filters and clean
room terminal filters there may be scope for sealing the leak until a new filter can be purchased and
installed, provided an intact seal can be produced. The time period between finding a leak and installing
the new filter must be kept to a minimum (as guidance this should not exceed 1 filling day).
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Contents
11. Microbiological Testing .................................................................................................... 142
11.1. Introduction to Aseptic Testing................................................................................ 142
11.2. Settling Plates........................................................................................................... 145
11.2.1. Introduction ......................................................................................................... 145
11.2.2. Equipment [Settling Plates] ................................................................................. 146
11.2.3. Sample Locations [Settling Plates] ....................................................................... 146
11.2.4. Frequency of Sampling [Settling Plates] .............................................................. 147
11.2.5. Method of Sampling (SOP outline) [Settling Plates] ............................................ 148
11.2.6. Action Levels [Settling Plates] .............................................................................. 149
11.3. Active Air Sampling .................................................................................................. 149
11.3.1. Introduction ......................................................................................................... 149
11.3.2. Equipment [Active Air Sampling] ......................................................................... 150
11.3.3. Sample Locations [Active Air Sampling] ............................................................... 151
11.3.4. Frequency of Sampling [Active Air Sampling] ...................................................... 151
11.3.5. Method of Sampling [Active Air Sampling] .......................................................... 152
11.3.6. Action Levels [Active Air Sampling] ...................................................................... 152
11.4. Finger Plates ............................................................................................................. 152
11.4.1. Introduction ......................................................................................................... 152
11.4.2. Equipment [Finger Plates] .................................................................................... 153
11.4.3. Sample Locations [Finger Plates] ......................................................................... 153
11.4.4. Frequency of Sampling [Finger Plates]................................................................. 153
11.4.5. Method of Sampling [Finger Plates] .................................................................... 153
11.4.6. Action Levels [Finger Plates] ................................................................................ 154
11.5. RODAC Plates ........................................................................................................... 154
11.5.1. Introduction ......................................................................................................... 154
QPS 140 of 163 Rev 1

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11.5.2. Equipment [RODAC Plates] .................................................................................. 155

11.5.3. Sample Locations [RODAC Plates] ........................................................................ 156
11.5.4. Frequency of Sampling [RODAC Plates] ............................................................... 156
11.5.5. Method of Sampling [RODAC Plates] ................................................................... 156
11.5.6. Action Levels [RODAC Plates]............................................................................... 157
11.6. Swabs ....................................................................................................................... 157
11.6.1. Introduction ......................................................................................................... 158
11.6.2. Equipment [Swabs] .............................................................................................. 158
11.6.3. Sample Locations [Swabs] .................................................................................... 160
11.6.4. Frequency of Sampling [Swabs] ........................................................................... 160
11.6.5. Method of Sampling [Swabs] ............................................................................... 160
11.6.6. Action Levels [Swabs] ........................................................................................... 162

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11. Microbiological Testing
A major consideration in the operation of clean room technology for aseptic dispensing is the
monitoring of viable contamination within clean environments. It is important to stress that statistically
microbiological monitoring is not as reliable as physical monitoring and that examination of trends or
patterns of contamination must be carried out.
11.1. Introduction to Aseptic Testing

Solid growth media (e.g. settling and RODAC plates [RODAC = Replicate Organism Detection and
Counting]), prepared according to a recognized formulation, may be purchased from a commercial
manufacturer. They will be manufactured to a suitable consistency and are available in a variety of
formats (e.g. irradiated). RODAC plates are surface contact plates often containing Trypticase Soy Agar
and Polysorbate 80. RODAC Plates are recommended for the detection of microorganisms on non-
porous surfaces. With the use of Rodac Plates a facility can monitor sanitation levels with before and
after colony counts. Commercially available materials are batch prepared to an appropriate specification
and quality controlled to test the growth capabilities of the media. Agar strips specific to certain test
devices should also be purchased. Certificates of analysis and sterility must be obtained for each batch
of product supplied. Growth media should also be demonstrated as ‘growth supporting’ for the time and
conditions under which exposure occurs.
The quality of test materials should be checked thoroughly prior to using them. Spurious high counts
may arise from the use of inadequately controlled microbiological test materials. Consideration should
be given to purchasing irradiated materials for use in critical zones. Irradiated materials need not be pre-
incubated and may be safely transferred to the critical zones before opening. If materials are obtained
that are not irradiated from a commercial source then consideration should be given to pre-incubating
to check media sterility prior to use.
It is preferable to use a growth medium with low selectivity i.e. capable of supporting a broad spectrum
of microorganisms including aerobes, anaerobes, fungi, yeast and molds. The media types listed below
have been found to be suitable:
• Nutrient agar
• Tryptone Soya Agar (TSA)
• Blood agar
• Columbia agar with horse/sheep blood

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When necessary to detect or search for a particular type of microorganism a selective culture medium
should be used - consult with a qualified microbiologist for advice and recommendations. The
recommended size of solid media is 90 mm in diameter (approximate internal area 64 cm²) for settling
plates and 55 mm (surface area 25 cm²) for RODAC plates. The media may be modified and contain
neutralizing agents to inactivate residual surface disinfectant present on the surface to be tested. Other
media may be used provided the growth supporting capability of the media has been tested.
Sampling should be undertaken at the frequencies and locations specified. It is preferable that sampling
be carried out in the operational state with any process equipment running and personnel performing
normal operations or in a specified condition. It should be stressed that such sampling should not
interfere with critical work zone protection or compromise the quality of any products prepared that
may be administered to patients. The operational condition for unidirectional airflow cabinets/ isolators
and transfer devices can be considered to be where an operator is working in any part of the clean air
device. Sampling in the at rest condition may be continued at an agreed frequency to monitor baseline
contamination levels. The operational conditions and the activities being performed at the time of
testing should be recorded.
A sampling plan should be prepared to provide on-going information on the maintenance of a stable and
suitable environment. Areas where a microbiologically controlled environment is specified should be
monitored. These include critical zones of clean air devices, transfer devices, background environments
for clean air devices and changing rooms. Sample locations are classified according to the risk to the
product during the aseptic preparation process.
Sample positions within background environments should include areas where there is personnel
activity or specific operations are carried out e.g. adjacent to bench areas where trays that have been
passed into the clean room are held before being transferred into a clean air device. Sample locations,
therefore, must be considered as part of the test and monitoring strategy.
Following testing the samples should be incubated as soon as possible (within 24 hours of sampling,
same day is preferred) and should be held at room temperature with the medium uppermost until
incubated or manipulated. If the medium is dropped or touched by an operator then this should be
reported, the sample should be marked accordingly and treated as usual. Under no circumstances
should samples that have been taken be refrigerated. Incubation of samples, inverted, at 30 - 35°C for
at least 7 days is suitable for the growth of bacteria. Incubation of samples, inverted, at 20 - 25°C for at
least 7 days is suitable for the growth of mold and fungi. Different media and regulations will change the
recommended exposure times. Often, much shorter incubation periods are used. Other incubation
conditions may be used if it can be shown that the conditions promote the growth of (all)
microorganisms that may have been recovered during the sampling procedure. Incubation conditions
should be monitored to ensure that the appropriate incubation temperature is maintained throughout
the incubation phase.

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After appropriate incubation microbiological contamination should grow into discrete macroscopic
colonies that can be enumerated and the number of discrete colony forming units (cfu) can be counted
on each sample. Record the number (per unit surface area) on the appropriate report. Separate colony
counts may be tabulated for mold and bacteria. Colony types may be identified if this is considered
appropriate. If cfu are not discrete (coincidence) entities or are Too Numerous To Count (TNTC - usually
greater than 300 cfu per sample), record the result as TNTC. If one type of cfu tends to grow in a
spreading manner, count this as "one spreading colony" and record it as such. All samples
(contaminated or not) should be disposed of according to local procedures.
The following details should be recorded:

1. Sample location.
2. Date sample taken (length of time plate exposed, if appropriate, for Settling plates).
3. Number of colony forming units (cfu) per sample.
4. Batch number and expiry of media.
5. Operator responsible for exposure of samples.
6. Operation being undertaken in clean room.
7. Operator reading the result and date read.
8. Person reviewing/approving/accepting results (Responsible Quality personnel)
It is important that there is knowledge of the ‘normal’ background flora of the clean room facility and
therefore a suitably qualified person should identify organisms. Any unusual organisms or deviation
from ‘normal’ flora may require action.
Due to the natural imprecision of the test method, the expected low levels of contamination and the
natural variability of the levels, the data obtained requires most careful analysis if it is to be of value in
assessing the level of control of critical processes. The use of statistical models to analyze monitoring
data requires considerable care and cannot be recommended unreservedly. Alternatively, trends in the
data should be identified. Recommended action levels are given in the individual test methods.
Exceeding action levels on isolated occasions may not require more action than examination of control
systems. However, the frequency of exceeding the limit should be examined and should be low. If the
frequency is high or shows an upward trend then action should be taken which may include an increase
in frequency of testing, observation of operator technique or investigation of cleaning procedures.
Identification of the causative microorganism(s) may aid tracing the source of the contamination.
Successive results greater than the action levels demand that appropriate action be taken to reduce
contamination to within limits. Where a problem has been observed the contaminating microorganisms
should be identified. Isolated instances require no more action than examination of control systems. If
repeated contamination appears an investigation into the problem should take place and corrective
action should be carried out which will rectify the problem. The corrective action should investigate the
root cause of the problem and identify steps, with time scale, that will be taken to reduce the
contamination levels to "normal". A record of all action taken should be made in an "Abnormal Event
Log". The following areas should be examined where action levels are repeatedly breached:
Identify

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1. Possible cause
2. Contaminating microorganisms
Investigate
1. Whether isolated sample or whole area involved
2. Personnel - operator status (grade), level of training, health, technique, wash up
3. Cleaning procedures
4. Changing procedure
5. HEPA filter integrity of room/clean air device
6. Processes carried out
7. Previous test results for trends or other identified problems.
Contact
1. Aseptic personnel
2. Microbiology personnel
3. QA/QC personnel
11.2. Settling Plates
Settling plate sampling is a direct method of assessing the likely number of microorganisms depositing
onto the product or surface in a given time. It is based on the fact that, in the absence of any kind of
influence, airborne microorganisms, typically attached to larger particles, will deposit onto open culture
plates. Microorganisms are usually found in the air of occupied rooms rafted onto skin cells with very
few present on their own. The average size microbial particle will fall, by gravity, onto surfaces at a rate
of approximately 1 cm/s. In settling plate sampling Petri dishes containing agar medium are opened and
exposed for a given period of time, thus allowing microbe-bearing particles to deposit onto them. Petri
dishes which are 90 mm in diameter (approximate internal area 64 cm2) are most commonly used. The
number of microbe bearing particles deposited onto the agar surface of the plate over the period of
exposure is ascertained by incubation of the plate and counting the number of microbial colonies, more
commonly known as colony forming units (cfu). The microbial deposition rate may be reported as the
number depositing in a given area per unit time.
Settling plates allow continuous sampling throughout a given work period, although they cannot
indicate variation of contamination levels throughout the sampling period. They are insensitive unless a
long exposure period is adopted in order to detect the low levels of airborne microorganisms. If this is

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not carried out the results are biased to give favorable data. If this is not practicable then plates should
be monitored for successive work sessions and the incidence of contamination analyzed. It is important
to know that the plates used are capable of sustaining the growth of microorganisms after the period of
exposure since drying of the medium surface may be a problem.
11.2.2. Equipment [Settling Plates]
Plates may be obtained from a general scientific supplier. For example from Fischer Scientific:
https://www1.fishersci.com/Coupon?gid=101623&cid=1333.
Media, Prepared, BBL® Sterile Pack Plated, Trypticase Soy Agar, 150 x 15 mm Settling Plate, Sterile for
Critical Environments. Catalog number = 08-757-189.
11.2.3. Sample Locations [Settling Plates]
All Grades, A, B, C, and D must be monitored, but the frequency can vary amongst them based on the
critical nature of the work performed. Areas where a microbiologically controlled environment is
specified should be monitored. An assessment of the controlled environment should identify the
processes taking place and all potential microbiological hazards, which may be expected to occur or to
be introduced. The critical work zone of clean air devices should be monitored as these are the areas
where previously sterilized materials are brought together and manipulated (processed) into finished
dosage forms. Sample locations of plates in the critical work zone should be selected with reference to
the actual work area and the position of filters. Monitoring critical areas should be carried out under
"worst case" conditions for contamination with process equipment running and personnel performing
normal operations. It is recommended that 2 settling plates are exposed for the whole working session.
One needs data from growth promotion studies to support the exposure time selected. Three hours is a
starting point. Six hours might be found acceptable. It is known that 30 minutes is too short to
statistically be of any value. Sample locations for settling plates in clean rooms should include areas
where there is little air movement (i.e. "dead spaces") or where airflows converge or are excessively
turbulent. Areas where these conditions are most likely to occur are:
- adjacent to doors
- in pass through hatches
- at low level return air grilles (but not on the floor)
- in corners of rooms
- on any tables or flat surfaces such as a filling machine table.
Areas within the clean room where there is personnel activity or specific operations are carried out
should also be subject to monitoring e.g. adjacent to bench areas where trays that have been passed
into the clean room are held before being transferred into a clean air device.

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11.2.4. Frequency of Sampling [Settling Plates]
11.2.4.1. Dynamic
The frequency of sampling should be at least seasonally - at all testing sites, and otherwise at some
established interval. For example, if the interval were established to be twice a month, then settling
plates would be put out at all designated sites every two weeks. If a filling event is taking place during
the two week period, then monitoring during that filling event would be sufficient to meet the
requirement of monitoring every two weeks.
Recommended frequency for dynamic monitoring:

Grade A: There are two Grade A areas, the laminar flow hood and the biological safety cabinet.
Sampling should be performed at every filling event.
Sampling should be done at every non-fill event which is scheduled to last for 3 or more hours and will
have open product exposed to the room environment.
It is not reasonable and is unnecessary to obtain dynamic data if no organized event is occurring in the
Grade A area. Only during initial validation would one simulate a dynamic situation in order to collect
data.
Grade B: There are no Grade B areas.
Grade C: Each Grade C areas should get settling plates one time per month during operations.
Grade D: Grade D is not an operational area. There is a specification for Grade D dynamic
operation and it will be the specification used since no effort should be made to limit
passage within the area after validation.
11.2.4.2. Static
At least one time per month, settling plates should be placed into the designated sites at a time when
no activity is planned (static conditions). That data can be used as reference point data to compare with
dynamic data in the event of an investigation.
Recommended Frequency for static monitoring:

Grade A: There are two Grade A areas, the laminar flow hood and the biological safety cabinet.
Static monitoring must be conducted one time per month and more frequently during initial validation.
A specific validation protocol will call out for more intense monitoring during the month immediately
following any “shut down” of the rooms.
Grade B: There are no Grade B areas.
Grade C: Each Grade C area should get static settling plates one time per month.

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Grade D: Grade D is not an operational area. There is a specification for Grade D dynamic
operation and it will be the specification used since no effort should be made to limit
passage within the area after validation. i.e. no requirement for routine static
monitoring. The only requirement is for validation monitoring statically and
dynamically, followed by routine (one time per month) dynamic monitoring.
11.2.5. Method of Sampling (SOP outline) [Settling Plates]
The following specimen procedure for use and exposure of settling plates may be adopted.
1. Examine the plates for contamination prior to use.
2. Assemble the plates required and ensure that the correct information is written on the base of the
plate (the part containing the media) with ink or other marker. Do not mark the lid of the plate, as there
is always a possibility of lids coming off and being replaced with an incorrect sample plate. The following
details may be marked on each
• plate or recorded separately:
• operator who collected sample
• date and time of day sample taken
• area/location of sample
• position/sample number
• A blank “________________________” to record exposure time.
3. Gown into the area to be tested.

5. Place the plates in the appropriate positions with the lids still on.
6. Raise lids to expose the surface of the medium, rest the lid on the very edge of the plate so that the
entire agar surface is completely exposed. Take care not to put fingers on plates. Avoid passing anything
over the top of plates being exposed.
7. Leave plates exposed for the full work session or for the full time permitted as a result of prior growth
promotion validation with these vendor’s plates. The exposure time should be recorded before sending
the plates for incubation. Exposures may be shorter than the “maximum allowable exposure time, thus
“time of exposure” must be recorded. Exposures of less than 45 minutes are of dubious value.
8. After exposure:
• Replace lids of plates.
• Record the exposure time on the plate base and/or in the appropriate record.
• Swab areas where plates have been exposed with a suitable disinfectant (e.g. sterile isopropyl
alcohol 70% solution) to remove any trace of media or condensation from the lids, which may
contaminate the clean room.
• Remove plates from the area.
• Collect all plates exposed, and return to QA or microbiology for incubation.

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• Ensure plates are secured in a suitable container.

• Incubation of plates must commence within 24 hours of exposure.
9. Complete and enclose the necessary documentation.

The SOP should also include:
• Outline process of vendor validation for settling plates.
• Settling plates for later use should be stored in the recommended storage conditions with the
medium uppermost. If refrigerated, the plates should be removed from the refrigerator not less
than half an hour before they are due to be exposed.
• Care should be taken to ensure that plates are used before the expiry date on the label. Plates
should not be used after this date.
• Outline details of how settling plates are returned to the Microbiology department, where used.
Details of transport and any other arrangements should be described. Also outline method of
destruction of exposed plates with/without growth.
11.2.6. Action Levels [Settling Plates]
EC Grade Static (cfu) Dynamic (cfu)

A 1 per 2 plates 1 per 2 plates
B <1* 5
C 5 50
D 50 100
* Multiple plates are required to establish less than 1 per plate. If only one plate, then zero.
11.3. Active Air Sampling
Control of viable particles or microorganisms, which are typical of certain types of manufacturing
environments or products, is essential. While there are finite limits and standard methods for the
evaluation of non-viable particles within the pharmaceutical industry, as yet there is no definitive
standardization of methods for the
Microbiological evaluation of airborne particles.
Active air samplers all work on the principle of sucking or blowing a stream of air at a sufficiently high
velocity to cause any microorganisms in the sample to be impacted against a chosen medium.

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11.3.2. Equipment [Active Air Sampling]
A variety of equipment and methods is available for active air sampling, however the two main types of
equipment in use are the centrifugal and the single sieve to agar samplers. In all cases after the specified
sampling time, the agar strip, plate or filter in the sampler is removed, incubated under appropriate
conditions then examined for microbiological growth. The number of colony forming units (cfu) is
recorded.
Active air sampling instrumentation falls into two main categories, inertial impactors and a group
consisting of impingers, rotating impactors, and filters. Most active air sampling methods rely on
impaction. The slit sampler and the cascade impactor are examples of inertial impactors and are
typically used for screening evaluations because they have short sampling times and accommodate a
wide variety of organisms to be collected. Collection can be performed over a short sampling time,
because the media can be examined directly under a microscope or a variety of strips or plates
containing different culture media can be used sequentially. Impingers collect material from the air in a
liquid medium that can be transported to the laboratory for appropriate analysis. Filter instruments
collect samples directly onto filter paper media.
Reuter Centrifugal Air Sampler: (RCS) Example:

The “Biotest RCS Isolator®” operates on the principle of impaction, whereby the air stream enters the
rotor from the front of the instrument and the airborne microbes are ejected onto the Agar Strip by
centrifugal force. The single air outlet passage is directed to the rear, parallel with the instrument.
These units may be conveniently located attached

to a tripod for sampling at various locations within
a room.
941105 (50 strips)

TC (Tryptic Soy Agar)
Tryptic Soy Agar is a standard USP formulation designed to promote the growth of bacteria, yeast and
mold.
Incubation is typically at 30º - 35ºC for 1 to 3 days.
Sieve to Agar Sampler

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Air is aspirated at a fixed speed for variable time through a cover, which has been machined with a
series of small holes of special design. The resulting airflow is directed onto the surface of a settle or
contact plate containing agar medium.
M Air T Tester (Millipore Corporation)

A sieve-impaction sampler with ready-to-use
agar cassettes
The sieve has nearly 1000 micro-perforations

that evenly distribute air over the agar
surface. Colony overlapping is minimized and
results are easier to read.
11.3.3. Sample Locations [Active

Air Sampling]
Areas where a microbiologically controlled environment is necessary should be specified in a sampling

plan and should be monitored. These include critical zones of clean air devices, transfer devices,
background environments for clean air devices and changing rooms and areas where there is personnel
activity or specific operations are carried out e.g. adjacent to bench areas where trays that have been
passed into the clean room are held before being transferred into a clean air device. The number and
volume of samples taken in each area should be considered. The number of samples will depend on the
size and the grade of the area being monitored. In total, a minimum volume of 1000 liters (1 m³) should
be sampled. This may be obtained from one sample or in larger areas from a number of samples.
11.3.4. Frequency of Sampling [Active Air Sampling]
At a minimum samples must be collected monthly. It is recommended that samples be collected during
every filling event at the start, middle, and end of the event.

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11.3.5. Method of Sampling [Active Air Sampling]
For full details of operation of equipment, consult the operating manual. SOP's should define how
sampling is performed in clean rooms and clean air devices, taking into account any limitations of test
equipment. Set out below are important points to be considered which should be included in SOP's for
carrying out sampling:
• media strips and plates should be examined prior to use for signs of contamination;
• equipment which is battery powered should be checked to ensure the battery status before use;
• equipment must be subject to routine calibration; volume of air passed into the device by
calculation (and measurement of rotations) from the mechanical workings of the device.
• all equipment must be sterilized or disinfected according to normal SOP's prior to transfer into
clean areas;
• all media strips and plates must be handled carefully to avoid inadvertent contamination;
• all media strips and plates must be appropriately labeled.
11.3.6. Action Levels [Active Air Sampling]

A Hoods / Cabinets <1 <1
B Not Applicable <1 10
C Fill Room 10 100
D Other Areas 100 200
11.4. Finger Plates

Finger dab plates can be used to show a breakdown in operator aseptic technique where the operator
touches a contaminated surface e.g. face when adjusting mask and contamination is transferred to the
operators hand and then to products or materials that are handled in the critical work zone. They may
also show a breakdown in the transfer process disinfection. Poor technique may lead to items being
insufficiently disinfected and transferred into the clean room or clean air device. The contamination may
then be transferred to the operators gloved hands and then from there to product and materials. In
addition finger dab plates can be used to evaluate operator training.
There are limitations in using settling or contact plates for finger dabs:
• only sample a small part of the operators hands (usually only operators finger tips);

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• efficiency of the sampling method is low (plates will lift only a percentage of the actual surface
contamination present);
• There is a problem with coincidence for certain microorganisms if agar is overly wet or the test
surface is wet;
• Residual medium must be removed from the operator’s gloves after testing.
11.4.2. Equipment [Finger Plates]
Finger dabs can be performed using either standard 90 mm settling plates or 55 mm contact plates (with
a raised media surface).
11.4.3. Sample Locations [Finger Plates]
Only sample employees in the Grade A zone and only when they purport to be sterile. Some sites
sample gloved fingers as well as other body locations, such as the top of the head, chest, or back.
11.4.4. Frequency of Sampling [Finger Plates]
Each operator should be tested at each filling event (left and right hand on separate plates). More
frequent monitoring may be carried out if the operator is new or a specific problem is identified through
investigations.
11.4.5. Method of Sampling [Finger Plates]
The sample technique will be similar irrespective of whether settling or contact plates are used. The
following specimen procedure for finger dab plates may be adopted.
2. Transfer suitably labeled plates to the operator to be tested. It may be convenient for these plates to
be supplied at the same time as materials for the last product to be made in that session or at the end of
a lengthy operation. This will also ensure that all disinfectant has evaporated from the external surface
of the plates prior to commencing the test.
3. Sampling should take place at the middle or end of a work session prior to the operator carrying out
any cleaning or tidying operations. Before performing the test the operator should ensure that gloves
are dry and free of any disinfectant.

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4. The operator should lift the lid of the plate, with the opposite hand to that being tested, and keeping
hold of the lid in the other hand, touch the agar surface with the tips of all fingers then the thumb (in
the gap on the plate behind where fingers were tested) on the hand being tested. A firm and even
pressure should be applied for approximately 5 to 10 seconds, taking care not to damage the agar
surface. Replace the lid of the plate.
5. Repeat the process for the other hand with a new plate.
6. Ensure that the glove surfaces tested are cleaned with a suitable disinfectant to remove any possible
traces of residual agar before performing any other operations, or institute a procedure for changing to
a new set of “over” gloves. [When operators wear two gloves on each hand, it is possible to remove the
outer glove and don a new pair.
7. Document the plate contents as previously indicated in section 2.2.5, Method of sampling for Settling
Plates.
11.4.6. Action Levels [Finger Plates]
Glove print 5 fingers, cfu/glove <1 (in Grade A zones)

The average should be less than 1. On an infrequent basis the limits may be exceeded.
11.5. RODAC Plates

Surfaces may become contaminated in a number of ways e.g. microorganisms settling out from the
environment or from the direct touch by an operator. One of the objectives of surface sampling is to
determine the efficiency of routine cleaning procedures in removing contamination. Therefore, sampling
should be done before and after cleaning to determine the effectiveness of the cleaning procedure.
Standard contact plates (RODAC: Replicate Organism Detection And Counting) should contain sufficient
agar growth media to create a raised media surface, and may have a grid scored on the base. The
convex agar meniscus allows direct application to test surfaces (e.g. walls, floors, equipment) for
hygiene control. The medium used may contain neutralizing agents, which inactivate any residual
disinfectants on the surface to be tested and therefore enable comparative results before and after
cleaning. Contact plates are best used for detecting microorganisms that may be present on flat surfaces
in relatively low numbers.
All test methods possess shortcomings, therefore the sampling regimen used and analysis of the
subsequent data is crucial. Below are details of some of the limitations in using contact plates:
• not suitable for inaccessible or irregular surfaces;
• must be used prior to drying of the agar;
• problem with coincidence with certain microorganisms if agar is overly wet or test surface is
wet;

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• residual medium must be washed away from surface area tested;

• contact plates have a low sample recovery and plates will lift only a percentage of the actual
surface contamination present;
• the test is only sampling a very small area (sample population)
11.5.2. Equipment [RODAC Plates]
Available from Fischer Scientific: [Example Product]

BD* BBL* Sterile Pack Plated Media
Plated media in gamma-irradiated packs are validated for performance and sterility according to
Association for the Advancement of Medical Instrumentation (AAMI) guidelines.
· Validated to 10-5 SAL
· Outer Tyvek*/polyethylene wrapping minimizes risk

of false contamination, provides fiber-free bacterial
barrier, and allows for gas exchange
· To use, remove the outer wrapping in the noncritical

environment; then remove the sterile inner wrapping
in the clean area—the included inner bag may be
used to transport plates from your critical environment
· Storage temperature: 2° to 8°C (36° to 46°F).
Available in many formulations and plate types
· Rodac* (Replicate Organism Detection and Counting) for

personnel and surface sampling

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· Settling for active and passive air sampling
· Finger Dab* for sampling gloved hands
· Contact
11.5.3. Sample Locations [RODAC Plates]
Sample locations should be selected on the basis of risk to the product during the aseptic filling process,
in accordance with the table below. For example, the sampling location in a grade A zone for high risk
aseptic operations will be the filling zone work surface of the clean air device. The number of plates used
will be related to the size and grade of the area under test.
Zone Grade Location/Area

A Local zone for high risk operations (filling cabinet).
In front of lyophilizer.
C/D Fill Room & Clean Prep
11.5.4. Frequency of Sampling [RODAC Plates]
For surfaces the recommended minimum frequency of testing is weekly and during fills in the Grade A
zone. Other grades should be tested with less frequent interval. The frequency of sampling can vary
and will depend on the scale of activity carried out in the aseptic facility. It should be tied in with other
monitoring techniques so that a snapshot of all aspects of the aseptic environment can be obtained.
Monitoring of specific zones may be carried out more frequently. It is preferred that all surfaces within
Grade A are tested to evaluate the level of their microbial cleanliness on a frequent basis according to
the scale of aseptic operations.
Grade Zone Test Frequency

A No less than weekly and with every fill.
B N/A Monthly
C/D Monthly or Quarterly (differing by location)
11.5.5. Method of Sampling [RODAC Plates]

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The following specimen procedure for use of contact plates may be adopted.
2. Assemble the plates required and ensure that the correct information is written on the base of each
plate (the part containing the media) using indelible ink. Do not mark the lid of the plate as there is a
possibility of lids coming off and being replaced on the incorrect sample plate. The following details may
be marked on each plate or recorded separately:
operator who collected sample
date and time of day sample taken
area/location of sample
position/sample number
3. Transfer the contact plates into the area to be tested, as described in an appropriate transfer
procedure.
4. Enter the area to be tested by the appropriate procedure, if required.
5. Before testing any surface ensure that it is dry.
6. When sampling surfaces with contact plates, remove the lid and place the agar surface in maximum
contact with the sampling site for 10 seconds by applying a constant force spread evenly over the whole
contact plate without twisting or sliding and avoiding the creation of bubbles.
7. After contact with the sample surface, remove and replace the lid, avoiding any unnecessary hand
movement near or over the agar surface.
8. Clean the surface tested with a suitable disinfectant to remove any possible traces of residual agar.
9. Repeat as required for each sampling location.
10. After all the samples are taken gather all sample plates together, remove from the area/room and
incubate as directed.
11. Complete and enclose the necessary documentation.
11.5.6. Action Levels [RODAC Plates]

Fill Room background <1 5
B Sterile Storage <1 5
D Hallways 25 50
11.6. Swabs

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Surfaces may become contaminated in a number of ways e.g. microorganisms settling out from the
environment or from the direct touch by an operator. One of the objectives of surface sampling is to
determine the efficiency of routine cleaning procedures in removing contamination. Therefore, sampling
should be performed before and after cleaning to determine the effectiveness of the cleaning
procedure. Swabs can be used to quantitatively analyze the level of contamination by using a template
of known dimensions e.g. a flat square aluminum plate (or stainless steel) with a 5 cm x 5 cm hole cut
out may be used. The inner edges of the hole should be beveled (no right angles) to allow complete
sampling of the surface. The area inside the template is swabbed thus giving a reproducible sample size.
For uneven surfaces or awkward areas e.g. door handles, curved surfaces, where contact plates or the
swab template cannot be used, swabs can still be used to give a qualitative analysis of the cleaning
procedure i.e. what type of microorganism might be present.
It must be noted that there are limitations with the use of swabs to validate cleaning, whether
quantitative or qualitative, the method is dependant on the ability of the swabbing technique to remove
microorganisms present on the sample surface.
11.6.2. Equipment [Swabs]
There are a variety of swab types available from different manufacturers. The type of swab used will
affect the sampling method and plating out technique employed. Ordinary dry sterile swabs which
require to be moistened with sterile 0.9% Sodium Chloride (NaCl) Injection BP before use are one type
and the transport swab is another type. The latter is a swab with a plastic shaft and a synthetic tip - with
Amies medium, which is capable of sustaining microbial growth until the swab can be plated out.
Of the two example products here, the second one is more common.
[EXAMPLE PRODUCT #1]

BBL™ CultureSwab™
Liquid Stuart Medium, Liquid Amies Medium, Cary-Blair Medium and Sterile Swabs
BBL™ CultureSwab™ devices are sterile ready-to-use systems intended for the collection, transport and
preservation of clinical specimens for bacteriological examination.
One of the routine procedures in the diagnosis of bacterial infections involves the collection and safe
transportation of a clinical specimen from the patient to the laboratory. This can be accomplished using
the BBL CultureSwab collection and transport device. Each BBL CultureSwab unit is comprised of a

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sterile peel pouch containing a rayon-tipped swab applicator used to collect the sample and a tube
containing transport medium into which the swab applicator is placed after sampling.
The BBL CultureSwab transport media (Amies Liquid Medium, Liquid Stuart Medium and Cary-Blair
Transport Medium) are non-nutritious, buffered with phosphate and provide a reduced environment
due to their formulation with sodium thioglycollate.1 Organisms in the sample material are protected
from drying by moisture in the transport medium. The medium is designed to maintain the viability of
organisms during transit to the laboratory. BBL CultureSwab pouches are made of a plastic film which
retards the penetration of atmospheric air into the product.
BBL CultureSwab media are contained in a constricted (venturi) tube. Nitrogen gas is flushed into the
transport tube during the media filling and capping process. During final packaging of the swab and
tube, air is removed from the pouch by vacuum and nitrogen gas is flushed inside.
[EXAMPLE PRODUCT #2]

Sterile Pack Swab BD
The BD Sterile Pack Swab is a ready-to-use sterile swab in a pre-filled tube of rinse solution for surface
and equipment sampling.
Product Summary: «
Surface and equipment sampling is routine practice in hospitals, pharmaceutical and food industries as
part of infection control, environmental monitoring and hygiene control programs. Collection of samples
from the same area before and after cleaning and/or treatment with a disinfectant permits the
evaluation of the efficacy of the sanitary procedures. Each Sterile Pack Swab unit is ready-to-use and
comprised of a peel open pouch containing a sampling swab in a tube with approximately 10 mL rinse
solution. The Sterile Pack Swab is designed to sample a variety of surface textures and equipment with
the advantage of sampling hard-to-reach areas such as equipment crevices and inside pipe work. The
pre-filled rinse solution is a general-purpose isotonic solution with neutralizing agents for the
maintenance of microorganisms and the neutralization of disinfectants. The double pouch design is
gamma-irradiated.
The swab tip is made of Dacron fiber on a polypropylene applicator within a polypropylene flat
bottomed tube. The rinse solution is a balanced isotonic solution to maintain organism viability. Four
neutralizers are incorporated to inactivate a variety of disinfectants and antiseptic chemicals. Sodium
thiosulfate inactivates working concentrations of stabilized blends of hydrogen peroxide and peracetic
acid (SporeKlenz). Lecithin inactivates quaternary ammonium compounds; and polysorbate 80

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inactivates substituted phenolic disinfectants. Engley and Dey reference the neutralizer sodium
thioglycollate for inactivation of mercurials. Sodium pyruvate helps recover injured microorganisms.
Because the doubled- bagged product is subjected to a sterilizing dose of gamma radiation, the contents
inside the outer bag are sterile. This allows the outer bag to be aseptically removed and its contents to
be brought into an environmentally controlled area without introducing contamination. Since this
product has been sterilized after packaging, the presence of microbial growth after sampling and
incubation can be relied upon to represent the presence of environmental contaminants and not pre-
existing microorganisms in the product that may have been introduce during manufacture.
11.6.3. Sample Locations [Swabs]
Sample locations should be selected on the basis of risk to the product during the aseptic preparation
process, in accordance with the table below. For example, the sampling location in a grade A zone for
high risk aseptic operations will be the filling zone work surface of the clean air device. The number of
swabs taken will be related to the size and grade of the area under test. Swabs are intended for
“difficult to access” locations, and places where RODAC media should not be used (inside of
lyophilizer).
11.6.4. Frequency of Sampling [Swabs]
Grade Zone Location Test Frequency

A Fill Machine Surfaces With every fill.
Hood Surfaces With every use.
Interior of Lyophilizer With every use.
B N/A Monthly or Quarterly
C/D Work Surfaces Not typically used in these grades.
11.6.5. Method of Sampling [Swabs]
The following method is suggested for using swabs.

Equipment for carrying out test
• Sterile transport swabs
• 10 ml syringe and needle
• 10 ml ampoule of 0.9% Sodium Chloride (NaCl) Injection BP
• Aluminum plate (with 5 cm x 5 cm hole)
• Disinfectant spray/sterile swabs (for cleaning plate/surfaces tested)

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• Settle plates for plating out swabs
Procedure for taking a swab sample using transport swabs

1. Assemble the swabs required and ensure that the correct information is written on the label of each
tube. The following details should be indicated:
operator who collected swab sample
date and time of collection
area/site of collection
position/sample number
2. Before testing any surface ensure that it is dry.
3. Where used, disinfect the aluminum plate and allow disinfectant to evaporate before placing on the
surface to be tested, if required.
4. Assemble the needle and 10 ml syringe. Open the sterile ampoule of 0.9% NaCl Injection and draw up
the contents.
5. Push a small amount of liquid (about 0.5 ml) onto the surface to be tested. If the surface to be tested
is uneven or not horizontal then it is permissible to apply a small amount of liquid from the syringe
directly onto the swab. Do not allow the needle tip to come into contact with the swab.
6. Open the swab package and take out the swab. Keep transport tube in hand.
7. Wipe the swab over the sample area in close parallel streaks, using firm even pressure and rotating
the swab between fingers to maximize sample pick-up. The swab should be held at a 30° angle to the
contact surface. With the same swab, repeat this process at right angles to the first streaks to ensure
that the entire sample area is swabbed.
8. Replace swab into transport tube ensuring that swab comes into contact with transport medium by
pushing down hard.
9. Remove the aluminum plate from the surface. Clean the surface tested and the aluminum plate,
where used, with a suitable disinfectant e.g. isopropyl alcohol 70% to remove any possible
contamination.
10. Repeat as required for each sampling position.
11. A negative control swab should be carried out at the end of sampling by applying a small amount of
liquid from the syringe directly onto the swab and immediately placed into the transport tube. A positive
control swab should be carried out in a similar way by sampling a known “dirty" surface.
12. Gather all swab samples together and manipulate further as directed below under “plating out
method for swabs”.
Note: The operational conditions at the time of testing should be recorded. In quantitative testing if a
nonstandard size of area is sampled an approximation of the area tested should be recorded so that the
colony count per approximate area obtained can be related to an action level.
Plating out method for swabs

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Examine the plates for contamination prior to use. A standard streaking out method should be used
when plating out the swab. The method should ensure that the swab is rotated as it is run over the
surface of the media to ensure that any microorganisms recovered from the surface sample are
deposited onto the surface of the plate.
Note: Swabs should be plated out as soon as possible after sampling. If there is a delay the swab should
be stored at room temperature.
11.6.6. Action Levels [Swabs]

Grade
A Hood or Fill Area <1 <1
Lyophilizer <1 <1
B Not Applicable <1 5
C Fill and Support Rooms 5 25
D Non Sterile Rooms 25 50

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Bethesda: 1 April 2009

Pharmaceutical Development Section

Validation Master Plan

First Draft (For review) 1 April 2009

Document: Revision Identification: Original Origination

Facility: Location: This Revision

Title: _____________________________ Title: _____________________________

1. Introduction, Policy and Scope

1.1. Project Description

401 Vial Washer

9 Holding Period for Sterile Items Prior to Use

1.3. Definition of the Term Validation

1.4. Living Document

2. Concept of Qualification / Validation

of operations. The Operational Qualification is normally conducted following the successful

Direct Product Contact Equipment

Indirect Product Contact Equipment

2.2.Validation Life Cycle & Revalidation

2.3.System Impact Assessment – What processes must be validated?

2.4.Elements of a Process Validation Protocol

2.5.Elements of an Equipment Validation Protocol

2.5.1. User Requirement Statement

2.5.4. Qualifications run by the Equipment Vendor

2.5.5. Standard Operating Procedures for Equipment

2.5.6. Calibrated Measurement Equipment

2.5.7. Documentation Use

2.5.8. Installation Qualification

2.5.9. Operational Qualification

• Utilities and services have been installed and qualified.

2.5.10. Performance Qualification

2.5.11. Product Process Validation

2.6.Documentation Format for Qualification Programs

2.6.2. Operational Qualification Protocol Format

2.6.3. Performance Qualification Protocol Format

2.6.4. SOP on SOPs

3. Organizational Responsibilities for Validation

3.1. Validation Department Personnel

3.2. Operations Department Personnel

• Review and approve the VMP

3.3. Quality Control Personnel

4. Description of the Area and Equipment Covered by this VMP ........................................................... 28

4.3.1.6. Nitrogen System .......................................................................................................... 34

Description of the Area and Equipment Covered by this VMP

4.1.2. Tablet Area Description

4.1.3. Capsule Manufacture

4.1.4.1. Filling Capacity

4.1.4.2. Tablet Capacity

Press Type 8(4x4) 8 13 16 19

4.1.4.3. Capsule Capacity

Model Zanasi/IMA 6/12 F model 6F

4.1.4.4. Fluid Bed Dryer

4.1.4.5. Lyophilization Capacity

Capacity Height Body OD

4.1.5. Packaging Capacity

4.2.2. Floor Plan – Product flow

4.2.3. Floor Plan – Waste flow

4.2.4. Floor Plan – Room Classification

4.2.5. Floor Plan – Room Pressurization

4.2.6. Routine Operation – Description

4.3.1.1. Chilled Water

4.3.1.2. Plant steam

4.3.1.4. Sanitary waste

4.3.1.5. Compressed air

Title: _ Title: _