STERILITY

TESTING OF

PHARMACEUTICAL
PRODUCTS

Tim Sandle
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CONTENTS

Introduction
1

xiii

STERILITY

Introduction
Sterility
Microorganisms and Microbial Growth
Types of microorganisms
Sterilization and microbial kill
Microbial viability
Microbial Contamination of Pharmaceutical Products
Air
People
Equipment and materials
Water
Sterile Products
Sterilization
Sterility Assurance
The Sterility Test
Conclusion
References

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iv
2

Sterility Testing of Pharmaceutical Products

HISTORY OF THE STERILITY TEST

37

Introduction
Origins of Sterility Test
1930s
1940s
1950s
1960s
1970s
1980s
1990s
2000s
The Future
Conclusion
References

37
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48
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53
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STERILITY TEST REGULATIONS

61

Introduction
Pharmacopeia: USP, Ph.Eur., and JP
International Pharmacopeia
Code of Federal Regulations
FDA
EU GMP
PIC/S
TGA
ICH
Conclusion
References

61
62
64
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STERILITY TEST: THE PHARMACOPEIA

CULTURE BASED METHOD

79

Introduction
Sterility Test Methods
Test Controls
Environmental Monitoring
Sample Incubation and Reading
Sterility Test Failures

79
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84
85
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89

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Contents

Culture Media
Fluid Thioglycollate Medium
Soy-bean casein digest medium
Test Environment
Method Validation (Method Suitability)
Validation method
Re-validation
GMP Requirements
Cellular Products
Radiopharmaceuticals
Gene Therapy Products
Products Containing Preservatives
Combination Products
Conclusion
References

90
90
91
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93
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96
97
97
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100
101
102
103
104

PRACTICAL APPROACHES TO
STERILITY TESTING

107

Introduction
Validation
Practical Approaches for Difficult Products
Membrane filtration
Type of membrane filter
Pump speed
Direct inoculation
Type of neutralizing agent
Dilution
Turbid samples
For both techniques
Summary
Some Examples of Testing Special Products
Antibiotics
Oily samples
Other ointments and creams
Anti-cancer treatments and radiopharmaceuticals
Implants
Sterile aerosols
Cell lines

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vi

Sterility Testing of Pharmaceutical Products

Fibrin sealant
124
Dressings
124
Solid articles that cannot be tested using the standard methods 124
Transfusion or infusion assemblies
125
Conclusion
125
References
126

CONTROLLED ENVIRONMENTS FOR

STERILITY TESTING
Introduction
UDAF Devices in a Conventional Cleanroom
The unidirectional airflow cabinet
The cleanroom
Gowning procedure
Conducting the test
Isolators for Sterility Testing
Basic concept
Sanitization of isolators
Pre-cycle disinfection
Preparing the isolator or gassing port
Sanitization cycle
Monitoring the sanitization cycle
Validation of isolators
Maintenance of isolators
Background environment
Environmental Monitoring of the Sterility Test
Sample types
Culture media
The monitoring plan
Microbial identification
Particle counting
The cleanroom
Conclusion
References

129
129
130
131
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134
134
135
135
136
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138
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146
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149
150
152
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Contents
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vii

CULTURE MEDIA FOR THE STERILITY TEST

161

Introduction
Media used for the Test for Sterility
Soybean-Casein Digest Medium (Trypticase Soy Broth/
Tryptone Soy Broth
Fluid Thioglycollate Medium
Rinse fluids
Environmental monitoring
Media used for Environmental Monitoring
Manufacture of Culture Media
Quality Control of Culture Media
Test Panel of Microorganisms
Preparation of Microorganisms
Microbial Challenge Level
Testing Culture Media
Post-inoculation Incubation
Purity of Cultures
Sterility Test of Culture Media
Frequency of Testing
Expiry Time Testing
Storage of Media
Conclusion
References

161
163

RAPID AND ALTERNATIVE METHODS FOR

STERILITY TESTING
Introduction
Rapid and Alternative Methods
Rapid Sterility Test Methods
Growth based technologies
Viability based
Cellular component methods
Overview
Pharmacopeial Guidance
Validation Requirements
Qualitative tests for presence/absence
Quantitative tests for enumeration
Implementing a Change

163
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166
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166
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169
170
172
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174
175
175
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177
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viii

Sterility Testing of Pharmaceutical Products

Barriers to Implementation
Conclusion
References

199
200
200

INVESTIGATING STERILITY TEST FAILURES

Introduction
Sterility Test Failure Investigations
Immediate Actions
Conducting Investigations
Test laboratory
Manufacturing
Sterility Test and Process Area Link
Genotypic microbial identification
Re-testing the Finished Product Batch
Concluding Sterility Test Failure Investigations
Product Impact Assessment
Conclusion
References
Appendix: Sterility Test Failure Investigation Checklist

10 AUDITING STERILITY TESTING

205
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208
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213
217
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225

233

Introduction
The Quality Audit
Types of Quality Audits
The Audit Process
Approaching Audits
Non-compliances
Audits as Preparations for Regulatory Inspections
The Use of Risk Assessment for the Audit Process
Auditing Sterility Testing: The Focal Points
Strategy for microbiological control
Sample receipt
Product sample testing and the sterility test
Sterility test facility
Media control and testing
Incubator, refrigerator and freezer control and monitoring

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240
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243
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Contents

ix

Culture collections
Environmental monitoring
Isolate identification, Gram stain techniques and
ID system validation
Autoclave control
Sterilization, cleaning and disinfection validation efficacy
Microbiological training of laboratory and production staff
Atypical results and OOS procedures
Other aspects of the laboratory
Quality systems
Change control
Annual product reviews
Reporting the Audit
Conclusion
References
Appendix: A Sterility Testing Audit Checklist

11 EVALUATING THE STERILITY TEST

Introduction
The Limitations of the Sterility Test
The Statistical Limitations of Sampling for the Sterility Test
Example 1: Selecting specific contaminated containers
from a batch
Example 2: Chance of passing a sterility test where
contamination is present in a batch
Example 3: Further cases examining the difficulty of
detecting non-sterility in a batch
The Inherent Weaknesses of the Pharmacopeia Culture
Based Method
Suitability of culture media
Physiological state of contaminating microorganisms
Viable but non-culturable microorganisms
Methodological limitations of the culture based method
Time to result
Environmental Control
Post-manufacturing Contamination: Container Seals and GDP
Conclusion
References

251
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259
261

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285

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Sterility Testing of Pharmaceutical Products

12 ALTERNATIVES TO STERILITY TESTING:

PARAMETRIC RELEASE
Introduction
Regulatory Documents for Parametric Release
US documents
EU and global documents
ISO standards
Sterile Product Suitability for Parametric Release
Risk Assessing the Manufacturing Process
Risk based application for parametric release
Ongoing risk assessment of the process
Requirements for Parametric Release
Quality Management Systems
Other quality documents
Validation
Biological indicator validation
Purity
Population
D-value
Process Definitions
Product Definition and Design Control
Equipment, Facility Design and Qualification
Process Steps
Cleaning and decontamination
Inspection and assembly
Packaging, including the materials and techniques
Sterilizer loading
Sterilization cycle
Storage and distribution
Record keeping
Microbial Control
Environmental control monitoring
Bioburden and endotoxin control
Pre-sterilization product bioburden
Personnel Training
Change Control
Release Procedure
Conclusion
References

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301
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Contents

xi

13 FINAL PRODUCT RELEASE

323

Introduction
Final Products
Terminally Sterilized and Aseptically Filled Products
The Microbiological Assessment
Review of processing
Materials
In-process bioburden
Sterilization
Equipment records
Cleanroom and facility operations
Water bioburden and endotoxin
Sterile filtration
Environmental monitoring
Quality control tests
Final product testing
Endotoxin and pyrogenicity
The sterility test
Conclusion
References

323
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327
328
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329
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331
331
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14 THE STERILITY TEST AND STERILE

PRODUCTS:THOUGHTS ON DEFINITION
AND APPLICATION

343

Introduction
Sterility: A Return to the Definition
The Manufacture of Sterile Products
The Sterility Test
Assessing the Sterility of Sterile Products
Conclusion

343
344
345
347
352
353

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