This is volume 7 of the Environmental Monitoring Handbook series. Each volume of this series discusses different aspects of environmental monitoring. One of the first topics discussed in this volume is the topic of cleanrooms and ways to prevent contamination. Subject matter experts Dr. Tim Sandle, Jan Eudy, Jim Polarine, John Lindsay and others describe new and/or better ways to do things. The second section of the book describes various environmental monitoring techniques and methods and includes informative chapters from Ryan Burke, Allan Marinelli, Dr. Andrew Sage, Tim Cser, Sean Toler, Claire Fritz Briglia and others. The third section of the book deals with changes to standards. Memarzadeh and DeBerandinis explain the changes that occurred for ANSI Standard 29.14 and how this is applicable in pharmaceutical environments and the last section of this volume talks about new technologies and aids that can be used in evaluating these methods with chapters from Dr. Sage and Dr. JP Jiang. There is a wealth of useful information that you can use in establishing, maintaining and updating your environmental monitoring program!

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2. ENVIRONMENTAL
MONITORING
A Comprehensive Handbook
JEANNE MOLDENHAUER
Editor
Volume Seven
PDA
Bethesda, MD, USA
DHI Publishing, LLC
River Grove, IL, USA
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3. 10 9 8 7 6 5 4 3 2 1
ISBN: 1-933722-85-1
Copyright © 2015 J. Moldenhauer
All rights reserved.
All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored
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otherwise, is made by reference to any such marks in the book. The authors have made every
effort to provide accurate citations. If there are any omissions, please contact the publisher.
While every effort has been made by the publisher and the authors to ensure the accuracy
of the information expressed in this book, the organization accepts no responsibility for errors
or omissions. The views expressed in this book are those of the editors and authors and may not
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4. CONTENTS
Introduction xiii
CLEANROOMS AND PREVENTING CONTAMINATION
1 INTRODUCTION TO CLEANROOM DESIGN
AND OPERATION 3
Tim Sandle
Introduction 3
Cleanrooms 3
Clean air devices 5
Cleanrooms and Contamination Control 6
Risk assessment 10
Cleanroom Classification 10
Particle counting 16
Cleanroom Operational Parameters 17
Air filtration 18
Air-patterns and air-movement 19
Airflow velocity and direction 20
Air changes 20
Clean up times 21
Separation concept and positive pressure differentials 21
Other factors 21
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5. Cleanroom Design 22
Energy saving 22
Cleaning and Disinfection 22
Environmental Monitoring 23
Auditing 24
Conclusion 25
References 26
About the Author 27
2 PROTECTING CLEANROOMS FROM CONTAMINATION 29
Jeanne Moldenhauer
Introduction 29
Types of Particulates 29
Size of Particulates 30
Recommended Practices 31
Cleanroom Standards 32
Cleanroom Design 33
Cleaning and Disinfection 34
Preventative Maintenance 34
Good Housekeeping Practices 35
Clean in Place, Steam in Place, Sterilize in Place Systems 35
Humans are the Biggest Source of Contamination 35
Cleanroom Garments 37
Personnel Behaviors 37
Cleanroom Personnel and Smoking 38
Conclusion 39
References 39
About the Author 41
3 CLEANROOM GOWNING AND GOWNING CERTIFICATION 43
Jan Eudy
Introduction 43
A Short Course in Particulation 43
Facility Design 44
Human Sourced Contamination 46
Behavior 47
Cleanroom Garments 48
Fabric and Findings 49
Type, Style and Construction of Garments 50
Garment Cleaning Process 51
Testing of Cleanroom Garments 52
Sterile Cleanroom Garments 55
Gamma Subcontractor Qualification 56
Routine Monitoring of Gamma Radiation 56
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6. Certificate of Sterility 56
Gowning for the Cleanroom 57
Sterile Gowning Procedure for ISO Class 3–5/Class A Cleanrooms 57
Donning 57
Doffing 58
Non-Sterile Gowning Procedure for ISO Class 3–5 Cleanrooms 58
Donning 58
Doffing 59
Gowning Procedure for Frocks in ISO Class 6–8 Cleanrooms 59
Donning 59
Doffing 60
Gowning Certification 60
Summary 61
References 61
About the Author 63
4 CLEANING AND DISINFECTING CLEANROOMS 65
Jim Polarine, Elaine Sartain, and Carol Bartnett
Introduction 65
Disinfectant Selection 66
References 72
About the Authors 73
5 STERIPLEX,A NEW SILVER-BASED DISINFECTANT
— NON-CORROSIVE, NON-TOXIC, SPORICIDAL:
DISINFECTANT EFFICACY EVALUATIONS 75
John M. Lindsay
Summary 75
Introduction 76
Materials and Methods 77
Neutralizer broth 77
Procedures 78
Results 83
In Situ Evaluations 85
Additional tests 86
Conclusions and Discussion 88
References 89
About the Author 90
Attachment 1 Project Protocol 91
Attachment 2 Enumeration of Challenge Microorganism Suspensions 100
Attachment 3 Preparation of Steriplex Neutralizer Broth 102
Attachment 4 Data Sheet for Steriplex Challenge 103
Attachment 5 Pour Plate or Spread Plate Method for
Total Aerobic Microbial Count 113
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7. 6 OZONE GAS: DECONTAMINATION, SANITIZATION
AND STERILIZATION 115
Brian G. Hubka and Jeanne Moldenhauer
Introduction 115
What is Ozone and how does it Work? 116
Ozone generators 116
Using ozone 116
Ozone to inactivate microorganisms 117
Safety concerns using ozone 117
Concerns using ozone technology 118
Ozone benefits 118
Compatibility of Ozone with Other Materials 119
Using Ozone as a Sterilant 120
Microbiological Aspects of Ozone Use 121
Pharmaceutical Applications for Gaseous Ozone Usage 123
Conclusion 123
References 123
About the Authors 125
7 STERILIZATION AND DECONTAMINATION WITH
NITROGEN DIOXIDE GAS 127
David Opie
Introduction 127
NO2 Physical Properties 127
Safety 128
Mechanism of Action 130
Response of microorganisms to NO2 131
Inactivation kinetics 132
Cycle Parameters 133
NO2 Cycle Descriptions 137
Vacuum NO2 cycles 138
Non-vacuum NO2 cycles 139
Material Compatibility 139
Chemical compatibility 140
Biocompatibility 141
Sterilizability on materials 142
Process Accessories 143
Biological indicators 143
Process challenge devices 144
Chemical indicators 144
NO2 concentration detectors 145
Humidity detector 145
Applications for the NO2 Technology 145
NO2 application to prefilled syringes and drug containers 145
Terminal sterilization of medical devices 147
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8. Decontamination of filling lines and isolators 148
Summary 149
References 149
About the Author 151
ENVIRONMENTAL MONITORING TECHNIQUES
AND METHODS
8 SETTING UP AN ENVIRONMENTAL MONITORING
PROGRAM FOR CONTROLLED ASEPTIC CLEANROOM
ENVIRONMENTS 155
Ryan Burke and Francis McAteer
Introduction 155
Design and Assessment of the Cleanroom Facility 157
How does a cleanroom accomplish control? 157
Determining Appropriate Sampling Methodologies 162
Environmental monitoring general overview 162
How the air is sampled in a cleanroom 163
How surfaces are sampled in a cleanroom 163
How personnel are monitored in a cleanroom 163
Monitoring and Sampling Techniques 164
Generating SOPs and Protocols 169
Sampling Plan Design 170
Data Management 172
Establishing Alert and Action Limits 173
Corrective Action and Preventative Action Process 176
Importance of Maintaining and Conducting the Environmental
Monitoring Program Properly 177
Conclusion 177
References 178
About the Authors 180
9 QUALITY RISK MANAGEMENT SAMPLING PLAN —
A VALIDATION ENGINEERING PERSPECTIVE 181
Allan Marinelli
Introduction 181
Purified Water System 182
Quality Risk Management 182
Rationale for Determining the PQ Sample Points 183
Conclusion 201
References 201
About the Author 202
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9. 10 PRACTICAL APPROACH ON THE EFFICIENCY OF
MICROBIOLOGICAL METHODS FOR ENVIRONMENTAL
MONITORING 203
Anne-Grit Klees and Reinder Hedderich
Fertility: Growth Promotion Test 205
Growth Promotion Tests in the Presence of Inhibitory Substances 208
Overall Efficiency of Surface Sampling Methods 212
References 216
About the Authors 218
11 OPTIMIZING TIME-TO-RESULTS FOR ENVIRONMENTAL
MONITORING APPLICATIONS FOR GROWTH-BASED
RAPID MICROBIOLOGY METHODS 219
Andrew Sage
Time-to-results and Incubation Profiles of Environmental
Monitoring Testing Applications 219
TTR: An Important Requirement for Growth Based Rapid
Micro Methods 220
Requirements for Performing a TTR Determination on
Growth-based RRMs 220
Experimental Strategy for Optimizing Incubation and Setting TTR 221
Determining Optimal Incubation Profile and TTR for EM Testing
on the Growth Direct System™: An Automated, Growth-based RRM 221
Optimal Incubation Regime for Detection of Bacteria and Mold
Species on the Growth Direct System 224
TTR Determination for Serial Incubation on the Growth Direct
System and Verification Testing 228
A Single Temperature Alternate Incubation Strategy may Replace
Serial Incubation 230
Summary 232
References 232
About the Author 234
12 REAL-TIME RISK ASSESSMENT CONTROL STRATEGY 235
Sean Toler
Real-time Risk Assessment Shaped from Quality by Design 235
QbD Risk Assessments to Develop RTRA 237
QbD Design Space Impact on RTRA 238
QbD Control Strategy of RTRA 239
Continuous Improvement from RTRA 241
Implementing the RTRA 244
Microbiologist Development for RTRA 252
Cultural Improvement from RTRA 253
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10. Summary 254
References 254
About the Author 256
13 ENVIRONMENTAL MONITORING OF COMPRESSED GASES 257
Tim Cser and Anne Connors
Introduction 257
Regulations and Guidance 257
Uses of Compressed Gas 259
Compressed Gas Testing 259
Bacteria Yeast and Mold 259
ISO 8573-4 Test Methods for Solid Particle Content 260
ISO 8573-7 Test Method for Viable Microbiological Contaminant Content 261
Alternative Viable Testing Methods 262
MAS-100 CG EX 262
RCS with CG adapter 262
Results Interpretation and Data Analysis 263
Investigations and Corrective Actions 263
References 264
About the Authors 264
14 ENVIRONMENTAL MONITORING FOR STERILITY
TEST ISOLATORS 265
Claire Fritz Briglia
History of Isolators for Sterility Testing 265
The Regulations 268
Installation Qualification/Operation Qualification 269
Performance Qualification 269
Why Establish an Environmental Monitoring Program
for a Sterility Testing Isolator? 270
What to Monitor and What are the Methods? 271
Non-viable Air 271
Viable Air 272
Surface Monitoring 272
Glove Monitoring and Testing 274
Monitoring Outside the Isolator 275
When? 275
Action and Alert Levels 276
Culture Media for Environmental Monitoring 276
Validation 277
Conclusion 278
References 278
About the Author 279
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11. CHANGES TO STANDARDS
15 TESTING AND VERIFICATION OF VENTILATION
SYSTEMS FOR BIOSAFETY LEVEL 3 (BSL-3) AND
ANIMAL BIOSAFETY LEVEL 3 (ABSL-3) FACILITIES 283
Farhad Memardazeh and Louis DiBerardinis
Introduction 283
Definitions 285
What is Laboratory Biocontainment? 286
History of ANSI/ASSE Z9.14 288
What is ANSI/ASSE Z9.14? 290
Findings of the Gap and Needs Analysis 291
National US and local laws, standards and guidelines 292
National initiatives outside the US 295
International initiatives 296
ANSI/ASSE Z9.14: The Standard 297
How to read the standard 297
Purpose 297
Scope 297
Definitions 298
Applicability and conformance 298
Procedure for entities using the ANSI/ASSE Z9.14 Standard 299
Overview 300
Roles and responsibilities 302
Risk assessment 303
Guidelines for Implementing Testing and Performance-Verification 305
Verification 305
Documentation overview 305
Visual inspection 307
Testing methodologies 307
References 318
About the Authors 322
NEW TECHNOLOGIES FOR ENVIRONMENTAL
MONITORING AND AIDS TO EVALUATING THEM
16 STRESSED MICROBIAL POPULATIONS: REAL-WORLD
CHALLENGE FOR VALIDATION OF NEW APPLICATIONS 325
Andrew Sage
Importance of Use of Stressed Microbial Populations
for Validation Testing in Industry 325
Microbes in the Environment Exist in a Stressed State 326
Stress and Injury Response in Microbes 327
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12. Preparation of Stressed Populations for Method Validation 329
Case study 1 Preparation of heat-injured spores
of A. brasiliensis spores 330
Case study 2 Preparation of an acid-stressed population
of Pseudomonas aeruginosa 332
Using the Growth Direct™ System to Measure the Effect of Stressed
Induced Injury on the Growth Dynamics of Microbial Populations 332
Stressed Populations Exhibit Attenuated Detection as Exemplified
by Heat-stressed A. Brasiliensis, and Acid-injured P. Aeruginosa 336
The Physiological State of a Test Organism can Influence
its Response to Stress 337
Conclusion 338
References 339
About the Author 340
17 REAL TIME MICROBIAL ANALYZER FOR WATER 341
J.P. Jiang
Technical Description of RMS-Water Analyzer 342
Validation Test of RMS-W Using BioBall® 344
BioBall quantitative samples 344
Validation Test Data and Method Descriptions 346
Ruggedness test procedure 350
Miscellaneous suggestions for conducting feasibility
study of RMS-W instrument 351
Application Example: Online Water Monitoring Using RMS-W 352
Example 1: RMS-W online monitoring of a high purity
water line 352
Example 2: RMS-W online monitoring at a mineral water
bottling plant 353
Application Example: Surface Sampling and Cleaning Validation 355
Example 1: surface sampling using RMS-W and Quantiswab® 355
Example 2: cleaning validation using RMS-W and Quantiswab® 357
Summary 358
References 358
About the Author 359
Appendix 361
Index 371
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14. INTRODUCTION
This is volume 7 of the Environmental Monitoring Handbook series. Each volume of this series
discusses different aspects of environmental monitoring. The appendix describes the various
topics and authors that can be found in volumes 1 through 6 of this series.
One of the first topics discussed in this volume is the topic of cleanrooms and ways to
prevent contamination. This is important — as an industry and we are changing the paradigm
from reacting to contamination to a mode of preventing contamination. Dr. Tim Sandle
provides a great guide to cleanrooms. This is followed by a discussion of methods to protect
cleanrooms from contamination. Jan Eudy describes the methods to be used for gowning and
gowning qualification to protect from contamination. Jim Polarine and his group have written
a discussion on the cleaning and disinfection of cleanrooms. As we are always looking for new
and/or better ways to do things, John Lindsay has a chapter on the qualification of a new
disinfectant. This disinfectant is effective with approximately one minute of contact time! Two
new methods of contamination control: Brian Hubka discuss the use of gaseous/vaporized
ozone and David Opie discusses the use of nitrous oxide.
The second section of the book describes various environmental monitoring techniques
and methods. As you have seen in various earlier volumes, Burke has a chapter on how to set
up an environmental monitoring program. It is useful to look at the various different methods
individuals have used to customize the program that will work best for your site. Another hot
topic in today’s compliance arena is sampling plans. Marinelli discusses the methods to use in
setting up a sampling plan for your site. Klees and Hedderich provide useful information on the
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15. efficiency of microbiological methods that are used for environmental monitoring. Many times
wrong assumptions are made about the environmental data, because we don’t understand the
efficiency of these methods. When using growth-based methods, a common complaint is the
long time it takes to get results. Dr. Sage has identified how to optimize the time you need to
get results using these types of methods. In recent years several companies have evaluated
and/or implemented newer environmental monitoring methods that are based upon the use of
viability methods. One on-going concern with these types of methods is how you could do an
investigation should you have a contamination event. Sean Toler describes a program
implemented that conducts real time risk assessment along with a viability-based methodology.
Tim Cser explains the conduct of environmental monitoring for compressed gases and Claire
Fritz-Briglia provides information on the conduct of environmental monitoring to support
sterility testing in an isolator environment.
The third section of the book deals with changes to standards. Memarzadeh and
DiBerardinis explain the changes that occurred for ANSI Standard 29.14 and how this is
applicable in pharmaceutical environments.
The last section of this volume talks about new technologies and aids that can be used in
evaluating these methods. Dr. Sage provides a defined program for creating and using stressed
organisms in the validation of alternative microbiological methods. Dr. J.P. Jiang explains how
the IbioScan works. This technology detects the presence of viable biological particles in water
monitoring in real time.
There is a wealth of useful information that you can use in establishing, maintaining and
updating your environmental monitoring program!
Environmental Monitoringxiv
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