www.pda.

org/bookstore

01FrontMatter:FrontMatter

27/10/14

08:56

Page 1

Cleanroom
Microbiology

Tim Sandle and R. Vijayakumar

PDA
Bethesda, MD, USA

DHI Publishing, LLC

River Grove, IL, USA

www.pda.org/bookstore

01FrontMatter:FrontMatter

27/10/14

08:56

Page 2

10 9 8 7 6 5 4 3 2 1
ISBN: 1-933722-00-0
Copyright 2014 Tim Sandle and R. Vijayakumar
All rights reserved.
All rights reserved. This book is protected by copyright. No part of it may
be reproduced, stored in a retrieval system or transmitted in any means,
electronic, mechanical, photocopying, recording, or otherwise, without
written permission from the publisher. Printed in the United States of
America.
Where a product trademark, registration mark, or other protected
mark is made in the text, ownership of the mark remains with the lawful
owner of the mark. No claim, intentional or otherwise, is made by
reference to any such marks in the book. Websites cited are current at the
time of publication. The author has 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
represent those of either Davis Healthcare International or the PDA, its
officers, or directors.

This book is printed on sustainable resource paper approved by the Forest Stewardship
Council. The printer, Gasch Printing, is a member of the Green Press Initiative and all paper
used is from SFI (Sustainable Forest Initiative) certified mills.
PDA Global Headquarters
Bethesda Towers, Suite 150
4350 East-West Highway
Bethesda, MD 20814
United States
www.pda.org/bookstore
001-301-986-0293

Davis Healthcare International Publishing, LLC

2636 West Street
River Grove
IL 60171
United States
www.DHIBooks.com

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page iii

(Black plate)

CONTENTS

INTRODUCTION TO
CLEANROOM MICROBIOLOGY

CLEANROOMS, CLEAN ZONES AND

CLEANROOM TECHNOLOGY

11

Introduction
Cleanrooms and Clean Air Devices
Isolators, glove boxes and hatches
Contamination Control
Particles
Particle counting
Classification of Cleanrooms and Clean Air Devices
The classification process
Designing Cleanrooms
Assessing Cleanroom Performance
Airflows
Air change rates
Clean-up times (recovery tests)
Pressure differentials
HEPA filters

11
12
14
15
20
22
25
31
32
34
34
35
36
37
38

iii

www.pda.org/bookstore

02Contents:Layout 1

iv

27/10/14

09:32

Page iv

(Black plate)

Cleanroom Microbiology
Leak testing of HEPA filters
Temperature, humidity, lighting and room design
On-going or routine monitoring
Conclusion
References

40
40
41
42
42

GLOBAL STANDARDS FOR CLEANROOMS

AND cGMPs

47

Introduction
History of Cleanroom Standards
Current Cleanroom Standards
ISO standards
EU GMP Annex 1
WHO GMP
FDA
Regulatory Differences
The Pharmaceutical Inspection Co-operation Scheme (PIC/S)
Pharmacopeia
cGMP
Conclusion
References

47
49
52
52
69
71
71
73
74
75
76
77
77

MICROBIOLOGICAL ENVIRONMENTAL
MONITORING OF CLEANROOMS:
PART 1: CONTAMINATION SOURCES
AND METHODS

83

Introduction
Microbiological Contamination: Sources and Point of Monitoring
Water
Air
Surface monitoring
Personnel monitoring
Media for Microbiological Environmental Monitoring
Culture media and incubation conditions
Methodology for Non-Viable Particles
Rapid and Alternative Methods
Conclusion
References

83
84
85
87
93
97
99
99
102
105
107
108

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page v

(Black plate)

Contents
5

MICROBIOLOGICAL ENVIRONMENTAL
MONITORING OF CLEANROOMS:
PART 2: CONSTRUCTING AN
ENVIRONMENTAL MONITORING PROGRAM
Introduction
Limitations of Environmental Monitoring
The Environmental Monitoring Program
Sample Types
Sampling Locations
Frequencies of Monitoring
Time of Monitoring
Sampling Responsibilities
Special Types of Monitoring
Monitoring During Construction and Start-up
Alert and Action Levels
Data Review
Conclusion
References

CLEANROOM MICROFLORA

115
115
117
118
119
121
124
126
127
127
128
129
135
137
138

141

Introduction
Growth Requirements of Microorganisms
Consumables (concentration of organic and
inorganic compounds)
The influence of environmental factors on growth
Measurement of Microbial Growth
The Pattern of Growth
Strategies for Microbial Survival in the Cleanroom Environment
Formation of bacterial spores
Changes in enzymatic and protein profiles
Changes in cell size
Biofilm formation
Effect of Survival Strategy in In-Vitro Results
Types of Microorganisms Found in Cleanrooms and Their Origins
Personnel
Air
Surfaces (equipment, walls and ceilings)
Raw materials
Water
Summary
Details of Cleanroom Microflora
Bacteria in Cleanroom Environments

141
142
143
144
147
148
149
150
151
151
152
154
155
155
156
157
158
159
160
161
163

www.pda.org/bookstore

02Contents:Layout 1

vi

27/10/14

09:32

Page vi

(Black plate)

Cleanroom Microbiology
Gram-positive Cocci
Micrococcus
Staphylococcus
Gram-positive Rods
Bacillus spp. spore-forming Bacillus
Paenibacillus sp.
Corynebacterium spp.
Clostridia
Gram-negative Bacteria
Pseudomonas aeruginosa
Pseudomonas oryzihabitans
Fungi in Cleanroom Environment
Viruses
Survey of Worldwide Available Reports
Conclusion
References

CHARACTERIZATION AND IDENTIFICATION

OF CLEANROOM MICROFLORA
Introduction
Cleanroom Microflora
Microbial Taxonomy
Identification Methods
Phenotypic Methods
Colony and cell morphology
Staining
Gram stain
Spore stain
Fungal staining
Differential media
Biochemical profiling
Biochemical tests
Selecting methods
Serological methods
Automated phenotypic systems
Fatty acid
Mass spectrometry
Flow cytometry
Genotypic Methods
Molecular diagnosis of microbial contamination
nucleic acid amplification techniques (PCR technology)
Multiplex PCR

163
163
165
167
167
168
168
168
169
169
170
171
175
176
176
179

189
189
192
193
193
197
198
199
200
203
204
204
205
207
207
215
215
217
219
219
220
221
223

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page vii

(Black plate)

Contents

vii

RNA and DNA homology

Validation of Microbiological Methods
USP chapter <1113>
EP Chapter 5.1.6
Comparing the Pharmacopeia
Conclusion
References

225
226
227
228
231
233
234

EVALUATION AND INTERPRETATION OF

ENVIRONMENTAL MONITORING DATA

243

Introduction
Distribution of Microorganisms
How do these Observations Apply to Control Charts?
Review
Trend Analysis and Results Reporting
Reporting
Displaying data
Histograms
Control Charts
Types of Control Chart
The Cumulative Sum Chart
Worked example: Grade B/ISO Class 7 cleanroom
The Shewhart Chart
Worked example: Grade C cleanroom
Using MS Excel
Alternative Approaches
Discussion
Computerized Systems
Conclusion

243
244
246
252
252
253
254
254
258
261
262
264
267
271
274
275
276
276
278

THE HUMAN MICROBIOME PROJECT AND

THE IMPLICATIONS FOR CLEANROOM
MICROBIOLOGY
Introduction
The Microbiome
The Human Microbiome Project
The Skin Microbiome
Implications for Pharmaceuticals
Implications for Cleanroom Microbiology
Environmental monitoring
Sterility testing

283
283
285
286
289
295
296
296
299

www.pda.org/bookstore

02Contents:Layout 1

viii

27/10/14

09:32

Page viii

(Black plate)

Cleanroom Microbiology
Disinfectant efficacy
Culture media testing
Cleanroom gowning
Microbial data deviations
Conclusion

300
301
301
303
303

10 IMPLICATIONS OF PHARMACEUTICAL
MICROBIAL CONTAMINATION

311

Introduction
Recall Classification of Pharmaceutical Products
Recall definition
Recall classification by FDA
Microbial Spoilage of Pharmaceutical Products
Review of Pharmaceutical Product Recalls
Recalls due to Bacterial Contamination
Burkholderia cepacia
Pseudomonas aeruginosa
Unusual Gram-negative bacterial contamination
Ralstonia pickettii
Elizabethkingia meningoseptica
Acinetobacter baumannii
Achromobacter xylosoxidans
Stenotrophomonas maltophilia
Gram-positive bacterial contamination
Unidentified bacterial contamination
Product Recalls due to Fungal Contamination
Risk Assessment of Objectionable Microorganisms
Conclusion
References

311
314
314
315
316
318
322
323
326
329
331
332
332
332
333
334
336
338
341
347
348

11 CONTAMINATION CONTROL IN CLEANROOMS:

PART 1: DISINFECTION PRACTICES IN
PHARMACEUTICAL CLEANROOMS
Introduction
Terminology
Types of Disinfectants
Alcohols
Aldehydes
Amphoterics
Phenolics
Quaternary Ammonium Compounds

357
357
358
360
361
364
366
366
367

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page ix

(Black plate)

Contents

ix

Oxidizing disinfectants
Halogens
Hydrogen peroxide and peroxygen compounds
Peracetic acid
Cleaning Methods in Cleanrooms
Cleaning
Disinfection
Cleaning Methods
Vacuuming
Wet wiping
Mopping
Tacky rollers
Developing and Implementing a Disinfection Program
SOP for cleaning and disinfection procedures
SOP for disinfectants in-house verification
Determination of test method
Frequency of cleaning and disinfection
Selection of disinfectants
Rotation of disinfectants
Qualification of Disinfectants
Disinfectant qualification methods
Classification of disinfection tests
First stage tests in vitro tests
Practical tests
In-use tests and field trials
Susceptibility Breakpoints of Disinfectants Against
Cleanroom Isolates
Antibacterial efficacy of common biocides
Antifungal efficacy of common biocides
Safety and Environmental Factors
Disinfectants for Fumigation and the Sanitization of Isolators
Conclusion
References

12 CONTAMINATION CONTROL IN CLEANROOMS:

PART 2: CLEANROOM DESIGN FACTORS
Introduction
Contamination Control and Airflow
Heating Ventilation and Air Conditioning
High Efficiency Particulate Air Filters
Standards for HEPA filters
Positioning HEPA filters

367
368
368
369
369
370
370
370
371
371
374
375
376
376
377
378
378
379
382
384
385
386
388
396
397
399
400
402
404
405
407
408

415
415
418
420
422
423
424

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page x

(Black plate)

Cleanroom Microbiology
Modification of HEPA filters
Function of Air Handling Systems and HEPA Filters
for Contamination Control
Air filtration
Air dilution
Air movement
Pressure differentials
Ongoing Assessment of Cleanrooms
Annual verification
Air velocity/supply
Air changes
Clean-up times
HEPA filter installation leak tests
Pressure differentials
Temperature and humidity
Microenvironments or minienvironments
Isolators
Energy Efficiency
Modular Cleanrooms
Conclusion
References

13 CONTAMINATION CONTROL IN CLEANROOMS:

PART 3: THE PERSONNEL FACTOR
Introduction
Staff Gowning and Behavior
Importance of clothing
Cleanroom garments
Cleanroom Personnel Behavior and General Rules
Basic Training
Cleanroom Entry
Changing into cleanroom garments
Personnel access
Materials access
Environmental Monitoring
Strategic questions
Background detail questions
Analytical questions
The Microbiology Laboratory
Relationship to the quality unit
Microbiology laboratory facility design

425
426
426
426
427
429
429
430
435
435
437
438
439
440
440
441
444
445
446
446

451
451
452
452
452
458
465
466
467
469
470
471
471
472
472
473
473
474

www.pda.org/bookstore

02Contents:Layout 1

27/10/14

09:32

Page xi

(Black plate)

Contents

xi

The Importance of Understanding Basic Microbiology

and Personal Hygiene
Personal Hygiene
Effect of improper hand wash
Hand Hygiene Technique in Cleanrooms
Hand washing protocol
Hand rubbing/glove sanitization
Conclusion
References

14 CONTAMINATION CONTROL IN CLEANROOMS:

PART 4: MEDIA SIMULATION TRIALS

476
479
480
482
482
484
485
486

489

Introduction
Aseptic Process Simulations
Media Fill Program
Filling speed
Filling volume
Design matrix
Process Parameters
Interventions Simulated During Test
Sterile air
Media Selection
Media Fill Frequency
Batch Size
Incubation Conditions
Acceptance Criteria
Failure Investigation and Corrective Action
Invalidation of a Media Fill
Conclusion
References

489
490
493
494
495
495
496
498
499
499
500
501
503
504
507
508
510
510

15 AUDITING CLEANROOMS FOR

THE CONTAMINATION CONTROL
PERSPECTIVE

513

Introduction
Quality Audits
The audit approach
Risk assessment
Auditors
Undertaking audits

513
514
515
516
517
518

www.pda.org/bookstore

02Contents:Layout 1

xii

27/10/14

09:32

Page xii

(Black plate)

Cleanroom Microbiology
Review of audits
Regulatory trends
Auditing Cleanrooms
Space and design
Process flow
Certification
Physical operations
Cleanroom systems
Personnel
Equipment
Change control
Environmental monitoring
Microbiological data
Aseptic filling
Cleaning and disinfection
Auditing cleanroom documentation
Electronic capture of data
Conclusion
References

16 CLEANROOMS AND MICROBIOLOGY:

THE IMPORTANCE OF RISK ASSESSMENT
Introduction
Risks, Risk Assessment and Cleanrooms
The value of risk assessment
Concluding Remarks
References

Index

522
522
523
523
525
525
525
529
530
531
534
535
540
540
543
546
548
549
549

553
553
553
562
563
566

569

www.pda.org/bookstore