www.pda.

org/bookstore
Front Matter:Front Matter copy 3/9/12 12:28 Page 1

Biofilm Control
in Drug
Manufacturing

Lucia Clontz and Carmen M. Wagner

Editors

PDA
Bethesda, MD, USA
DHI Publishing, LLC
River Grove, IL, USA

www.pda.org/bookstore
Front Matter:Front Matter copy 3/9/12 12:28 Page 2

10 9 8 7 6 5 4 3 2 1

ISBN: 1-933722-64-9
Copyright © 2012 Lucia Clontz and Carmen M. Wagner
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. 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 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 Davis Healthcare International Publishing, LLC

4350 East West Highway 2636 West Street
Suite 200 River Grove
Bethesda, MD 20814 IL 60171
United States United States
www.pda.org/bookstore www.DHIBooks.com
001-301-986-0293

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page iii (Black plate)

CONTENTS

PREFACE xv

1 THE CONTAMINATION CONTROL BY DESIGN

(CCbD©) MODEL — A PROACTIVE APPROACH
TO CONTAMINATION CONTROL 1
Carmen M.Wagner
Control of Microbial Contamination in Drug Manufacturing 1
GMP regulations and contamination control 3
Development of the (CCbD©) concept 5
Relationship between CCbD©, QbD, RM and PAT 7
Quality management and QbD 10
Quality risk management 11
Process Analytical Technology (PAT) 15
The Contamination Control by Design (CCbD©) Model 16
Composition of the CCbD© model 17
CCbD© model elements 18
Knowledge and understanding 19
Risk assessment 21
Quality systems and documentation 22
Process Analytical Technology 22

iii

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page iv (Black plate)

iv Biofilm Control in Drug Manufacturing

Controlling the sources of contamination 23
Personnel controls 23
Raw materials controls 24
Process controls 24
Facility and clean utilities controls 25
Equipment controls 25
Monitoring and continuous improvement 26
Conclusion 27
References and Additional Reading 29
About the Author 32
Appendix: Regulatory Framework 33

2 BIOFILM BASICS 37
Paul J. Sturman
Introduction 37
Biofilm Formation, Growth and Maturity 40
Conditioning film deposition 42
Initial cell migration to surface and attachment 42
Permanent cell attachment and growth 43
Biofilm maturation and detachment 43
Biofilm Processes 44
Concentration gradients 45
Microbial community development 45
Genetic transfer in biofilms 46
Quorum sensing in biofilms 47
The Biofilm Matrix 47
Extracellular matrix composition 48
Biofilm morphology 49
The viscoelastic nature of biofilms 49
Detecting and Measuring Biofilms 50
Planktonic organism sampling 51
Biofilm sampling 51
Indirect assessment of biofilm 52
Why Biofilms are Hard to Kill 52
Slow penetration of antimicrobials 53
Altered microenvironments 54
Stress response and antimicrobial treatment 54
Persister cells 55
Biofilm Control Strategies 56
Physical control strategies 56
Chemical control strategies 57
Oxidizing biocides 58

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page v (Black plate)

Contents v
Non-oxidizing biocides 59
Biofilm removal agents 59
Quorum-sensing blockers 60
Anti-biofilm coatings/imbedded antimicrobials 61
Biological control strategies 61
Nutrient removal 63
Application method and design strategies 63
Conclusion 64
References 65
About the Author 72

3 MICROBIAL CONTAMINATION CONTROL

CONSIDERATIONS IN BIOPHARMACEUTICAL
PRODUCTION 75
Lucia Clontz
Introduction 75
Microbial Contamination Control 77
Biofilm Formation and Dispersion 80
Initial cell adhesion to surfaces 81
Surface roughness 81
Surface chemical composition 82
Biofilm formation in liquid environments 84
Biofilm dispersion 85
Equipment — Contamination Risks and Control 86
Equipment design 86
Sanitary design 87
Cleaning and decontamination 88
Cleaning validation 88
Cleaning verification 90
Microbial cleaning specifications 91
Sanitization and sterilization 91
Equipment soft parts 92
Bioprocessing equipment 94
UF/DF systems 96
Chromatography systems 101
Contamination control strategies 104
Single-use technology 107
Biofilm Impact on Products and Business 109
Conclusion 112
References 113
About the Author 116

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page vi (Black plate)

vi Biofilm Control in Drug Manufacturing

4 ESTABLISHING MICROBIAL CONTAMINATION

CONTROL IN DRUG MANUFACTURING
THROUGH RISK MANAGEMENT 117
Karen Zink McCullough
Introduction 117
Risk Management 119
Product Design and Development: Establishing Control
Measures for Bioburden and Endotoxin 121
The importance of product requirements in the
initial assignment of microbial limits 121
Microbiological risk assessment 125
Risk assessment tools 125
Method development 128
Building design 130
Process development 136
Validation/Qualification of Systems/Processes:
Demonstrating Control of Bioburden and Endotoxin 136
Facility cleaning and disinfection 137
Equipment cleaning and sanitization 138
Facility air and compressed gases 139
Personnel 140
Water systems 142
Process Validation: Critical Control Points for Bioburden
and Endotoxin Control 143
In-process limits or levels 147
Routine Monitoring: Demonstrating Continued
Control of Bioburden and Endotoxin 148
Operators 148
Monitoring of air and surfaces 149
Testing of raw materials and in-process samples 151
Data trending 153
Responding to nonconformances and excursions 155
Post Production Assessment of Microbiological Control 156
Summary 157
References 157
About the Author 163

5 CONTROLLING BIOFILMS IN DRUG

MANUFACTURING EQUIPMENT 165
Mark Pasmore and Robert Fry
Introduction 165
Biofilm Development 166

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page vii (Black plate)

Contents vii
Ranking Biofilm Control 168
Manufacturing Systems 169
Systems and equipment design 171
Water distribution systems 171
Pharmaceutical/biopharmaceutical processing and
filling equipment 172
Mixing/filling tanks 172
Pumps 175
Piping 176
Filters (microbial retentive) 177
Particulate filters/screens 178
Fill nozzles 178
Additional design considerations 179
Equipment cleanability 179
Materials of construction 180
Facility air quality 181
Single-use/disposable systems and components 181
Biofilm Prevention — System/Equipment Maintenance 182
Biofilm Control — Microbial Detection 184
Current methods for microbial monitoring 185
Biofilm detection 185
Biofilm Control — Equipment Cleaning 188
Cleaning/sanitization program 190
Trough/automatic parts washer cleaning 192
Storage of cleaned equipment 193
Equipment passivation 194
Effects of cleaning on materials 194
Biofilm Control — Differences/Similarities in Non-Sterile,
Terminally Sterilized, and Aseptic Manufacturing 195
Biofilm Control — Validation 197
Biofilm Remediation 198
Conclusion 200
References 200
About the Authors 203

6 INVESTIGATING THE LINK BETWEEN

CONTAMINATION IN THE MANUFACTURING
ENVIRONMENT AND PATIENT SAFETY 205
Mark Hunter, Michelle Luebke and Mark Pasmore
Patient 1 206
Patient 2 206
Infection 207

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page viii (Black plate)

viii Biofilm Control in Drug Manufacturing

Sepsis 209
Signs and symptoms of infection/sepsis 210
Patient risk factors 214
Infection/sepsis — the perfect storm 216
Investigation — the causal continuum 218
Intrinsic continuum 220
Extrinsic continuum 221
Manufacturing contamination impacts on patients 225
Microbial Contamination in the Manufacturing Environment 225
Non-sterile, terminally sterilized, aseptically filled 227
Proliferation 228
Ingress 229
Biofilms as an elevated risk 230
Conclusions 231
References 232
About the Authors 236

7 DETECTION, PREVENTION AND CONTROL

OF BIOFILMS IN PHARMACEUTICAL
WATER SYSTEMS 239
Teri C. Soli
Introduction 239
Biofilm Impact 241
Biofilm Detection 242
Purpose 242
Historical approach/confusion 243
New understanding of biofilm 244
Planktonic proportionality 244
Localized effects 245
Impracticality of surface monitoring in pharmaceutical
water systems 246
Flow interruption, system opening, and new
component installation 246
Non-similar flow and topography 248
Process control and ideal planktonic sampling 250
Challenges of planktonic sampling permanent connections 252
Speciation and objectionable microorganisms 253
Biofilm Prevention 258
Water system design 258
Materials of construction 259
Tank level controls, vent filters, and rupture disks 261

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page ix (Black plate)

Contents ix
Loops 261
Dead legs 263
Point-of-use valves 265
Point-of-use connectors 267
Sampling ports 268
Flush water disposal 270
Water system unit operations 271
In-line ultraviolet units 271
TOC reducers 274
Ozone destruction 275
Micro-retentive filters 276
Ultrafilters and nanofilters 278
Polishing deionizers 279
In-line UV unit in combination with downstream filter 279
Biofilm Control — Water System Maintenance 280
Multi-media or sand filters 281
Water softeners 281
Activated carbon beds 282
Ultraviolet lights 283
Filters 285
Reverse osmosis units 286
Deionizers 288
Twin bed deionizers 290
Mixed bed deionizers 290
Electro-deionizers 291
Ultrafilters 292
In-line/on-line instrumentation 293
Biofilm Control — Routine Sanitization 293
Hot water sanitization 293
Chemical sanitization 296
Heating/“pasteurization” 297
Continuous tank ozonation 298
Conclusion 298
References 299
About the Author 301

8 BIOFILM DETECTION AND DATA MANAGEMENT 303

Teri C. Soli
Introduction 303
Biofilm Detection 304
Conventional cultivation-dependent methods 306
Metabolic condition of bacteria 307

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page x (Black plate)

x Biofilm Control in Drug Manufacturing

Effect of growth medium composition 308
Alternative microbiological methods 315
Cultivation-dependent RMM methods 316
Non-cultivation dependent methods 319
Efficacy Monitoring 322
The value of data trending 323
Alert levels, action levels, and specifications 323
Control/Remediation 325
Root cause investigation 326
CAPA 328
Conclusion 330
References 331
About the Author 332

9 EARLY DETECTION AND PREVENTION

OF BIOFILMS IN PROCESS EQUIPMENT 333
Mark Fornalik
Introduction 333
Fouling/Biofouling: A Primer 335
Detecting and Characterizing Biofilms 339
Removable coupons: fouling cell technology 339
Assessment tools: surface analytical science 341
Detecting wall fouling 344
Preventing and Remediating Biofilms 345
Mechanical Cleaning: Water Flush Optimization 346
Chemical Cleaning: Optimizing CIP 347
Choice of chemical cleaner 348
Chemical cleaner concentration and temperature 351
Surface tension and incorporating surfactants
into chemical cleaning 353
Case Studies by the Author 354
High-purity water biofouling 355
Industrial salt water system 357
Bioprocessing operation 360
Large-scale brewery 362
Conclusion 365
References 369
About the Author 372

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page xi (Black plate)

Contents xi

10 SANITIZATION APPROACHES FOR

BIOFILM CONTROL 373
Teri C. Soli
Introduction 373
Heat Sanitization 374
Hot water 374
Pure steam 377
Chemical Sanitization 380
Sanitizer efficacy 382
Oxidizing Sanitizers 383
Chemical concentration 385
Chlorine 386
Alkaline solution 387
Partially neutralized solution 388
Precautions and issues 388
Non-chlorine halogens 389
Ozone 389
Precautions and issues 391
Peracetic acid 393
Precautions and issues 394
Hydrogen peroxide 394
Precautions and issues 395
Chlorine dioxide 396
Precautions and issues 397
Proprietary Mixtures 397
Hydrogen peroxide + peracetic acid + acetic acid 397
Precautions and issues 398
Alkaline peroxide + quat + chelator 399
Precautions and issues 400
Cleanroom disinfectants 401
Quaternary ammonium compounds (quats) 401
Phenolics 402
Formaldehyde 402
Glutaraldehyde 402
Acids and caustics 402
Passivating/derouging agents 403
Overview of Keys to Effective Sanitization 403
Sanitize frequently 404
Kill and remove biofilm 404
Use an effective sanitizer 404
Use an effective procedure 405
Minimize recolonization 405

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page xii (Black plate)

xii Biofilm Control in Drug Manufacturing

Conclusion 406
References 406
About the Author 408

11 DISINFECTANT EFFICACY STUDIES

USING BIOFILM REACTORS 409
Lucia Clontz
Introduction 409
Flow-through Biofilm Reactors 412
The Flow Cell System 413
The Drip Flow Biofilm Reactor 413
The Rotating Disc Reactor 414
The Annular Reactor 415
The CDC Biofilm Reactor 416
Static Biofilm Reactors 417
Colony biofilm assay 418
Microtiter plate biofilm assay 418
The MBEC™ assay 419
The HTP Assay™ 420
Factors that Impact Disinfectant Efficacy Studies 421
Choosing a Biofilm Reactor 423
Testing Disinfectants Using the CDC Biofilm Reactor 424
Setting up the CDC Biofilm Reactor 425
Inoculating the CDC Biofilm Reactor 426
Operating the CDC Biofilm Reactor 426
Exposure of biofilm to disinfectants 427
Harvesting and enumerating biofilm cells 428
Testing Disinfectants Using Microtiter Plates 429
Biofilm assay 430
Crystal violet reporter assay 430
Viable cell density determination 431
Biofilm inhibition determination 432
Disinfectant efficacy determination 432
Determination of biofilm removal 432
Determination of antimicrobial efficacy 433
Disinfectant Efficacy Evaluation 433
Method Validation 434
Conclusion 435
References 436
About the Author 438

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page xiii (Black plate)

Contents xiii

CONCLUSION 441
Lucia Clontz and Carmen M.Wagner
Future Trends in Biofilm Prevention, Control, and Remediation 442
References 444
About the Authors 445

APPENDIX 447
Organizations with Special Focus on Biofilm
Education/Discussion 447
Companies that Provide Consulting, Products, and/or
Services in the Area of Biofilms 450

Glossary 457

Index 469

www.pda.org/bookstore
Contents:Layout 1 11/9/12 19:59 Page xiv (Black plate)

www.pda.org/bookstore
Preface:Layout 1 11/9/12 15:46 Page xv (Black plate)

PREFACE

Microbial contamination of pharmaceutical products continues to be

frequently cited as a reason for product recalls, manufacturing
problems, and product rejection. Microbial contamination is costly to
manage, time consuming to investigate and often difficult to
determine root cause. The frequency of such problems, as well as the
cost and investment of time and resources required to address them,
support the need to develop and implement better microbial control
programs that can more effectively prevent contamination from
occurring.

As you will learn in various chapters in this book, biofilms are a

preferred way of life for microbes and they are not only responsible
for contamination in pharmaceutical and biopharmaceutical
processing but also for many human pathogeneses including chronic
wounds, device-associated infections, and various nosocomial
infections and diseases in patients with compromised immune
systems (Wilson, 2001). In fact, as reported in Infection Research
(Kerksiek, 2008) estimates of the frequency of infections caused by
biofilms (bacterial and/or fungal) lie between 65% (Centers for
Disease Control and Prevention/CDC) and 80% (National Institute

xv

www.pda.org/bookstore
Preface:Layout 1 11/9/12 15:46 Page xvi (Black plate)

xvi Biofilm Control in Drug Manufacturing

of Health). In drug manufacturing, biofilms are known to impact

equipment and systems, such as those used for water and product
purification (e.g., filtration and chromatography). In addition,
biofilms may lead to product adulteration, which can potentially
cause patient harm. Therefore, understanding how microorganisms
develop into biofilm cells, and the mechanisms of antimicrobial
resistance present in biofilm communities, is critical for successfully
controlling microbial contamination in drug manufacturing.

Over the years, many studies have been carried out in the areas
of biofilm eradication, remediation, and prevention. However, the
realization that biofilms are ubiquitous in nature and extremely
difficult to destroy resulted in a shift in paradigm — from microbial
eradication and remediation to biofilm prevention. Chapters 1, 4, 6,
and 7 specifically address the importance of risk assessment and a
proactive, rather than reactive, microbial control program.

On a molecular level, studies have been performed to better

understand biofilm development. For example, in an article
published in the Journal of Bacteriology (Branda et al., 2004), studies
with Bacillus subtilis provided valuable information regarding the
genetic control of biofilm formation, and showed that spore
formation, long thought to be a process involving only single cells,
is actually closely associated with the development of multicellular
communities. There is particular interest on gene expression
responsible for production of extra polymeric substances (EPS) and
cell signaling/quorum sensing, both associated with biofilm
establishment and growth (Hansen et al., 2007; Gonzales and
Keshavan, 2006). Other studies include research with cyclic-di-
GMP (c-di-GMP), which is a key player in the decision between
motile planktonic and sessile microbial lifestyle. One study
evaluated a chemosensory system that regulates biofilm formation
through modulation of cyclic diguanylate levels (Hickman et al.,
2005). Data suggested that increased c-di-GMP levels enhance
biofilm formation, while decreased c-di-GMP levels prevent
initiation of biofilm development. Researchers are also attempting
to gain greater understanding of “persister” cells, which are
specialized dormant cells proposed as the main reason for the
refractory nature of biofilm infections (Keren et al., 2004). Chapters
2, 3, and 8 explain in further detail some of these ideas.

www.pda.org/bookstore
Preface:Layout 1 11/9/12 15:46 Page xvii (Black plate)

Preface xvii

In terms of contamination control, the literature describes

approaches to biofilm removal and prevention that range from the
use of enzymes that can remove most, but not all, types of biofilms
(Orgaz et al., 2007) to antimicrobial surfaces (Danese, 2002).
Chapters 10 and 11 address sanitization approaches and studies in
biofilm remediation and control.

Given the fact that the first step in biofilm formation is actually
the ability to attach to a surface, and not microcolony formation or
production of an EPS matrix, it is critical to investigate biological
pathways used by bacteria to detect the presence of surfaces.
Although the initial contact with a surface is not necessarily
regulated and may happen by chance, there is evidence that
formation of a stable cell-surface interaction may be genetically
regulated (Stanley and Lazazzera, 2004). This is an important
finding, because if surface attachment can be controlled, or even
prevented, then all the other pathways to complete biofilm
formation would be negated. Chapters 2, 5, and 9 help shed some
light on these topics.

It is clear that biofilms are responsible for a number of serious

contamination challenges in pharmaceutical/biopharmaceutical
manufacturing, and the cause of several medical problems including
chronic infections and medical device related infections. These
complex and highly structured communities are difficult to prevent
and even more difficult to eradicate. This book reviews the status of
biofilm knowledge, management and control in pharmaceuticals,
and is the first attempt to gather this type of information.

We close the book with an Appendix on resources that can be

used by readers as a guide to general information about biofilms
and related products.

A special note of gratitude goes to the authors who worked

diligently to help make this book an excellent source of information
for the pharmaceutical professional working in this area. Special
thanks as well to our colleagues, associates, vendors and others
who encouraged us and provided direct or indirect input to our
work. We also would like to recognize Amy Davis — without her
constant guidance and encouragement, this book would not be

www.pda.org/bookstore
Preface:Layout 1 11/9/12 15:46 Page xviii (Black plate)

xviii Biofilm Control in Drug Manufacturing

possible. Last but not least, we would like to thank our spouses, Jim
Clontz and Brian Wagner, for their constant support, endurance
and patience as we deprived them of our company many nights,
holidays and weekends, to complete the book in a timely manner.
Thank you, all.

www.pda.org/bookstore
Lucia Clontz and Carmen M. Wagner

REFERENCES
Branda, S.S., González-Pastor, J.E., Dervyn, E, Ehrlich, S.D., Losick,
R., Kolter R. (2004) Genes involved in formation of structured
multicellular communities by Bacillus subtilis. Journal of
Bacteriology 186(12), 3970–3979.

Danese, P.N. (2002) Antibiofilm approaches: Prevention of catheter

colonization. Chemistry & Biology 9, 873–880.

Gonzáles, J.E. and Keshavan, N.D. (2006) Messing with bacterial

quorum sensing. Microbiology and Molecular Biology Reviews
70(4), 859–875.

Hansen, S.K., Rainey, P.B., Haagensen, J.A., Molin, S. (2007)

Evolution of species interactions in a biofilm community.
Nature 445, 533–536.

Hickman, J.W., Trifea, D.F., Harwood, C.S. (2005) A chemosensory

system that regulates biofilm formation through modulation
cyclic diguanylate levels. Proceedings of the National Academy of
Sciences USA 102: 14422–14427.

Keren, I., Shah, D., Spoering, A., Kaldalu, N., Lewis, K. (2004)
Specialized persister cells and the mechanism of multidrug
tolerance in Escherichia coli. Journal of Bacteriology 186(24),
8172–8180.