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CleanroomMicrobiology

Tim Sandle and R. Vijayakumar

PDABethesda, MD, USA

DHI Publishing, LLCRiver Grove, IL, USA

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ISBN: 1-933722-00-0Copyright © 2014 Tim Sandle and R. VijayakumarAll rights reserved.

All rights reserved. This book is protected by copyright. No part of it maybe reproduced, stored in a retrieval system or transmitted in any means,electronic, mechanical, photocopying, recording, or otherwise, withoutwritten permission from the publisher. Printed in the United States ofAmerica.

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While every effort has been made by the publisher and the authors toensure the accuracy of the information expressed in this book, theorganization accepts no responsibility for errors or omissions. The viewsexpressed in this book are those of the editors and authors and may notrepresent those of either Davis Healthcare International or the PDA, itsofficers, or directors.

This book is printed on sustainable resource paper approved by the Forest StewardshipCouncil. The printer, Gasch Printing, is a member of the Green Press Initiative and all paperused is from SFI (Sustainable Forest Initiative) certified mills.

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CONTENTS

1 INTRODUCTIONTOCLEANROOM MICROBIOLOGY 1

2 CLEANROOMS, CLEAN ZONES ANDCLEANROOM TECHNOLOGY 11Introduction 11Cleanrooms and Clean Air Devices 12

Isolators, glove boxes and hatches 14Contamination Control 15Particles 20

Particle counting 22Classification of Cleanrooms and Clean Air Devices 25

The classification process 31Designing Cleanrooms 32Assessing Cleanroom Performance 34

Airflows 34Air change rates 35Clean-up times (recovery tests) 36Pressure differentials 37HEPA filters 38

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Leak testing of HEPA filters 40Temperature, humidity, lighting and room design 40On-going or routine monitoring 41

Conclusion 42References 42

3 GLOBAL STANDARDS FOR CLEANROOMSAND cGMPs 47Introduction 47History of Cleanroom Standards 49Current Cleanroom Standards 52

ISO standards 52EU GMP Annex 1 69WHOGMP 71FDA 71

Regulatory Differences 73The Pharmaceutical Inspection Co-operation Scheme (PIC/S) 74Pharmacopeia 75cGMP 76Conclusion 77References 77

4 MICROBIOLOGICAL ENVIRONMENTALMONITORING OF CLEANROOMS:PART 1: CONTAMINATION SOURCESAND METHODS 83Introduction 83Microbiological Contamination: Sources and Point of Monitoring 84

Water 85Air 87Surface monitoring 93Personnel monitoring 97

Media for Microbiological Environmental Monitoring 99Culture media and incubation conditions 99

Methodology for Non-Viable Particles 102Rapid and Alternative Methods 105Conclusion 107References 108

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5 MICROBIOLOGICAL ENVIRONMENTALMONITORING OF CLEANROOMS:PART 2: CONSTRUCTING ANENVIRONMENTAL MONITORING PROGRAM 115Introduction 115Limitations of Environmental Monitoring 117The Environmental Monitoring Program 118Sample Types 119Sampling Locations 121Frequencies of Monitoring 124Time of Monitoring 126Sampling Responsibilities 127Special Types of Monitoring 127Monitoring During Construction and Start-up 128Alert and Action Levels 129Data Review 135Conclusion 137References 138

6 CLEANROOM MICROFLORA 141Introduction 141Growth Requirements of Microorganisms 142

Consumables (concentration of organic andinorganic compounds) 143The influence of environmental factors on growth 144

Measurement of Microbial Growth 147The Pattern of Growth 148Strategies for Microbial Survival in the Cleanroom Environment 149

Formation of bacterial spores 150Changes in enzymatic and protein profiles 151Changes in cell size 151Biofilm formation 152

Effect of Survival Strategy in In-Vitro Results 154Types of Microorganisms Found in Cleanrooms and Their Origins 155

Personnel 155Air 156Surfaces (equipment, walls and ceilings) 157Raw materials 158Water 159Summary 160

Details of Cleanroom Microflora 161Bacteria in Cleanroom Environments 163

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Gram-positive Cocci 163Micrococcus 163Staphylococcus 165

Gram-positive Rods 167Bacillus spp. — spore-forming Bacillus 167Paenibacillus sp. 168Corynebacterium spp. 168Clostridia 168

Gram-negative Bacteria 169Pseudomonas aeruginosa 169Pseudomonas oryzihabitans 170

Fungi in Cleanroom Environment 171Viruses 175Survey ofWorldwide Available Reports 176Conclusion 176References 179

7 CHARACTERIZATION AND IDENTIFICATIONOF CLEANROOM MICROFLORA 189Introduction 189Cleanroom Microflora 192Microbial Taxonomy 193Identification Methods 193Phenotypic Methods 197

Colony and cell morphology 198Staining 199Gram stain 200Spore stain 203Fungal staining 204Differential media 204Biochemical profiling 205Biochemical tests 207Selecting methods 207Serological methods 215Automated phenotypic systems 215Fatty acid 217Mass spectrometry 219Flow cytometry 219

Genotypic Methods 220Molecular diagnosis of microbial contamination —nucleic acid amplification techniques (PCR technology) 221Multiplex PCR 223

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RNA and DNA homology 225Validation of Microbiological Methods 226

USP chapter <1113> 227EP Chapter 5.1.6 228

Comparing the Pharmacopeia 231Conclusion 233References 234

8 EVALUATION AND INTERPRETATION OFENVIRONMENTAL MONITORING DATA 243Introduction 243Distribution of Microorganisms 244How do these Observations Apply to Control Charts? 246Review 252Trend Analysis and Results Reporting 252

Reporting 253Displaying data 254

Histograms 254Control Charts 258Types of Control Chart 261The Cumulative Sum Chart 262

Worked example: Grade B/ISO Class 7 cleanroom 264The Shewhart Chart 267

Worked example: Grade C cleanroom 271Using MS Excel 274Alternative Approaches 275Discussion 276Computerized Systems 276Conclusion 278

9 THE HUMAN MICROBIOME PROJECT ANDTHE IMPLICATIONS FOR CLEANROOMMICROBIOLOGY 283Introduction 283The Microbiome 285The Human Microbiome Project 286The Skin Microbiome 289Implications for Pharmaceuticals 295Implications for Cleanroom Microbiology 296

Environmental monitoring 296Sterility testing 299

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Disinfectant efficacy 300Culture media testing 301Cleanroom gowning 301Microbial data deviations 303

Conclusion 303

10 IMPLICATIONS OF PHARMACEUTICALMICROBIAL CONTAMINATION 311Introduction 311Recall Classification of Pharmaceutical Products 314

Recall definition 314Recall classification by FDA 315

Microbial Spoilage of Pharmaceutical Products 316Review of Pharmaceutical Product Recalls 318Recalls due to Bacterial Contamination 322

Burkholderia cepacia 323Pseudomonas aeruginosa 326Unusual Gram-negative bacterial contamination 329Ralstonia pickettii 331Elizabethkingia meningoseptica 332Acinetobacter baumannii 332Achromobacter xylosoxidans 332Stenotrophomonas maltophilia 333Gram-positive bacterial contamination 334Unidentified bacterial contamination 336

Product Recalls due to Fungal Contamination 338Risk Assessment of Objectionable Microorganisms 341Conclusion 347References 348

11 CONTAMINATION CONTROL IN CLEANROOMS:PART 1: DISINFECTION PRACTICES INPHARMACEUTICAL CLEANROOMS 357Introduction 357Terminology 358Types of Disinfectants 360

Alcohols 361Aldehydes 364Amphoterics 366Phenolics 366Quaternary Ammonium Compounds 367

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Oxidizing disinfectants 367Halogens 368Hydrogen peroxide and peroxygen compounds 368Peracetic acid 369

Cleaning Methods in Cleanrooms 369Cleaning 370Disinfection 370Cleaning Methods 370Vacuuming 371Wet wiping 371Mopping 374Tacky rollers 375

Developing and Implementing a Disinfection Program 376SOP for cleaning and disinfection procedures 376SOP for disinfectants in-house verification 377Determination of test method 378Frequency of cleaning and disinfection 378Selection of disinfectants 379Rotation of disinfectants 382

Qualification of Disinfectants 384Disinfectant — qualification methods 385Classification of disinfection tests 386First stage tests — in vitro tests 388Practical tests 396In-use tests and field trials 397

Susceptibility Breakpoints of Disinfectants AgainstCleanroom Isolates 399

Antibacterial efficacy of common biocides 400Antifungal efficacy of common biocides 402

Safety and Environmental Factors 404Disinfectants for Fumigation and the Sanitization of Isolators 405Conclusion 407References 408

12 CONTAMINATION CONTROL IN CLEANROOMS:PART 2: CLEANROOM DESIGN FACTORS 415Introduction 415Contamination Control and Airflow 418Heating Ventilation and Air Conditioning 420High Efficiency Particulate Air Filters 422

Standards for HEPA filters 423Positioning HEPA filters 424

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Modification of HEPA filters 425Function of Air Handling Systems and HEPA Filtersfor Contamination Control 426

Air filtration 426Air dilution 426Air movement 427Pressure differentials 429

Ongoing Assessment of Cleanrooms 429Annual verification 430Air velocity/supply 435Air changes 435Clean-up times 437HEPA filter installation leak tests 438Pressure differentials 439Temperature and humidity 440

Microenvironments or minienvironments 440Isolators 441

Energy Efficiency 444Modular Cleanrooms 445Conclusion 446References 446

13 CONTAMINATION CONTROL IN CLEANROOMS:PART 3: THE PERSONNEL FACTOR 451Introduction 451Staff Gowning and Behavior 452

Importance of clothing 452Cleanroom garments 452

Cleanroom Personnel Behavior and General Rules 458Basic Training 465Cleanroom Entry 466

Changing into cleanroom garments 467Personnel access 469Materials access 470

Environmental Monitoring 471Strategic questions 471Background detail questions 472Analytical questions 472

The Microbiology Laboratory 473Relationship to the quality unit 473Microbiology laboratory facility design 474

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The Importance of Understanding Basic Microbiologyand Personal Hygiene 476Personal Hygiene 479

Effect of improper hand wash 480Hand Hygiene Technique in Cleanrooms 482

Hand washing protocol 482Hand rubbing/glove sanitization 484

Conclusion 485References 486

14 CONTAMINATION CONTROL IN CLEANROOMS:PART 4: MEDIA SIMULATION TRIALS 489Introduction 489Aseptic Process Simulations 490Media Fill Program 493

Filling speed 494Filling volume 495Design matrix 495

Process Parameters 496Interventions Simulated During Test 498

Sterile air 499Media Selection 499Media Fill Frequency 500Batch Size 501Incubation Conditions 503Acceptance Criteria 504Failure Investigation and Corrective Action 507Invalidation of a Media Fill 508Conclusion 510References 510

15 AUDITING CLEANROOMS FORTHE CONTAMINATION CONTROLPERSPECTIVE 513Introduction 513Quality Audits 514

The audit approach 515Risk assessment 516Auditors 517Undertaking audits 518

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Review of audits 522Regulatory trends 522

Auditing Cleanrooms 523Space and design 523Process flow 525Certification 525Physical operations 525Cleanroom systems 529Personnel 530Equipment 531Change control 534Environmental monitoring 535Microbiological data 540Aseptic filling 540Cleaning and disinfection 543Auditing cleanroom documentation 546Electronic capture of data 548

Conclusion 549References 549

16 CLEANROOMS AND MICROBIOLOGY:THE IMPORTANCE OF RISK ASSESSMENT 553Introduction 553Risks, Risk Assessment and Cleanrooms 553

The value of risk assessment 562Concluding Remarks 563References 566

Index 569

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