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Biopharmaceutical Processing
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Biopharmaceutical Processing
Development, Design, and Implementation of
Manufacturing Processes

Edited by

Günter Jagschies
Eva Lindskog
Karol Łącki
Parrish Galliher
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States

© 2018 Elsevier Ltd. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, ­including photocopying,
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Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research
methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds,
or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including
parties for whom they have a professional responsibility.

To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to
persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or
ideas contained in the material herein.

Library of Congress Cataloging-in-Publication Data

A catalog record for this book is available from the Library of Congress

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A catalogue record for this book is available from the British Library

ISBN: 978-0-08-100623-8

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Publisher: John Fedor

Acquisition Editor: Kostas KI Marinakis
Editorial Project Manager: Michelle Fisher
Production Project Manager: Maria Bernard
Cover Designer: Victoria Pearson

Typeset by SPi Global, India

Contents

Contributors xxiii 1.6.2 Regulatory Prioritization Initiatives 24

Proverb xxvii 1.6.3 Specific WHO Priority—
Preface xxix Antimicrobial Resistance (AMR) 25
1.6.4 Global Priority Proposals for
Medicine Development and Disease
Research 26
Section I 1.7 Additional Drivers and Challenges in
Healthcare 27
Healthcare and Biopharma 1.7.1 Cost of Treatment 27
Industry, Products and Processes 1.7.2 Sharing Information About Health
and Disease 28
1. Disease and Healthcare Priorities 1.7.3 Conflicts of Interest vs Symbiotic
Günter Jagschies Collaboration 28
References 28
1.1 Introduction 3 Further Reading 31
1.1.1 Two Core Scenarios for Healthcare 3
1.1.2 Poverty Scenario—Essential
Healthcare Not Available or 2. Brief Review of the
Unaffordable 4 Biopharmaceutical and Vaccine
1.1.3 Prosperity Scenario—Essential Industry
Healthcare Available and Affordable 5
Günter Jagschies
1.2 Status and Measurement of Global Health 6
1.2.1 Life Expectancy and Mortality 6 2.1 Industrial Context 33
1.2.2 Morbidity and Disease Burden 7 2.2 Brief History of the Biopharma
1.3 The World is Changing—Demographic Industry 33
Transition 9 2.2.1 Vaccine Industry 34
1.3.1 Factors Driving Population Growth 2.2.2 Diabetes Therapeutic Industry 37
and Ageing 9 2.2.3 Plasma Protein Industry 38
1.3.2 Momentum, the Impact of the 2.2.4 Recombinant Protein Industry 40
Current Age Structure 13 2.3 Business Summary 41
1.4 The World is Changing—Disease 2.4 Biopharma Business Challenges 43
Challenge Transition 13 2.4.1 Patient Treatment—High Prices 43
1.4.1 General Observations on Disease 2.4.2 Vaccines Risk for Market Failure—
Patterns 15 Low Prices 46
1.4.2 Communicable Diseases 15 2.4.3 Biosimilar Competition—Low
1.4.3 Noncommunicable Diseases 17 Prices 47
1.4.4 Nonfatal Conditions 19 2.4.4 Biopharma R&D—Between
1.5 Prevention—Health Risks and Their Unique Opportunity and
Mitigation 20 Soaring Cost 53
1.5.1 Identified, Preventable Risks to 2.5 Biopharma Pipeline—The Promise 55
Develop Disease 20 2.5.1 Conclusions for the Future of
1.5.2 Prevention via Vaccination 21 Bioprocessing 56
1.6 Priorities for Medicine Development 23 References 57
1.6.1 Private Industry Prioritization 23 Further Reading 58

v
vi  Contents

3. Selected Biotherapeutics Overview 4.6.2 Multiproduct Facilities 93

References 94
Günter Jagschies Further Reading 94
3.1 Introduction 59
3.2 Marketed Biopharmaceuticals by
Molecule Category 60 Section II
3.2.1 Monoclonal Antibodies 61 Upstream Processes Principles
3.2.2 Insulins 64
3.3 Brief Business Review of
and Methods
Biopharmaceutical Therapeutics 65
3.3.1 Blockbuster Drugs (Revenue > $1 5. Upstream Bioprocessing: Basic
Billion Per Year) 66 Concepts
3.3.2 Revenue Trends by Category of Eva K. Lindskog
Molecule 66
3.3.3 Revenue Trends for Selected 5.1 Introduction 97
Therapeutic Proteins 67 5.2 Kinetic Models 98
3.4 Current R&D Pipeline of Biotherapeutics 5.3 A General Mass Balance 98
and Vaccines 67 5.4 Cell Growth 99
3.5 Basic Processing Characteristics of 5.5 Cell Death 101
Biotherapeutics 69 5.6 Metabolic Concepts 102
3.5.1 Use of Production Organisms 70 5.7 Glucose, Glutamine, Lactate and
3.5.2 Use of Purification Technology 71 Ammonia 102
References 72 5.8 Oxygen 104
5.9 Carbon Dioxide 105
4. Process Capability Requirements 5.10 Product Formation 105
5.11 Appendix 106
Günter Jagschies, Karol M. Łącki 5.11.1 Experimental Determination of
4.1 Introduction 73 kLa and OUR 106
4.2 Regulatory Requirements 74 References 108
4.2.1 Timing of Efforts to Achieve Full Further Reading 110
cGMP Status 74
4.3 Producing the Required Quality 76 6. Host Cells
4.3.1 Heuristics: Requirements on the Eva K. Lindskog, Simon Fischer, Till Wenger,
Upstream Process 76 Patrick Schulz
4.3.2 Heuristics: Requirements on the
Downstream Process 77 6.1 Cells in Biopharmaceutical
4.3.3 Risk Mitigation: Impurities That a Production 111
Process Needs to Remove 79 6.1.1 Introduction 111
4.3.4 Process Integration 82 6.1.2 Sourcing of Cultures 114
4.4 Producing the Required Quantity 85 6.2 Animal Cell Culture 115
4.4.1 How Much Product Needs to 6.2.1 Culture Types 115
Be Made? 85 6.2.2 Non-Human Mammalian
4.4.2 Special Case: Biosimilars 86 Systems 116
4.4.3 Special Case: Manufacturing for 6.2.3 Human Cells 120
Clinical Trials 87 6.3 Microbial Systems 122
4.5 Product Yield 87 6.3.1 Bacteria 122
4.5.1 Upstream Processing Yield 6.3.2 Yeast 124
and Scale Requirements 87 6.4 Other Production Systems 124
4.5.2 Downstream Processing Yield and 6.4.1 Insect Cells 125
Scale Requirements 89 6.4.2 Plant Cells 126
4.6 Pre-Clinical, Phase I to III Manufacturing, 6.5 Outlook and Perspective 126
and Regular Production 92 Appendix I The Hybridoma Technology
4.6.1 Manufacturing Throughout the Life and Humanization of Antibodies 127
Cycle of Products 92 References 127
 Contents  vii

7. Cell Line Development 8.7 Outsourcing Versus In-House

Development and Manufacturing of Cell
Andreas Castan, Patrick Schulz, Till Wenger, Culture Media 159
Simon Fischer 8.7.1 Choosing the Right Media Supplier 160
7.1 Introduction 131 8.8 Summary 160
7.2 Generation of Stable CHO Cell Lines 131 References 161
7.2.1 Vector Design and Production of
Recombinant DNA 131
7.2.2 Gene Delivery 131
7.2.3 Pool Selection and Enrichment/ Section III
Bulk Sorting 133 Recovery Processes, Principles,
7.2.4 Single Cell Cloning 134 and Methods
7.2.5 Importance of Clonality of
Production Cell Lines 136 9. Industry Review of Cell Separation
7.2.6 Clone Development and
and Product Harvesting Methods
Characterization 137
7.3 Host Cell Engineering 138 John P. Pieracci, John W. Armando, Matthew Westoby,
7.3.1 Strategies to Develop Biosuperiors/ Jorg Thommes
Biobetters (Glyco-Engineering) 140
9.1 Introduction 165
7.4 Conclusions and Future Perspectives 140
9.1.1 CHO Systems 165
Appendix I Biosafety Analysis for
9.1.2 General CHO Harvest Process 165
Cell Banks 141
9.1.3 Development of CHO Harvest
Appendix II Transient Transfection 142
Processes 166
References 142
9.1.4 Centrifugation 169
Further Reading 146
9.1.5 Depth Filtration 173
9.1.6 Microfiltration TFF (MF-TFF) 175
8. Cell Culture Media in Bioprocessing
9.1.7 Analytical Tools 177
William G. Whitford, Mats Lundgren, 9.1.8 Economic Considerations 177
Alain Fairbank 9.2 E. coli System 178
9.2.1 Overview 178
8.1 Introduction 147
9.2.2 Cell Disruption 181
8.2 Cell Culture Media 147
9.2.3 Inclusion Body Recovery and
8.2.1 Cell Culture Media Categories 147
Separation 188
8.3 Cell Culture Media Components and
9.2.4 Inclusion Body Solubilization 190
Supplements 150
9.2.5 Inclusion Body Renaturation 193
8.3.1 Commonly Used Media
9.3 Future Developments 201
Components and Supplements 150
9.3.1 Acoustic Wave Separation 201
8.3.2 Other Components and
9.3.2 Magnetic Cake Filtration 201
Supplements 152
References 201
8.3.3 Supplementation 153
8.3.4 Cell Culture Feeds 154
8.3.5 Animal Sera 154 10. Overview of Alternative
8.4 Serum-Free Media Selection, Separation Methods in Relation to
Development and Optimization 155 Process Challenges
8.4.1 Media Selection 155
James M. Van Alstine, Günter Jagschies,
8.4.2 Media Development/Optimization 156
Karol M. Łącki
8.5 Manufacturing and Supply of Cell
Culture Media 157 10.1 How To Become Knowledgeable of
8.5.1 Employing Liquid Versus Alternative Separations in One Hour 207
Powdered Media in Bioprocess 10.2 Overview of Alternative Bioprocessing
Manufacturing 157 Operations 207
8.6 Quality Systems for Cell 10.2.1 Introduction 207
Culture Media 158 10.2.2 General Challenges in Modern
8.6.1 Risk Management Programs 159 Bioprocessing 210
viii  Contents

10.2.3 General Reviews on Classic and 11.5 Examples of Contaminant Precipitation 233
Alternative Separation Methods 212 11.5.1 Contaminant Precipitation With
10.3 General Comparison of Alternative Polyelectrolytes 233
Separation Methods 213 11.5.2 Impurity Precipitation With
10.3.1 Preliminary Comparison of Caprylic Acid 233
Alternative Separation Methods 213 11.6 Sequential and Continuous Precipitation 234
10.4 Alternative Separation Operations 215 11.6.1 Sequential Precipitation 234
10.4.1 Separation Factors and Partition 11.6.2 Batch Versus Continuous
K Values 215 Precipitation 234
10.4.2 Process and Cost-of-Goods 11.7 Process Economics Notes 235
Modeling of Standard and AS 11.7.1 Introduction 235
Methods 215 11.7.2 Simple Cost of Goods
10.4.3 Comparisons Related to mAb Comparison of Caprylic Acid
Processing 216 and Protein A 235
10.5 Acknowledgements, Notices, and 11.7.3 Precipitation Versus Protein
Disclaimers 218 A—Hammerschmidt, Jungbauer 235
References 218 11.8 Conclusions 236
Further Reading 220 Acknowledgments, Notices, and
Disclaimers 236
11. Alternative Separation Methods: References 237
Flocculation and Precipitation
12. Alternative Separation Methods:
James M. Van Alstine, Günter Jagschies,
Crystallization and Aqueous
Karol M. Łącki
Polymer Two-Phase Extraction
11.1 Introduction 221
James M. Van Alstine, Günter Jagschies,
11.2 Clarification and Primary Recovery
Karol M. Łącki
Challenges 221
11.2.1 Self-Associating and 12.1 Introduction 241
Nonassociating Flocculation/ 12.1.1 Two Classic Separation Methods
Precipitation Agents 225 Based on Phase Transfer 241
11.3 Cell Debris Reduction and Clarification 226 12.1.2 Related Notes From Previous
11.3.1 PEI, Chitosan, or CaCl2 Plus Chapters in This Book 241
K2PO4-Based Cell Flocculation 226 12.2 Crystallization 242
11.3.2 Cell Flocculation Using Poly 12.2.1 Introduction 242
(Diallyldimethylammonium 12.2.2 Preparative Small Protein
Chloride) (pDADMAC) 226 Crystallization 246
11.3.3 Cell Flocculation Using 12.2.3 Monoclonal Antibody Fragment
Benzylated Poly(Allylamine) and Antibody Crystallization 246
Phosphate Responsive Polymer 227 12.2.4 Monoclonal Antibody
11.3.4 Cell Flocculation Using EOPO Crystallization Phase Diagrams 246
Temperature-Responsive Polymer 227 12.2.5 Crystallization From Mixtures of
11.4 Precipitation and Flocculation Proteins 248
of Target 229 12.2.6 Crystallization of mAb From
11.4.1 Ammonium Sulfate Precipitation Clarified Feed 249
of Clarified NS0 Cell mAb Feed 229 12.2.7 Scale-Up of Protein Crystallization 249
11.4.2 Protein A Chromatography 12.2.8 Discussion and Conclusions 249
Versus Polyacid Target 12.3 Aqueous Polymer Two-Phase Extraction 249
Precipitation in an mAb Process 229 12.3.1 Introduction 250
11.4.3 Selective Precipitation of 12.3.2 Phase Systems, Phase Diagrams
Polyclonal Ig by Polyacrylic and Partition Coefficients 251
Acid and Kosmotropic Salts 230 12.3.3 Recent Trends in ATPE 256
11.4.4 Sequential Precipitation of 12.3.4 Key References on the
Protein Mixture Components Partitioning of Monoclonal
With NaPAA 231 Antibodies 256
 Contents  ix

12.3.5 General Considerations Related 14.9 Summary 292

to Scouting and Choice of References 292
Phase System 256
12.3.6 Clarification in Single-Polymer 15. Filtration Methods for Use in
EOPO Phase System 257 Recovery Processes
12.3.7 Partition of Proteins in Two-
Polymer Phase Systems Jonathan Royce, Jakob Liderfelt,
Containing Polyacrylate 258 Craig Robinson
12.3.8 Partition of Recombinant 15.1 Introduction 295
Proteins in EOPO Two-Polymer 15.2 Normal-Flow Filtration 295
Phase Systems 259 15.2.1 Formats 295
12.3.9 Single-Polymer ATPE Clarification 15.2.2 Use (Modes of Operation) 299
Followed by Polyacid Precipitation 259 15.2.3 Disposal 299
12.3.10 ATPE Versus Classical Protein 15.2.4 Process Development 299
A-Based Bioprocessing of mAbs 259 15.2.5 Scale-Up 302
12.3.11 Elimination of Perceived 15.2.6 Other Design Considerations 304
Weaknesses of ATPE 262 15.3 Cross-Flow Filtration 305
12.3.12 ATPE Discussion and 15.3.1 Formats 305
Conclusions 262 15.3.2 Operation 306
12.4 Crystallization and ATPE—Summing Up 263 15.3.3 Process Development 309
Acknowledgements, Notices, and 15.3.4 Scale-up 313
Disclaimers 264 15.3.5 Other Design Considerations 314
References 264 15.4 Summary 314
Further Reading 267 References 315
Further Reading 315
13. Expanded Bed Adsorption
Kine A.-K. Frej, Rolf A. Hjorth
Section IV
13.1 Introduction and Background 269
13.2 Principles of Operation 269
Purification Processes, Principles
13.2.1 Evaluation of Bed Stability 270 and Methods
13.3 Adsorbents for EBA 272
13.4 Columns for EBA 272 16. Introduction to Preparative
13.5 Examples 273 Protein Chromatography
13.6 Cleaning and Sanitization 275
Karol M. Łącki
13.7 Capabilities and Challenges 276
References 276 16.1 Introduction 319
16.2 Chromatographic Principles 320
14. Filtration Principles 16.2.1 Porosity, Permeability, and Time
Constant 322
Jakob Liderfelt, Jonathan Royce
16.3 Chromatography Stationary Phase:
14.1 Introduction 279 Properties, Classification, and Basic
14.2 Types of Filters and Materials of Concepts 324
Construction 281 16.3.1 Chemical and Physical Properties 324
14.3 Nominally-Rated Filters 284 16.3.2 Classification of
14.4 Sterilizing-Grade Filters 284 Chromatography Stationary
14.5 Microfiltration Versus Ultrafiltration 285 Phases 325
14.6 Retention 286 16.3.3 Description of Stationary Phases 327
14.7 Fouling 287 16.4 Mass Transfer Effects in Protein
14.8 Modeling of Filtration Operations 288 Chromatography 328
14.8.1 Microfiltration Operations 288 16.4.1 Sorption Kinetics 330
14.8.2 UF/DF Operations 290 16.4.2 External Mass Transfer 331
14.8.3 Critical Flux 291 16.4.3 Intraparticle Mass Transfer 332
14.8.4 Advanced Filtration Models 292 16.4.4 Rate-Limiting Step 333
x  Contents

16.5 Modes of Chromatography Operations 334 18. Ion Exchange Chromatography

16.5.1 Elution Chromatography 335
16.5.2 Frontal Chromatography 335 Anna Grönberg
16.5.3 Displacement Chromatography 336 18.1 Introduction 379
16.5.4 Flow-Through and Bind/Elute 18.2 Application Areas 380
Modes 336 18.3 Examples 380
16.6 Performance Descriptors for 18.3.1 Capture of Interferon α-2a Using
Preparative Protein Chromatography 336 a High-Capacity CIEC Resin 381
16.6.1 Column Efficiency 336 18.3.2 Polishing of mAbs Using CIEC 381
16.6.2 Flow Resistance of Packed Beds 338 18.3.3 Purification of Type 5
16.6.3 Resolution and Selectivity 339 Adenovirus Using AIEC 382
16.6.4 Capacity 341 18.4 IEC Principle and Standard Methods 382
16.6.5 Reusability and Security of Supply 342 18.5 Buffers 384
16.6.6 Throughput and Productivity 343 18.6 Cleaning in Place and Sanitization 386
16.7 Scale-up of Preparative Chromatography 344 18.7 Process Development Workflow 388
16.7.1 Chromatographic Factors 344 18.8 Critical and Key Process Parameters 391
16.7.2 Non-Chromatographic Factors 345 18.9 Methodology 391
16.7.3 Other Scale-up Factors 346 18.9.1 Retention in IEC 392
16.7.4 Scale-up Based on Modeling 347 18.9.2 Zone Broadening in IEC 392
16.8 Economy of Preparative Chromatography 348 18.10 Optimization 392
16.9 Purification Strategies 350 18.11 Productivity and Economy 395
16.9.1 Capture Stage 350 18.12 Future 397
16.9.2 Intermediate Stage 352 References 397
16.9.3 Polishing Stage 352
16.9.4 Chromatography Resin 19. Hydrophobic Interaction
Selection Process 353 Chromatography
16.10 Introduction to Process
Development and Optimization 354 Kjell O. Eriksson
16.10.1 Rational Design of 19.1 Application Areas 401
Chromatography Step  354 19.2 Examples 401
16.10.2 Cycling Operations 356 19.2.1 Purification of Recombinant
16.10.3 Flow Programming Method 358 Hepatitis B Surface Antigen
16.10.4 Elution 360 (r-HBsAg) 401
16.11 Multicolumn Operations 362 19.2.2 Removal of Aggregate From a
16.12 Conclusions 362 mAb Preparation 402
References 363 19.3 Standard Methods 403
19.3.1 Buffers, Salts, and Other Additives 403
17. Affinity Chromatography 19.4 CIP 404
19.5 PD Workflow 405
Åke Danielsson
19.6 QbD Perspective 405
17.1 Application Areas 369 19.7 Methodology 405
17.2 Examples 370 19.8 Economy 407
17.3 Standard Methods 371 19.9 Productivity 407
17.4 Buffers 372 References 407
17.5 CIP 372
17.6 PD Workflow 373 20. Multimodal Chromatography
17.7 Scale-Up 374
Eggert Brekkan
17.8 Critical Process Parameters 375
17.9 Economy 375 20.1 Application Areas 409
17.10 Future 376 20.2 Examples 409
Notes 376 20.2.1 Optimization of Loading
References 376 Conditions on Capto Adhere
Further Reading 378 Using DoE 411
 Contents  xi

20.2.2 Process Development for 23. Filtration Methods for Use

Capture of Recombinant Pro- in Purification Processes
Insulin From E. coli 413 (Concentration and Buffer Exchange)
20.2.3 Purification of Influenza
A/H1N1 Using Capto Core 700 413 Jakob Liderfelt, Jonathan Royce
20.3 Standard Methods 415 23.1 Application Areas 441
20.4 Buffers 415 23.2 Process Development 441
20.5 CIP/SIP 416 23.3 Examples 445
20.6 PD Workflow and Optimization 416 23.3.1 mAb Purification 445
20.7 Critical Process Parameters 417 23.3.2 Vaccine Purification 447
20.8 Economy and Productivity 417 23.4 Advanced Methods 447
20.9 Future 417 23.4.1 Cascade of UF/DF Units 447
References 418 23.4.2 Single-Pass Tangential Flow
Filtration 447
21. Size Exclusion Chromatography (SEC) 23.4.3 Continuous Cross-Flow Filtration 448
Martin Hall 23.4.4 High-Performance Tangential-
Flow Filtration 450
21.1 Introduction 421 23.5 Critical Process Parameters (CPP) 451
21.2 Basic Theory 421 23.6 Key Process Parameters 451
21.3 Application Areas 425 23.7 Economy/Optimization 451
21.4 Examples 425 23.7.1 Productivity (Flux, Time,
21.4.1 Group Separation—Buffer Membrane Area, Buffer
Exchange/Desalting 425 Consumption) 451
21.4.2 Group Separation—Large Proteins 426 23.7.2 COGS Discussion 452
21.4.3 Group Separation—Virus 426 23.8 Future 452
21.4.4 Fractionation—Monoclonal References 452
Antibody (mAb) 427
21.5 Standard Methods 428
21.6 Buffers 428
21.7 CIP/SIP 428 Section V
21.8 Process Development (PD) Workflow 428 Bioprocessing Equipment
21.9 Critical and Key Process Parameters
(CPP and KPP) 430 24. Upstream Processing Equipment
21.10 Methodology 430
21.11 Economy/Optimization 430 Kenneth P. Clapp, Andreas Castan, Eva K. Lindskog
21.12 Productivity 431 24.1 Introduction 457
21.13 Future 431 24.2 Common Design Aspects in
References 432 Bioprocessing Equipment 457
Further Reading 432 24.2.1 Process-Contacting Components 458
24.2.2 Nonprocess Contacting
22. Reversed Phase Chromatography Components 462
24.3 The Bioreactor 464
Kjell O. Eriksson
24.3.1 General Design Principles 464
22.1 Application Areas (Goals and Objectives) 433 24.3.2 Gas and Liquid Management 465
22.2 Example: Insulin Purification 434 24.3.3 Mechanical Design 465
22.3 Standard Methods 435 24.4 Available Bioreactor Technology 467
22.4 CIP/SIP 436 24.4.1 Stainless-Steel Bioreactors 468
22.5 Process Development Workflow 436 24.4.2 Autoclavable Glass Bioreactors 468
22.6 Critical and Key Process Parameters 437 24.4.3 Single-Use Stirred Tank Reactors 469
22.7 Methodology 437 24.4.4 Rocking Bioreactors 470
22.8 Economy 438 24.5 Vessels for Adherent Processes 471
22.9 Productivity 439 24.5.1 Roller Bottles 471
References 439 24.5.2 Packed-Bed Reactors 472
xii  Contents

24.5.3 Bioreactors for Microcarrier 26.2.3 Design for Chromatographic

Culture 473 Performance 497
24.6 High-Throughput Process 26.3 Column Packing 502
Development Bioreactors 474 26.3.1 General Considerations for
24.7 Modeling and Simulation 475 Column Packing 503
References 476 26.3.2 Packing Methods 505
26.3.3 Preparation of Column and
25. Downstream Processing Equipment System for Packing 507
26.4 Assessing Column Performance:
Mikael I. Johansson, Martin Östling,
Efficiency Testing 508
Günter Jagschies
26.4.1 Pulse Method 509
25.1 Introduction 477 26.4.2 Step Method 510
25.2 Critical Aspects of the User 26.5 Conclusions and Outlook 510
Requirement Specification 478 References 511
25.2.1 Chemical Compatibility and
Hygienic Aspects 478
25.2.2 Hygienic Design and Cleaning 480 27. Simplification of Buffer
25.3 Common Components in Equipment Formulation and Improvement
for Bioprocessing 481 of Buffer Control with In-Line
25.3.1 Monitors, Meters, and Sensors 481 Conditioning (IC)
25.3.2 UV Monitoring 481
25.3.3 Conductivity Monitoring 481 Enrique N. Carredano, Roger Nordberg,
25.3.4 pH Monitoring 482 Susanne Westin, Karolina Busson,
25.3.5 Temperature 482 Tomas M. Karlsson, Torbjörn S. Blank,
25.3.6 Flow Meters 482 Henrik Sandegren, Günter Jagschies
25.3.7 Air Sensors and Air Traps 482 27.1 Introduction 513
25.3.8 Pressure Sensor 483 27.2 Buffers for Downstream Processing 513
25.4 System Flow Path 483 27.2.1 Basic Buffer Specifications 514
25.4.1 General Design Criteria for 27.2.2 Buffer Calculations 514
Flow Paths 484 27.3 In-Line Dilution (ILD)—Addressing the
25.4.2 Sloped Pipes 484 Footprint Issue 517
25.5 Pumps 484 27.4 In-Line Conditioning—Controlled
25.5.1 Pulsations 485 Production of Any Buffer 518
25.5.2 Suction Lines to the Pump 485 27.4.1 IC System Layout 519
25.5.3 Cavitation 485 27.4.2 IC Control Modes 519
25.5.4 Pump Types 486 27.5 Testing and Verifying the IC Buffer
25.6 Valves 487 Preparation Capabilities 520
25.6.1 Valve Characteristics 488 27.5.1 Variable Input and Reproducibility 520
25.6.2 Valve Details 488 27.5.2 Gradient Delivery 521
25.7 In-Line Filtration (Sterile Filtration and 27.5.3 Model Albumin Process 522
Particle Filtration) 489 27.5.4 Model Monoclonal Antibody
25.8 Engineering Documents 490 Process 523
25.8.1 Process Flow Diagrams and P&I 27.6 Straight-Through Processing (STP), an
Diagrams 490 Extension of IC Use 524
References 492 Acknowledgments 525
References 525
26. Chromatography Columns Further Reading 525
Klaus Gebauer, Johan Tschöp 28. Continuous Capture of mAbs—
26.1 Introduction 493 Points to Consider and Case
26.1.1 Application Requirements 493 Studies
26.2 Column Design 493
Günter Jagschies
26.2.1 Column Types 493
26.2.2 Mechanical Design 496 28.1 Introduction 527
 Contents  xiii

28.2 The Rationale for Continuous 29.1.5 Single Use Technologies

Processing in Biopharma 528 Evolving From Support Systems
28.2.1 Upstream Process 528 to Production Systems 559
28.2.2 Purification Process 529 29.1.6 Increasing Need for Flexibility,
28.3 Technical Options for Continuous Agility and Economy—
Purification 530 Increased Drug Diversity and
28.3.1 Utilization of Resin Capacity 530 Emerging Markets 559
28.3.2 Parallel Execution of Load and 29.1.7 Maturation From Development
Turnaround Cycles 532 to GMP Clinical and
28.4 Systems for Continuous Purification 534 Commercial Manufacturing 560
28.4.1 Overview of the Commercial 29.2 Overview of Single Use Technologies 561
Offering for SMB/PCC Systems 29.2.1 Long History of Use of
in Biopharma 534 Plastics in the Medical
28.4.2 Aspects of Column Selection for Field and Stainless Steel
PCC or SMB Systems 536 Biomanufacturing Facilities 561
28.4.3 PAT Approach to Process 29.2.2 Potential Toxicity and Effects
Control in Periodic Counter- of Leachables From Polymeric
Current Chromatography 537 Materials on Cells and Product 561
28.5 Process Development Guidance 540 29.2.3 Best Practices for Qualification
28.5.1 Manufacturing Scenario and and Use of Single-Use
Objective 540 Technologies 562
28.5.2 Key Information to be 29.2.4 Regulatory Agency Guidelines
Developed for PCC 540 for Validation of Extractables
28.6 Case Studies—Capture and Polishing 544 and Leachables From Single-
28.6.1 Capture—PCC to Increase Use Technologies 562
Chromatography Media 29.2.5 Leachables in
Capacity Utilization 544 Manufacturing—Risk
28.6.2 Polishing Steps Using Straight- Assessment of Potential
Through Processing 546 Product Exposure 563
28.7 Selected Economic Considerations 548 29.2.6 Leachables in the Upstream
28.7.1 Management Review 548 Process—Risk Assessment and
28.7.2 Points to Consider in Financial Mitigation 563
Comparisons 549 29.2.7 Leachables in the Downstream
28.7.3 Example Calculations 550 Process—Risk Assessment and
28.7.4 Summary—Putting Things in Mitigation 563
Perspective 553 29.2.8 Mitigation of Overall
28.8 Acknowledgments 554 Risk—Produce the
References 555 Toxicological Batches in Small
Scale Single Use Systems 564
29. Single Use Technology and 29.2.9 Commercial Licensure Viability
Equipment of Single Use Technologies 564
29.2.10 Broad Impact on Operations,
Parrish M. Galliher Flexibility, Agility, Process
29.1 Introduction 557 Economics, Product Quality
29.1.1 History of Bioprocessing— and the Environment 565
1970–80s 557 29.3 Single Use Material of Construction,
29.1.2 Industry Drivers and Developing Componentry, Assembly, Sterilization,
Trends—1990–2010 557 Integrity and Use 566
29.1.3 Perfect Storm: Industry 29.3.1 Materials of Construction and
Pressures, Changing Markets, Assembly 566
and New Technologies 558 29.3.2 Sterilization of Single-use Films
29.1.4 Cost, Quality, Speed, and Components 566
Flexibility—Agile and Flexible 29.3.3 Assurance of Single-Use Bag
Single-Use Manufacturing 558 and Assembly Integrity 566
xiv  Contents

29.4 Description of SU Unit 30.7 Exception Handling 606

Operations and General User 30.7.1 Alarms 606
Requirement Specifications for a 30.7.2 Interlocks 607
Typical Monoclonal 30.7.3 Fail State 607
Antibody Suspension 30.7.4 Strategies for HOLD, STOP,
Cell Process 567 ABORT 608
29.4.1 Upstream Mammalian Cell 30.8 Computer System Validation 608
Operations 567 30.8.1 Lifecycle 608
29.4.2 Downstream Purification 30.8.2 21 CFR Part 11 611
Systems for mAb 30.9 System Backup, Archival and Disaster
Processing 569 Recovery 612
29.4.3 General User Requirement 30.9.1 Mitigation Strategies 613
Specifications for Other Single 30.10 Data Historian 613
Use Systems 574 30.11 Other Automation Standards 614
29.4.4 Single Use Facility Design 575 30.11.1 IEC 61131 Standard for
29.5 Gaps and Disadvantages of SUT 575 Programmable Controllers 614
29.6 Conclusions and the Future of Single 30.11.2 IEC-62443: Network and
Use Technologies 575 system security for industrial-
References 576 process measurement and
Further Reading 577 control 615
30.11.3 ISA-95 Standard for Enterprise-
30. Process Control and Automation Control System Integration 617
30.12 Further Reading 617
Solutions
30.12.1 Infrastructure Virtualization 617
Trevor J. Marshall, Yvonne A. Brady 30.12.2 Simulated Versus Plant
Environment 618
30.1 Introduction 580
30.12.3 Smart and Self-Verifying
30.2 Instruments and Input & Outputs 581
Instruments 618
30.2.1 Instruments 581
30.12.4 Data Analytics 619
30.2.2 Inputs & Outputs 582
30.12.5 Building Management and
30.2.3 Common Control
Environmental Monitoring
Algorithms 583
Systems 619
30.3 Automation Hardware 584
30.12.6 Non-GMP Critical Plant
30.3.1 Overview and Introduction 584
Utilities 620
30.3.2 I/O Cards and Bus
References 620
Systems 585
30.3.3 Controllers 586
30.3.4 Servers and Workstations 586
30.4 Programmable Logic Controller &
Section VI
Supervisory Control Industrial Process Design
and Data Access 587
30.4.1 PLC Components 588 31. The Upstream Process: Principal
30.4.2 PLC Program 588 Modes of Operation
30.4.3 Human Machine Interfaces 589
Eva K. Lindskog
30.4.4 Batch 592
30.5 Distributed Control System 593 31.1 Introduction 625
30.5.1 Configuration 594 31.2 Process Development 625
30.5.2 Different Manufacturers 595 31.3 Principal Modes of Operation 627
30.5.3 PLC/SCADA Versus DCS 595 31.3.1 Batch Culture 628
30.6 The ISA-88 Standard for Batch Control 595 31.3.2 Fed-Batch 628
30.6.1 Physical Model 596 31.3.3 Perfusion 629
30.6.2 Procedural Model 598 31.3.4 Continuous Fermentation 632
30.6.3 Single Use Bioreactor S88 31.4 The Seed Train 632
Implementation Example 602 31.5 Process Intensification 633
30.6.4 Variations in S88 31.6 Discussion and Outlook 634
Implementation 604 References 635
 Contents  xv

32. Downstream Process Design, 33.4 Cleaning and Sanitization of

Scale-Up Principles, and Process Chromatography Resins 683
Modeling 33.4.1 Chromatography Resin Lifetime 683
33.4.2 Compatibility of
Karol M. Łącki, John Joseph, Kjell O. Eriksson Chromatography Resins With
32.1 Introduction 637 Cleaning and Sanitization Agents 686
32.2 Process Design Landscape 638 33.4.3 Chromatography Resin Cleaning
32.3 The Core Elements of Process Design 638 Efficiency 687
32.3.1 Process Development 638 33.4.4 Resin Sanitization Efficiency 690
32.3.2 Control Strategy 639 33.5 Cleaning and Sanitization Validation 690
32.4 A Process Design Framework 640 33.5.1 Cleaning Validation 691
32.4.1 Example of Process Design 33.5.2 Assays and Testing Techniques 692
Workflow 640 33.5.3 Sanitization Validation 693
32.4.2 Platform Processes 642 33.6 Preventive Steps and Bioburden
32.5 Downstream Process Design Management 693
Methodologies and Tools 643 33.7 Economic and Environmental Aspects 695
32.5.1 Product in-Process Stability References 697
and Impurity Profiles 644 34. The Search for Process
32.5.2 Basics of Downstream Intensification and Simplification:
Process Development 646
Alternative Approaches versus
32.5.3 Introduction to Risk Analysis,
Design Space Concept, and
Current Platform Processes for
Process Control 647
Monoclonal Antibodies
32.6 Combining Steps for an Efficient Process 650 Robert S. Gronke, Alan Gilbert
32.7 Scale-up as Part of Process Design 651
34.1 Overview 701
32.7.1 Upstream 651
34.1.1 Platform Approach for mAbs 701
32.8 Downstream 655
34.1.2 Changing Needs of the
32.8.1 Liquid Handling 655
Platform: Higher Demand
32.8.2 Membrane Filtration 658
Requirements and Balancing
32.8.3 Chromatography 661
Between Maintaining a Platform
32.8.4 Chromatography Systems 665
Versus Innovation 704
32.9 Process Modeling and Optimization 665
34.1.3 Process Intensification 706
32.9.1 Modeling of Biotech Processes 666
34.1.4 Process Simplification 706
32.10 Summary 671
34.1.5 Examples of Intensification and
References 671
Simplification: Cell Culture 706
34.1.6 Examples of Intensification or
33. Cleaning-in-Place and Sanitization Simplification: Purification 709
Anna Grönberg, Rolf A. Hjorth 34.1.7 Solving Problems at the
Interface Between Upstream
33.1 Introduction 676 and Downstream 716
33.2 Basic Considerations 676 34.2 Discussion/Conclusions 717
33.2.1 Impurities and Contaminants in Acknowledgments 718
the Bioprocess 676 References 718
33.2.2 Cleaning Agents 677
33.2.3 Sanitization Agents 678 35. Single-Use Technology
33.3 Cleaning and Sanitization of Implementation For Biologics and
Hardware 680 Vaccines Production
33.3.1 Compatibility of Hardware
David J. Pollard, Alain Pralong
Materials With Cleaning and
Sanitization Agents 680 35.1 Summary 721
33.3.2 Hardware Cleaning and 35.2 Benefits of Implementing Single-Use
Sanitization Efficiency 681 Technology 722
33.3.3 Examples: Cleaning and 35.2.1 Impact of Single-
Sanitization of a Column and a Use Technology on
Chromatography System 682 Biopharmaceutical Manufacturing 722
xvi  Contents

35.2.2 Economic Cost Analysis of 36.4.4 Disposable Equipment and

Single-Use Technology 722 Devices 751
35.3 Designing and Implementing a Single- 36.4.5 Right-Sizing Batch Size and Lot
Use Technology Process 725 Size 751
35.3.1 Creating a Single-Use 36.5 Summary 752
Technology Based Process 725 References 752
35.3.2 Basis of Process Design and
Equipment Selection 726 37. Perfusion N-1 Culture—
35.3.3 Regulatory Requirements for Opportunities for Process
SUT Implementation 726 Intensification
35.3.4 Process Architecture and John M. Woodgate
the Control Strategy for
Maintaining Product Quality 726 37.1 Introduction 755
35.3.5 End User Expectations of SUT 37.2 N-1 Perfusion Seed Culture 756
Suppliers 728 37.3 Available Technology 757
35.3.6 Extractable & Leachables of SUT 729 37.4 Bioreactor Types 757
35.3.7 Particulate Matter With SUT 730 37.4.1 Rocking Bioreactors 757
35.3.8 Standards for Single-Use 37.4.2 Stirred Bioreactors 758
Technology 730 37.5 Perfusion Filtration Systems 759
35.3.9 Securing the Single-Use 37.5.1 Floating Filter 759
Technology Supply Chain and 37.5.2 Alternating Flow Filtration (ATF) 759
Change Control 731 37.5.3 Tangential Flow Filtration (TFF) 761
35.3.10 Single-Use Technology 37.5.4 Equipment Conclusions 761
Reliability and Improvement 731 37.6 Process Development 762
35.3.11 Biosafety Applications 733 37.6.1 Environmental Stresses 763
35.4 Case Studies of Next Generation 37.6.2 Development Strategies 764
Processes Enabled by SUT 733 37.7 Use of Process Analytical
35.4.1 Vaccine Manufacturing 733 Technology (PAT) 765
35.4.2 Monoclonal Antibody Production 736 37.7.1 Bioreactor Weight 766
35.5 Future State Summary 738 37.7.2 Flow Control 766
Acknowledgments 738 37.7.3 Viable Cell Concentration 766
References 738 37.8 Conclusions 767
37.9 Future Prospects 767
References 767
36. Points to Consider for Design and
Control of Continuous Bioprocessing 38. Process Development and
Intensification for a Recombinant
Oliver Kaltenbrunner
Protein Expressed in E.coli
36.1 Introduction 741
Shuang Chen, William B. Wellborn, John T. Cundy,
36.2 Development and Implementation 742
Ratish Mangalath-Illam, Scott A. Cook, Matthew J.
36.3 Design of Unit Operations 743
Stork, Joseph P. Martin, Maire H. Caparon,
36.3.1 Production Cell Culture 743
Stephen E. Sobacke, Sriram Srinivasan,
36.3.2 Continuous Cycling Capture
Joost P. Quaadgras
Chromatography 744
36.3.3 Virus Inactivation 746 38.1 Introduction 769
36.3.4 Continuous Polishing 38.2 Microbial (E. coli) Expression System
Chromatography 747 and Culture Process Overview 770
36.3.5 Virus Filtration 747 38.3 Cell Line Development 771
36.3.6 Continuous Concentration and 38.4 Culture Process Development and
Formulation 748 Optimization 772
36.4 Integration of Unit Operations 749 38.5 Microbial (E. coli) Downstream
36.4.1 Bioburden Control 749 Process Overview 774
36.4.2 Equipment and Plant Utilization 749 38.6 The Baseline Downstream Process and
36.4.3 Fault Recovery 750 Process Intensification Goals 775
 Contents  xvii

38.7 Downstream Process Intensification 40.3 ADC Conjugation Equipment 832

and Improvement 776 40.3.1 Conjugation Scale-Down
38.7.1 IB Wash and Recovery 776 Model 832
38.7.2 High Concentration High 40.3.2 GMP Manufacturing 834
Efficiency Refolding 777 40.4 Conclusions 834
38.7.3 Clarification by Acid References 834
Precipitation 780
38.7.4 Chromatography Development: 41. Process Design for Bispecific
Increasing Throughput, Yield, Antibodies
and Improving HCP Removal 781
38.8 Summary 785 Ambrose J. Williams, Glen S. Giese, Andreas
38.9 Materials and Methods 786 Schaubmar, Thomas von Hirschheydt
38.9.1 Materials 786 41.1 Introduction 837
38.9.2 Methods 786 41.1.1 Evolution of Next-Generation
Acknowledgments 790 Formats 837
References 790 41.1.2 Clinical Applications for
Bispecifics 837
39. Next-Generation Process Design 41.1.3 Bispecific Formats 838
for Monoclonal Antibody Purification 41.1.4 Knob-Hole Assembly Approach 839
41.1.5 CrossMab Bispecific Approach 840
Krunal K. Mehta, Ganesh Vedantham
41.2 Process Designs for Bispecific
39.1 Introduction 793 Antibodies 841
39.2 Current Practices and Emerging 41.2.1 In Vitro Assembly of
Process Alternatives for Downstream Individually Expressed Knob
Unit Operations 794 and Hole Half-Antibodies 841
39.2.1 Harvest Recovery 795 41.2.2 Process Development for Knob
39.2.2 Capture Step 799 and Hole Bispecifics 846
39.2.3 Viral Inactivation and Depth 41.2.3 Process Development for
Filtration 801 CrossMabs 848
39.2.4 Polishing Step 802 41.3 Conclusion 854
39.3 Enabling Technologies for Next- References 854
Generation Manufacturing Facilities 805
39.3.1 Single-Use Technology 805
42. Current Manufacturing of Human
39.3.2 Continuous Processing 805
Plasma Immunoglobulin G
39.3.3 Process Analytical
Technology (PAT) 806 Andrea Buchacher, John M. Curling
39.4 Concluding Remarks 807
42.1 Introduction 857
Acknowledgments 808
42.2 Plasma Fractionation Technologies 858
References 808
42.2.1 Ethanol Fractionation 858
42.2.2 Caprylate Fractionation 862
40. Process Development and 42.2.3 Polyethylene Glycol
Manufacturing of Antibody-Drug Fractionation 862
Conjugates 42.2.4 Chromatographic Fractionation 862
42.2.5 Current Hybrid Methods of
Matt H. Hutchinson, Rachel S. Hendricks,
Plasma Fractionation 863
Xin Xin Lin, Dana A. Olsson
42.3 Processing Technologies to Assure
40.1 Introduction 813 Viral Safety 864
40.1.1 Design of an ADC 814 42.3.1 Solvent/Detergent
40.2 Process Development and Treatment 864
Manufacturing Considerations 816 42.3.2 Caprylate Treatment 864
40.2.1 ADC Quality Attributes 816 42.3.3 Pasteurization 864
40.2.2 ADC Process Overview 816 42.3.4 Nanofiltration (Viral Filtration) 864
40.2.3 ADC Conjugation Process 819 42.3.5 Chromatography 865
xviii  Contents

42.3.6 Precipitation 865 43.10 Vaccine Economics 889

42.3.7 Low pH 866 43.11 Closing Remarks 890
42.3.8 Neutralizing Antibodies 866 References 890
42.3.9 Critical Quality Attributes of IV
and SC Immunoglobulins 866 44. Bioprocesses for Cell Therapies
42.3.10 Distribution of Molecular Size 866
Suzanne S. Farid, Michael J. Jenkins
42.3.11 Anticomplementary Activity 866
42.3.12 Prekallikrein Activator and 44.1 Introduction 899
Kalikrein 867 44.2 Cell Therapy Bioprocess Economics 901
42.3.13 Hemagglutinins (Anti-A, B, D) 867 44.2.1 Capital Investment 901
42.3.14 Immunoglobulin A 867 44.2.2 Cost of Goods 902
42.3.15 Pyrogens and Endotoxins 867 44.2.3 Process Economic Drivers 903
42.3.16 ß-Glucans From Depth 44.3 Bioprocess Flowsheet Variations
Filters 867 Across Cell Therapies 903
42.3.17 Virus Inactivation Reagents 868 44.3.1 Autologous vs Allogeneic Cell
42.3.18 Additional Biochemical and Therapy Bioprocess Flowsheets 903
Biological Characterization 868 44.3.2 Adult Cell Therapy
42.3.19 Subclass Distribution 868 Bioprocesses vs Pluripotent
42.3.20 Fc-function 868 Stem Cell-Derived
42.3.21 Aggregate Formation 868 Bioprocesses 904
42.3.22 Antibody Titres 869 44.4 Culture Strategies for Cell Therapies 906
42.4 Low level Plasma Protein Impurities in 44.4.1 Planar Culture Systems for
Immunoglobulin Products 869 Therapeutic Cell Culture 906
42.4.1 Albumin and Transferrin 869 44.4.2 Three-Dimensional Culture
42.4.2 IgG Fragments and Other Systems for Human
Proteinaneous Impurities 869 Cell Culture 907
42.4.3 Factor XIa 870 44.5 Differentiation of hPSCs 911
42.5 Formulation 870 44.5.1 Planar Strategies for hPSC
42.6 Conclusions 870 Differentiation 911
References 871 44.5.2 Bioreactor-Based Systems for
hPSC Differentiation 911
43. Modern Production Strategies in 44.6 Genetic Engineering of Cells for Cell
the Vaccine Industry Therapies 913
Hari Pujar, Mats Lundgren 44.7 Downstream Processing of Cell
Therapies 914
43.1 Introduction 877
44.7.1 Cell Harvesting, Washing, and
43.2 Outline 877
Concentration 914
43.3 Whole-Organism Vaccines 877
44.7.2 Purification of Cells for Cell
43.3.1 Viral Vaccines 879
Therapies 915
43.3.2 Cell Substrates 879
44.8 Integrated and Continuous Bioprocess
43.4 Bacterial Vaccines 882
Strategies for Cell Therapies 917
43.5 Parasite Vaccines 882
44.9 Manufacturing and Distribution
43.6 Subunit Vaccines 882
Models for Cell Therapies 920
43.6.1 Recombinant Protein and VLP
44.10 Concluding Remarks 922
Vaccines 882
References 923
43.6.2 Alternate Expression Systems for
Protein Subunit Vaccines 885
43.7 Polysaccharide and Protein-
Section VII
Polysaccharide Conjugate Vaccines 886 Facility Design and Operation
43.8 Nucleic Acid Vaccines 886
43.9 Influenza Vaccines 887 45. Facility Design and Process Utilities
43.9.1 Egg-Based Influenza Vaccine
John Joseph
Production 887
43.9.2 Cell Culture-Based Influenza 45.1 Introduction 933
Vaccine Production 888 45.2 The Manufacturing Landscape 934
43.9.3 Universal Influenza Vaccines 889 45.3 Capacity Planning 934
 Contents  xix

45.4 Facility Functional Needs 936 46.3.3 Equipment, Utilities, and Facility 991
45.5 cGMP Facility Design 938 46.3.4 Materials and Components 991
45.5.1 Regulatory Compliance 938 46.3.5 Process Controls 992
45.5.2 The Concept of Segregation 938 46.3.6 Change Management 993
45.5.3 Application of Segregation 46.3.7 GMP Operations 993
Practices in Facility Design 944 46.3.8 Future Trends 996
45.5.4 Shrinking the Cleanroom 948 46.4 Economics 997
45.5.5 Biological Hazard 46.4.1 Scale 997
Containment 950 46.4.2 Capacity Utilization 997
45.6 Multiproduct Manufacturing 954 46.4.3 Materials 998
45.6.1 Considerations for Flexible 46.5 Outsourcing 998
Facility Design 956
45.7 Process Support Functional
Considerations 957 Section VIII
45.7.1 Fluid Transfer 958
45.7.2 Cleaning and Sterilizing Systems 958
Analytics, Regulatory, Quality, and
45.7.3 Heating Ventilation and Air Safety Aspects
conditioning (HVAC) 963
45.7.4 Technical and Clean Utility 47. Analytical Methods
Systems 966 Matthew J. Traylor, Peter Bernhardt, Bruce S.
45.7.5 Electrical Power 971 Tangarone, Johnson Varghese
45.7.6 Waste Treatment 972
45.7.7 Planning of Utility Systems 975 47.1 CQA 1002
45.7.8 Facility Control Systems 977 47.1.1 Introduction 1002
45.8 Planning for the Facility Support 47.1.2 Overview of Risk Assessment to
Systems on Site 978 Identify CQAs 1004
45.8.1 Warehousing 978 47.1.3 Structure/Function Studies to
45.8.2 Quality Control Building & Aid CQA Risk Ranking 1006
Laboratories 979 47.1.4 Analytical Methods for
45.8.3 Central Utilities Building 979 Measuring CQAs 1007
45.8.4 Fill –Finish for Drug Product 47.2 Analytical Control Strategy 1007
Production 979 47.2.1 Introduction 1007
45.8.5 Location 980 47.2.2 Developing the Analytical
45.9 Facility Construction 980 Control Strategy 1007
45.9.1 Execution Steps 980 47.2.3 Case Study—Streamlining
45.9.2 Construction 981 the Testing Panel of a Highly
45.10 Summary: Considerations When Glycosylated non-mAb Protein 1014
Planning a New Facility 982 47.3 Analytical Method Development and
Appendix A 982 Qualification 1017
A.1 Clean Room Classifications 982 47.3.1 Introduction 1017
A.2 Ancillary Control Systems 982 47.3.2 Target Identification and
References 984 Technology Selection 1017
Further Reading 986 47.3.3 Method Development 1017
47.3.4 Method Qualification 1018
46. Points to Consider in 47.3.5 Lifecycle Management of
Manufacturing Operations Analytical Methods 1019
47.4 Analytical Methods 1019
Geoff Hodge 47.4.1 Introduction 1019
46.1 Introduction 987 47.4.2 Protein Concentration 1023
46.2 Non-GMP Manufacturing 987 47.4.3 Titer 1024
46.2.1 Technology Transfer 988 47.4.4 Process-Related Impurities 1024
46.2.2 Toxicology Manufacturing 989 47.4.5 Product-Related Impurities 1028
46.3 GMP Manufacturing 989 47.4.6 Product Heterogeneity 1029
46.3.1 GMP Systems and Documentation 989 47.4.7 Multiattribute LC-MS Methods 1030
46.3.2 Training 991 47.4.8 Higher-Order Structure 1032
xx  Contents

47.5 Analytical Support of Process 49. Pathogen Safety

Development 1034
47.5.1 Introduction 1034 Albrecht Gröner
47.5.2 Improving Developability 49.1 Introduction 1075
During Sequence Selection 1034 49.2 Raw Material 1077
47.5.3 Analytical Support of Cell Line 49.2.1 Selection and Testing of Raw
Development 1035 Material 1078
47.5.4 Analytical Support of Upstream 49.3 Starting Material 1078
Processing 1039 49.3.1 Testing and Release of Starting
47.5.5 Analytical Support of Material 1078
Downstream Processing 1041 49.3.2 Human Derived Starting Material 1079
47.5.6 Feasibility of In-Line/At-Line 49.3.3 Cell Culture 1080
Monitoring and Real Time 49.3.4 Animal Derived Material 1084
Control 1042 49.4 Capacity of the Manufacturing Process
47.6 Cost of Operating a Modern, High- to Inactivate and Remove Pathogens 1084
Throughput Analytical Laboratory 1043 49.4.1 Principle of Virus Validation 1085
47.6.1 Typical Cost of Automated/ 49.4.2 Product Specific Requirements
High Resolution Analytical Regarding Virus Validation 1088
Instrumentation 1043 49.4.3 Advanced Therapy Medicinal
47.6.2 Typical Cost of Analytical Full Products 1092
Time Employees (FTE) 1045 49.5 Transmissible Spongiform
47.6.3 Estimated Cost of Performing Encephalopathy 1093
Analytics for Drug Substance/ 49.5.1 TSE and Starting/Raw Materials 1093
Drug Product Manufacturing 49.5.2 Removal and Inactivation of
Support 1045 TSE Agent by the Manufacturing
References 1047 Process of Biologicals 1094
49.6 Cleaning and Sanitization of
48. Implementation of QbD for Equipment and Material 1094
Manufacturing of Biologics—Has It 49.7 Assessment of Risk of Virus
Met the Expectations? Transmission 1095
49.8 Conclusion 1097
Anurag S. Rathore, Sumit K. Singh, Jashwant Kumar,
Glossary 1100
Gautam Kapoor
References 1101
48.1 Introduction 1052
48.2 What is QbD? 1052 50. Chemistry, Manufacture and Control
48.3 Framework for Assessing the Benefits
Kim R. Hejnaes, Tom C. Ransohoff
of QbD Application in Manufacturing
of Biologics 1053 50.1 Introduction 1105
48.4 Impact of QbD on Biopharmaceutical 50.2 Part A: Planning 1106
Life Cycle 1054 50.2.1 Introduction 1106
48.4.1 Molecule Selection 1054 50.2.2 Target Product Profile 1107
48.4.2 Process Development and 50.2.3 Drug Product Profile 1107
Characterization 1056 50.2.4 Drug Substance Profile 1107
48.4.3 Evaluation of Safety and Efficacy 50.2.5 Target Protein Profile 1107
of a Biologic 1062 50.2.6 Expression Systems 1108
48.4.4 Technology Transfer 1066 50.2.7 Process Design 1110
48.4.5 Marketing Application and 50.2.8 Quality 1111
Commercial Production 1067 50.2.9 Safety 1115
48.4.6 Role of Knowledge 50.2.10 Freedom to Operate 1115
Management in QbD 50.2.11 Timelines 1115
Implementation 1067 50.2.12 Project Plan 1116
48.5 Summary 1068 50.2.13 Master Validation Plan 1116
Acknowledgments 1069 50.2.14 Cost of Goods Sold 1116
References 1069 50.3 Part B: Tech Transfer 1116
Further Reading 1073 50.3.1 Description 1116
 Contents  xxi

50.3.2 GAP Analysis 1117 51.5.3 Dual-Sourced Resins &

50.3.3 Risk Profile for Project Execution 1117 Membranes 1146
50.4 Part C: Execution 1118 51.5.4 Unrealized Potential:
50.4.1 Introduction 1118 Precipitation, Crystallization,
50.4.2 Specifications 1119 Membrane Adsorbers 1146
50.4.3 Analytical Support and Validation 1120 51.5.5 Factories of the Future 1147
50.4.4 Reference Standards 1120 51.5.6 Continuous Processing 1147
50.4.5 Cell Line Development 1122 51.6 Conclusions 1148
50.4.6 Cell Banks 1122 Acknowledgments 1148
50.4.7 Process Development 1123 References 1148
50.4.8 Formulation Development 1124
50.4.9 Scale-Up 1125 52. Navigating the Regulatory Maze
50.4.10 GMP Manufacture 1127 Upon Process Changes
50.4.11 Quality Control 1128
50.4.12 Stability Studies 1129 E. Morrey Atkinson, Michael A. Rubacha
50.4.13 Virus Reduction 1129 52.1 Introduction: What is Fragmentation? 1151
50.4.14 Validation 1130 52.2 Why Changes Happen: The Business
50.4.15 Documentation 1131 Case for Changes to Approved
50.5 Part D: Common Technical Document 1133 Processes 1151
Glossary 1134 52.3 Lack of Common Practice: Impact of
References 1135 Divergent Regulations 1153
52.4 A Call to Action: Against Future
51. Post-Licensure Purification Process Fragmentation 1155
Improvements for Therapeutic References 1156
Antibodies: Current and Future States Further Reading 1157

Brian D. Kelley, Annika Kleinjans, Philip Lester

53. Security of Bioprocess
51.1 Introduction 1137 Consumables Supply
51.2 History and Current Status of
Jeffrey R. Carter, Daniel Nelson, David G. Westman
Therapeutic mAb Production and
Purification 1137 53.1 Introduction 1159
51.2.1 Cohn Fractionation for IgIV 1137 53.2 Material and Product Selection 1160
51.2.2 First Generation Processes for 53.3 Supplier Selection 1160
mAbs 1138 53.4 Risk Management 1163
51.2.3 Platform Processing Evolution 53.4.1 Supplier Auditing 1163
and Current Status 1138 53.4.2 Multi-Sourcing 1163
51.3 Strategies of Post-Licensure Changes 1138 53.4.3 Manufacturing Facility Risk 1165
51.3.1 Examples of Drivers for Post- 53.4.4 Supplier Agreements 1167
Licensure Changes 1138 53.5 Communication 1167
51.3.2 Regulatory Considerations 1139 53.6 Conclusion 1168
51.3.3 Platform Evolution 1140 References 1168
51.3.4 Virus Removal and Inactivation 1140
51.3.5 Costs 1141
51.4 Case Studies 1142 Section IX
51.4.1 Genentech mAbs (Licensed
Before 2005): Rituxan,
Financial Management and Process
Herceptin, Xolair, Raptiva, Avastin 1142 Economics
51.4.2 Remicade 1144
51.4.3 Enbrel 1144 54. Basics of Financial Management
51.4.4 Humira 1144
Brian Montgomery
51.4.5 Gazyva 1145
51.5 Future Embodiments and Options 1145 54.1 Finance Management Principles &
51.5.1 New Platforms: Evolution or Terminology 1171
Revolution? 1145 54.2 Financial Goals and Objectives of
51.5.2 Detergent Inactivation 1146 Business Enterprises 1171
xxii  Contents

54.2.1 Typical Ownership Structures: 55.5.2 Level 2 Guidance—Scale Down

Types of Companies, Interests, and Single-Use Technology 1210
Typical Opportunities and 55.5.3 Level 3 Guidance—Uptime in
Constraints 1171 the Facility and Automation 1215
54.3 Explanation of Financial Key Terms 1176 55.5.4 Level 4 Guidance—Leaving the
54.3.1 Balance Sheet 1176 Comfort Zone, New Designs
54.3.2 Income Statement 1177 and Approaches 1216
54.3.3 Cash Flow Statement 1178 55.6 Financial Analysis—Consider Bias and
54.3.4 Cost of Capital (WACC), Net Transparency 1217
Present Value and Return on 55.6.1 Risk for Bias in Assumptions and
Investment 1180 Preferences Applied to Financial
54.3.5 Financial Planning and Analysis 1183 Analysis 1218
54.3.6 Rationale for Financial 55.6.2 Upstream vs Downstream
Decision Making 1183 Processing Cost—Points to
54.3.7 Generating Cash 1184 Consider With Alternative
54.3.8 Growing Profitability 1185 Technology 1218
54.3.9 Relationship Between Price 55.6.3 Introducing New Technology—
and COGS 1185 With Financial Benefits in the
54.3.10 Risk Management 1186 Argument 1219
54.3.11 Positioning for Acquisitions 55.6.4 Sourcing of Materials for
and Dispositions 1187 Production—Cost Reduction
54.4 Measuring Success 1188 Versus Value Generation 1220
54.4.1 Wrap 1188 55.7 Conclusions 1221
References 1189 Acknowledgments 1222
References 1222
55. Management of Process
Economy—Case Studies
Günter Jagschies
Section X
Appendices
55.1 Introduction 1191
55.2 Billion-Dollar Questions 1192
56. Appendix 1—The Ideal-World
55.2.1 Access to Manufacturing
Research Portfolio
Capacity—Managing the
Network 1192 Günter Jagschies
55.2.2 Financing of R&D Activities,
References 1231
Creating Efficiency 1194
55.3 The Objective, the Baseline, and the
Return of Improvements 1197
57. Appendix 2—History of
55.3.1 The Objective—Best-in-Class Biomedical Research
Performance Metrics 1197 Günter Jagschies
55.3.2 Manufacturing Cost Baseline—
Reading the Income References 1236
Statement 1198
55.3.3 Efficiency in Improvement 58. Appendix 3—Marketed
Efforts—The Rule of Biotherapeutics
Diminishing Returns 1201 Günter Jagschies
55.4 Facility and Process Cost Break-Down,
Outlining the Options 1203 References 1252
55.4.1 Facility Cost Case Study—Drug
Substance Bioprocessing
Facility 1203
55.5 Million-Dollar Questions 1206 Index 1253
55.5.1 Level 1 Guidance—Processing
Tools 1207
Contributors
Numbers in parentheses indicate the pages on which the authors’
contributions begin.
John W. Armando (165), Biogen Inc., Cambridge; Visterra
Inc., Cambridge, MA, United States; john.armando@
biogen.com
E. Morrey Atkinson (1151), Bristol–Myers Squibb, New
Brunswick, NJ, United States; morrey.atkinson@bms.com
Peter Bernhardt (1001), Shire, Lexington, MA; Celgene,
Summit, NJ, United States; pbernhardt@shire.com
Torbjörn S. Blank (513), GE Healthcare Life Sciences,
Uppsala, Sweden; torbjorn.blank@ge.com
Yvonne A. Brady (579), Zenith Technologies, Dublin,
Ireland; yvonne.brady@zenithtechnologies.com
Eggert Brekkan (409), GE Healthcare Life Sciences,
Uppsala, Sweden; eggert.brekkan@ge.com
Andrea Buchacher (857), Octapharma Pharmazeutika
Produktions GmbH, Vienna, Austria; andrea.buchacher@
octapharma.at
Karolina Busson (513), GE Healthcare Life Sciences,
Uppsala, Sweden; karolina.busson@ge.com
Maire H. Caparon (769), Pfizer Inc., Chesterfield, MO,
United States
Enrique N. Carredano (513), GE Healthcare Life Sciences,
Uppsala, Sweden; enrique.carredano@ge.com
Jeffrey R. Carter (1159), GE Healthcare Life Sciences,
Marlborough, MA, United States; jeffreycarter@
ge.com
Andreas Castan (131,457), GE Healthcare Life Sciences,
Uppsala, Sweden; andreas.castan@ge.com
Shuang Chen (769), Pfizer Inc., Chesterfield, MO, United
States; shuang.chen@pfizer.com
Kenneth P. Clapp (457), GE Healthcare Life Sciences,
Marlborough, MA, United States; ken.clapp@ge.com
Scott A. Cook (769), Pfizer Inc., Chesterfield, MO, United
States; scott.cook@pfizer.com
John T. Cundy (769), Pfizer Inc., Chesterfield, MO, United
States; john.cundy@pfizer.com
John M. Curling (857), John Curling Consulting AB,
Uppsala, Sweden; john@consultcurling.se
Åke Danielsson (367), GE Healthcare Life Sciences,
Uppsala, Sweden; jagschies@gmail.com
Kjell O. Eriksson (401,433,637), Gozo Biotech Consulting,
Gozo, Malta; kjelleriksson@gozobiotech.com
Alain Fairbank (147), Biotechnology Consultant, Logan,
UT, United States; alainfairbank@gmail.com
Suzanne S. Farid (899), University College London,
London, United Kingdom; s.farid@ucl.ac.uk
Simon Fischer (111,131), Boehringer Ingelheim, Biberach,
Germany; simon.fischer@boehringer-ingelheim.com
Kine A.-K. Frej (269), GE Healthcare Life Sciences,
Uppsala, Sweden; kine.frej@ge.com
Parrish M. Galliher (557), Xcellerex, Inc., A GE
Healthcare Life Sciences Company, Marlborough, MA,
United States; parrish.galliher@ge.com
Klaus Gebauer (493), GE Healthcare Life Sciences,
Uppsala, Sweden; klaus.gebauer@ge.com
Glen S. Giese (837), Purification Development, Pharma
Technical Development, Genentech (Roche), South San
Francisco, CA, United States; giese.glen@gene.com
Alan Gilbert (701), Biogen Inc. Cambridge, MA, United
States; alan.gilbert@biogen.com
Anna Grönberg (379,675), GE Healthcare Life Sciences,
Uppsala, Sweden; Anna.Gronberg@ge.com
Albrecht Gröner (1075), PathoGuard Consult, Seeheim-
Jugenheim, Germany; info@patho-guard.com
Robert S. Gronke (701), Biogen Inc. Cambridge, MA,
United States; rob.gronke@biogen.com
Martin Hall (421), GE Healthcare Life Sciences, Uppsala,
Sweden; martin.hall@ge.com
Kim R. Hejnaes (1105), Hejnaes Consult AB,
Simlaangsdalen, Sweden; kim@hejnaes.dk
Rachel S. Hendricks (813), Genentech Inc., South San
Francisco, CA, United States; hendricks.rachel@
gene.com
xxiii
xxiv  Contributors
Rolf A. Hjorth (269,675), CERVUS Biotech Consulting,
Uppsala, Sweden; rolf@cervusbiotech.se
Geoff Hodge (987), Unum Therapeutics, Cambridge, MA,
United States; geoff.hodge@unumrx.com
Matt H. Hutchinson (813), Genentech Inc., South San
Francisco, CA, United States; hutchinson.matthew@
gene.com
Günter Jagschies (3,33,59,73,207,221,241,477,513,527,1191,
1227,1233,1237), GE Healthcare Life Sciences, Freiburg
im Breisgau, Germany; jagschies@gmail.com
Michael J. Jenkins (899), University College London,
London, United Kingdom; michael.jenkins@ucl.ac.uk
Mikael I. Johansson (477), GE Healthcare Life Sciences,
Uppsala, Sweden; mikael.i.johansson@ge.com
John Joseph (637,933), GE Healthcare Lifesciences,
Amersham; GE Healthcare, Little Chalfond, United
Kingdom; johnjoseph@ge.com
Oliver Kaltenbrunner (741), Amgen, Thousand Oaks, CA,
United States; oliverk@amgen.com
Gautam Kapoor (1051), Indian Institute of Technology,
New Delhi, India
Tomas M. Karlsson (513), GE Healthcare Life Sciences,
Uppsala, Sweden; tomas.m.karlsson@ge.com
Brian D. Kelley (1137), Genentech, South San Francisco,
CA, United States; bkelley@vir.bio
Annika Kleinjans (1137), Roche, Penzberg, Germany;
annika.kleinjans@roche.com
Jashwant Kumar (1051), Indian Institute of Technology,
New Delhi, India
Karol M. Łącki (73,207,221,241,319,637), VP Technology
Development, Avitide, Inc., United States (formerly
at Karol Lacki Consulting AB, Höllviken, Sweden);
k.lacki@icloud.com
Philip Lester (1137), Genentech, South San Francisco, CA,
United States; lester.phillip@gene.com
Jakob Liderfelt (279,295,441), GE Healthcare Life
Sciences, Uppsala, Sweden; jakob.liderfelt@ge.com
Xin Xin Lin (813), Genentech Inc., South San Francisco,
CA, United States; lin.xin-xin@gene.com
Eva K. Lindskog (97,111,457,625), Lonza Pharma &
Biotech, Basel, Switzerland; eva.lindskog@lonza.com
Mats Lundgren (147,877), GE Healthcare, Life Sciences,
Uppsala, Sweden; MatsLundgren@ge.com
Ratish Mangalath-Illam (769), Pfizer Inc., Chesterfield,
MO, United States; ratish.krishnan@pfizer.com
Trevor J. Marshall (579), Zenith Technologies, Dublin,
Ireland; Trevor.Marshall@zenithtechnologies.com
Joseph P. Martin (769), Pfizer Inc., Chesterfield, MO,
United States; joseph.p.martin@pfizer.com
Krunal K. Mehta (793), Bioprocess Sciences and
Technology, Amgen, Cambridge, MA, United States;
kmehta@amgen.com
Brian Montgomery (1171), GE Healthcare, Chicago, IL,
United States; brian.c.montgomery@ge.com
Daniel Nelson (1159), GE Healthcare Life Sciences,
Marlborough, MA, United States; daniel.nelson@ge.com
Roger Nordberg (513), GE Healthcare Life Sciences,
Uppsala, Sweden; roger.nordberg@ge.com
Dana A. Olsson (813), Genentech Inc., South San Francisco,
CA, United States; olson.dana@gene.com
Martin Östling (477), Martin Östling Konsult AB, Uppsala,
Sweden; jagschies@gmail.com
John P. Pieracci (165), Biogen Inc., Cambridge; Visterra
Inc., Cambridge, MA, United States; john.pierracci@
biogen.com
David J. Pollard (721), Merck & Co., Inc., Kenilworth, NJ,
United States; david_pollard@merck.com
Alain Pralong (721), Pharma-Consulting ENABLE GmbH,
Solothurn, Switzerland; alain.pralong@yahoo.com
Hari Pujar (877), Moderna Therapeutics, Cambridge, MA,
United States; hari.pujar@modernatx.com
Joost P. Quaadgras (769), Pfizer Inc., Chesterfield, MO,
United States; joost.p.quaadgras@pfizer.com
Tom C. Ransohoff (1105), BioProcess Technology
Consultants, Inc., Woburn, MA, United States;
transohoff@bptc.com
Anurag S. Rathore (1051), Indian Institute of Technology,
New Delhi, India; asrathore@biotechcmz.com
Craig Robinson (295), GE Healthcare Life Sciences,
Westborough MA, United States; jagschies@gmail.com
Jonathan Royce (279,295,441), GE Healthcare Life
Sciences, Uppsala, Sweden; jonathan.royce@ge.com
Michael A. Rubacha (1151), Bristol-Myers Squibb, East
Syracuse, NY, United States; michael.rubacha@bms.com
Henrik Sandegren (513), GE Healthcare Life Sciences,
Uppsala, Sweden; henrik.sandegren@ge.com
Andreas Schaubmar (837), Large Molecule Research,
Pharma Research and Early Development (pRED),
Roche Innovation Center Munich, Penzberg, Germany;
andreas.schaubmar@roche.com

Patrick Schulz (111,131), Boehringer Ingelheim,
Biberach, Germany; patrick_1.schulz@boehringer-
ingelheim.com
Sumit K. Singh (1051), Indian Institute of Technology, New
Delhi, India
Stephen E. Sobacke (769), Pfizer Inc., Chesterfield, MO,
United States; stephen.e.sobacke@pfizer.com
Sriram Srinivasan (769), Pfizer Inc., Chesterfield, MO,
United States; sriram.srinivasan@pfizer.com
Matthew J. Stork (769), Pfizer Inc., Chesterfield, MO,
United States; Matthew.Stork@pfizer.com
Bruce S. Tangarone (1001), Shire, Lexington, MA, United
States; btangarone@shire.com
Jorg Thommes (165), Biogen Inc., Cambridge; Visterra
Inc., Cambridge, MA, United States; jthommes@
VISTERRAINC.COM
Matthew J. Traylor (1001), Shire, Lexington, MA, United
States; mtraylor@shire.com
Johan Tschöp (493), GE Healthcare Life Sciences,
Uppsala, Sweden; johan.tschop@ge.com
James M. Van Alstine (207,221,241), JMVA Biotech AB;
Royal Institute of Technology, Stockholm, Sweden; jim.­
vanalstine@telia.com
Johnson Varghese (1001), Shire, Lexington, MA; Celgene,
Summit, NJ, United States; jsvarg@gmail.com
Contributors  xxv
Ganesh Vedantham (793), Drug Substance Process
Development, Amgen Manufacturing Ltd., Juncos,
Puerto Rico; vedanthg@amgen.com
Thomas von Hirschheydt (837), Large Molecule Research,
Pharma Research and Early Development (pRED),
Roche Innovation Center Munich, Penzberg, Germany
William B. Wellborn (769), Pfizer Inc., Chesterfield, MO,
United States; william.wellborn@pfizer.com
Till Wenger (111,131), Boehringer Ingelheim, Biberach,
Germany; till.wenger@boehringer-ingelheim.com
Susanne Westin (513), GE Healthcare Life Sciences,
Uppsala, Sweden; susanne.westin@ge.com
David G. Westman (1159), GE Healthcare Life Sciences,
Uppsala, Sweden; David.Westman@ge.com
Matthew Westoby (165), Biogen Inc., Cambridge;
Visterra Inc., Cambridge, MA, United States; matthew.
westoby@biogen.com
William G. Whitford (147), GE Healthcare, Bioprocess,
Logan, UT, United States; Bill.Whitford@ge.com
Ambrose J. Williams (837), Purification Development,
Pharma Technical Development, Genentech (Roche),
South San Francisco, CA, United States; williams.
ambrose@gene.com
John M. Woodgate (755), GE Healthcare Life Sciences,
Marlborough, MA, USA; john.woodgate@ge.com
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 “Today the world’s biggest problems
are the world’s biggest market opportunities.
Want to become a billionaire? Solve a billion-person problem.”

Peter Diamandis, XPRIZE Foundation

xxvii
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Preface

“Biopharmaceutical Processing” tells the story of making biotherapeutics and vaccines available for patients anytime, and
anywhere in the world. We tell it from the perspective of those who develop the means, design the processes, and operate
the plants where these medicines are manufactured. Helping to make these medicines affordable is a key motivator for us al-
ways to refocus our story from just technical or scientific descriptions. The safe and economically viable worldwide spread
of biologic medicines from production facilities is dependent on improving the methods, their productivity and robustness
in delivering quality, and on making the best decisions leading to an integrated strategy.
The book is intended for everyone who works with process development or manufacturing-related aspects in the bio-
pharma or vaccine industry, all the way from senior management to team leaders and supervisors. For academic readers,
this handbook is a key source for a comprehensive education in the field of bioprocessing and preparation of students for
work in their industry careers. We will go beyond the production of this book and make the story and its content available
for education and training around the globe.
In assembling the parts of this book, we have endeavored to provide answers to questions and solutions to the many
challenges faced by the industry. It has been a complex task of combining literally hundreds of different aspects of the
biomanufacturing field into one well-understood guidebook. The end result is driven by those concerned with the quality
of the medicinal product, the regulation of the industry and the goal of always producing a robust, economic manufacturing
process for a biologic medicine.
Instead of writing a cookbook with exact instructions and “do this first, then do that” work recipes, we give you a firm
understanding of your toolbox and the rules of play when applying those tools to your task at hand. There are references
on where to look for the recipes, and also what to expect when it comes to future developments. More importantly, when
you have worked through any of the chapters in this book you should be well equipped to recognize a great recipe. You will
learn how to combine different recipes into the best meal, a well-integrated, cost efficient, and sustainable process, which
tastes good to both the management of your business and to the regulators who have to approve the product coming out of it.
When we decided to write this new edition of the 2007 Handbook of Process Chromatography, we realized that we
could no longer limit ourselves to purification technology. The time when different parts of a process were developed on
islands, such as the one called “Upstream Processing” or “Downstream Processing,” is over. We have finally arrived in the
age of “The Process is the Product.” Continuing along this line of thought, we realized that successful “integration” must
be based on good understanding of the environment we do this in, the world with its disease and healthcare challenges and
its businesses with their microcosms of priorities and financial performance drivers.
Compiling a story such as this is not a trivial challenge, and I struggle to find enough words of gratitude for the many
friends in our industry and my company GE Healthcare who agreed to spend some extra time outside their daily duties at
work, stealing time from their private lives to author a chapter, and more importantly, provide their fantastic expertise and
experienced judgment for the benefit of this book. My co-editors Karol Łącki, Parrish Galliher, and Eva Lindskog have
been a tremendous support in getting this all organized and processed once the 55 chapter manuscripts started to come in.
None of us assumed that the better part of 3 years would pass from the initial idea to completion of the manuscript drafts.
A special thought goes to Karol who not only has been my “scientific conscience” (and advisor) over the many years we
worked together, but who continued to provide his loyalty with the project and his great attention to detail of the science
after he moved on to pursue his career outside of GE Healthcare. A special apology goes to those authors who were very
early with their deliveries. Not the least, I owe my former manager and mentor Nigel Darby a big thank you for allowing
the time I spent on this book, which was perhaps a little underestimated at the beginning.
The story of the book is organized into 10 sections beginning with a review of the global burden of disease and the
priorities coming from that perspective for the development of novel medicines. Reviews of the companies active in bio-
pharmaceuticals, their current products and future portfolio, and conclusions related to the processing capabilities required
to deliver those medicines complete Section I.

xxix
xxx  Preface

Sections II through IV discuss the methods used for bioprocessing in the three stages upstream, product recovery, and
downstream processing, from cells and cell culture to bulk drug substance at the end of the purification methods section.
The upstream processing Section II has chapters discussing basic principles, the variety of industrial producer cells and the
rationale for selecting them, the development of a mammalian cell line, and cell culture media. In the Product Recovery
section, you will find an industry review of approaches successfully applied to isolate both products from mammalian cell
culture and from bacterial producer cells. We have given much room to a thorough discussion of what is often referred to as
“alternative processing methods” in the space between the bioreactor and the purification train. Four chapters look at pre-
cipitation, flocculation, crystallization, extraction, and expanded bed adsorption (EBA) and their capabilities in large-scale
processing prior to the first column chromatography step. Principles and science of filtration as well as filtration methods
used for product recovery are explained in separate chapters. In Section IV, all major chromatographic methods used for
protein purification are described in detail (affinity, ion exchange, hydrophobic interaction and reversed phase, size exclu-
sion, and multimodal concepts of chromatography). This part is introduced by an extensive discussion of the principles and
science of chromatography. Filtration methods used for purification conclude this section.
Section V covers processing equipment used (bioreactors, chromatography and filtration skids, and process chro-
matography columns) for large scale operations as well as automation principles and strategies. One chapter is dedi-
cated to single-use technology (SUT) as an essential element in many concepts for the manufacturing facilities of the
future. Automated preparation of buffers for chromatography and filtration, avoiding most of the large floor space for
storage of these process liquids, is emphasized in a separate chapter. Continuous purification with periodic counter-
current chromatography (PCC) is discussed as one of the current main hopes for the next level of productivity in
bioprocessing.
Section VI first discusses concepts for modern process design in upstream and downstream process development, in-
cluding a major aspect of process integrity and product safety, the cleaning-in-place (CIP) of processing equipment and
consumables. This is followed by a series of industrial reviews on simplification and intensification of processes, the
implementation of single-use technology, continuous processing with its pros and cons, and high-density cell culture as a
candidate to shorten the seed train leading to production bioreactors. Product (and process) specific case studies follow in
dedicated chapters and discuss a project to optimize a microbial process, the design of next-generation processes for mono-
clonal antibodies (mAbs), antibody drug conjugate (ADC), and bi-specific antibody processing, and the bioprocessing of
human plasma IgG, vaccines, and cell therapy products.
Section VII is dedicated to the design of facilities for bioprocessing and their operation. A detailed study of design con-
cepts used today and in the foreseeable future is complemented with key points to consider in manufacturing operations.
Section VIII introduces selected topics from the wide field of analytical and regulatory aspects of bioprocessing. An
extensive chapter presents modern analytical concepts and methods for biotherapeutics and their application to support
different aspects of development and manufacturing. This is complemented with a case study on quality by design (QbD)
and process analytical technology (PAT). Pathogen safety is central to all bioprocessing and has its own review in this sec-
tion. Towards the first consolidation of a process development project, the Chemistry, Manufacturing, and Controls (CMC)
document is discussed in a dedicated chapter and describes the information gathered about the process and the designs
chosen. Two different chapters discuss post-licensure process changes, the considerations when evaluating them, and the
potential consequences for the marketing of a drug. In the final chapter, we cover an aspect that has received more and more
attention in recent years, the security of supply.
In Section IX, we focus on the economics of biopharmaceutical companies from two different angles: first, the reader
is introduced to the language and some key concepts of financial management and second, a review of the management of
process economics. These offer insights into key drivers and priorities of improving financial performance in biotherapeu-
tics processing. We are not telling you exactly how much it will cost to make a gram of antibody (even though we quote
a few papers trying to calculate that). Instead, we walk you through the winding path of considerations leading to the key
aspects of a successful financial improvement program. A hierarchy of financially relevant decisions with regard to both
cost and revenue is developed for your orientation. As with the process recipes, we believe that this enables you best to
develop your own program and to recognize important considerations. The last chapter rounds off and summarizes many
of the ideas developed through the preceding chapters on the economic aspects of methods or approaches taken in develop-
ment and manufacturing.
Finally, in three appendices, you can find what we call an “ideal world R&D portfolio,” a brief history of innovations
that enabled our industry and extend into its foreseeable future, and a listing of approved biotherapeutics with some key
features for each of them.
 Preface  xxxi

It has been a tremendously exciting journey to produce “Biopharmaceutical Processing.” Let me as the final thought of
this introduction thank our families who have endured a long period of irregular working habits. We love them, and we owe
them a big hug and thank you for all their support and patience in the last couple of years. Our readers will hopefully find
our efforts worth their while.

On behalf of the editing team,

Günter Jagschies.
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Section I

Healthcare and Biopharma

Industry, Products and Processes
This page intentionally left blank
Chapter 1

Disease and Healthcare Priorities

Günter Jagschies
GE Healthcare Life Sciences, Freiburg im Breisgau, Germany

1.1 INTRODUCTION
Most readers will either be directly involved with developing or operating bioprocesses, be managing teams busy with do-
ing so, or be interested in the topic for educational reasons. While textbooks covering the field typically provide detail about
the technical aspects of bioprocessing, this chapter is offering a discussion and background of why many of us come to
work every morning: the fact that bioprocessing is a key activity in making much needed medicines and vaccines available
to people! This first chapter provides a view on the current status and the dynamics of health around the globe and devel-
ops a set of priorities for medicines development by reviewing work performed, e.g., by teams within the United Nations,
the World Health Organization (WHO), the European Union, and the Seattle, WA based Institute for Health Metrics and
Evaluation (IHME) led by Christopher J.L. Murray. We have condensed the priority discussion into an “ideal R&D port-
folio” covering medicines, diagnostics, and studies into effective preventive measures (see Appendix I). This chapter may
serve as background to understand the strategic moves businesses in healthcare make and the environment within which we
all perform our work in the field of biopharmaceutical processing. It is by far not all about medicines!
Human disease may be defined as any harmful change that interferes with the normal appearance, structure, or function
of the body or any of its parts. Most diseases are becoming increasingly common around the globe. This is true, in general, as
prosperity with its positive and negative side-effects on health is gradually spreading and, for infectious diseases, because of
global travel and, potentially, climate change leading to migration of infectious pathogens and their carriers. The main excep-
tion to the rule is the still higher, in some cases almost exclusive, prevalence of certain infectious diseases in the developing
world, putting a double burden of disease onto these areas. The health status is very different for particular populations and
dependent to a large degree on which geographical region one lives in and the level at which healthcare is accessible and
affordable in that same region. We all have disease in common, but the impact on an individual's health is a different matter.
The United Nations' Millennium Development Goals (MDG) set the framework for global priorities on health promot-
ing programs including some major selected disease challenges. The MDGs comprise eight goals (Table 1.1) and a set of
measurable time-bound targets, and have established a blueprint for tackling the most pressing development challenges of
our time [2]. Four of these goals are directly addressing disease and health (# 4, 5, 6, 7) and three more are substantially
related to developing better chances for a healthy life over time (# 1, 2, 3). Very significant progress has been made, but
there is also a lot left to achieve.
Table 1.1 summarizes a few of the achievements relevant to healthcare. Access to clean water and basic sanitation,
prevention of early child mortality and improved maternal health, reduction of extreme poverty with its causes and conse-
quences, such as chronic hunger are the most fundamental aspects. The reduction of disease, particularly the “big-three”
infectious disease HIV/AIDS, Malaria, and Tuberculosis are also ranking high in priority among the health related MDGs.

1.1.1 Two Core Scenarios for Healthcare

There are effectively two very different scenarios for a disease and healthcare priorities discussion: the situation in more
developed parts of the world (high income countries) and the one in poor regions of the world (low income countries). Many
areas are on their way to prosperity and show features of both scenarios (middle income countries). And there are excep-
tions, i.e., with significantly more advanced healthcare than their level of wealth would suggest (e.g., Cuba).
The main distinction between the two scenarios is defined by poverty related issues such as exposure to environmental
hazards, including compromised water quality, air pollution both in-house and in the open, lack of sanitation, insufficient
nutrition, and lack of access to medical services or medicines. This may either directly increase the risk for infectious

Biopharmaceutical Processing. https://doi.org/10.1016/B978-0-08-100623-8.00001-3

© 2018 Elsevier Ltd. All rights reserved. 3
4 SECTION | I Healthcare and Biopharma Industry, Products and Processes

TABLE 1.1 Millennium Development Goals: Selected Targets of Relevance for Health Status and Comments
on Achievements [1]
# Millennium Goal Selected Targets Achievements and remaining challenges
1 Eradicate extreme 50% reduction of extreme poverty 1 billion people lifted out of extreme poverty
poverty and hunger and chronic hunger Young people, esp. women at high risk for vulnerable
Full and productive employment employment

2 Achieve universal Full course of primary schooling, boys 91% enrolment in developing regions
primary education and girls alike 57 million children still out-of-school

3 Promote gender equality Eliminate gender disparity at all levels Primary education improved; gender disparity remains
and empower women of education at higher level

4 Reduce child mortality Reduce <age 5 mortality by 2/3 More children can be saved from preventable deaths
(e.g., measles)
Focus more on new-borns

5 Improve maternal health Reduce maternal mortality ratio by 75% 56% of births in rural areas vs 87% in urban areas
Universal access to reproductive health attended by skilled staff
Adolescent child bearing still high

6 Combat HIV/AIDS, Halt and reverse spread of HIV/AIDS New HIV infections fell 40%; 13.6 million receive
Malaria and other Halt and reverse malaria and other antiretroviral therapy
diseases major diseases incidence 6.2 million Malaria deaths averted; 900 million
insecticide-treated mosquito nets distributed; 85% of
newly diagnosed tuberculosis cases successfully treated

7 Ensure environmental 50% reduction of population without Drinking water target achieved 5 years ahead of schedule
sustainability access to safe water and basic sanitation 2.1 billion people gained access to improved sanitation;
2.4 billion still without sanitation

8 Global partnership for Access to affordable, essential drugs Evidence suggests improvement, data limited
development in developing countries

disease in general and certain noninfectious disease in particular (e.g., chronic lung disease), or may generally worsen the
chances to live a healthy life. Lack of access to vaccination, lack of access to medical services during pregnancy and birth
or medical care for the new-born, and increased risks for injuries are just a few examples for the complex relation between
a low level of wealth and the compromised health status of a population (Table 1.2).
At a finer granularity, the burden from out-of-pocket payments for healthcare or, in reverse, the availability of health
insurance, the spread of medical care centers, and eventually any dominant cultural influence that discriminates parts of the
population (often girls and women or certain minority groups) are factors that may overlap with the two main scenarios.
The noninsured may lack access to essential healthcare even in an otherwise prosperous environment, a possible example
being rural areas versus urban environments in the same country or a loss of health insurance for millions in the United
States as a consequence of policy decisions by the Trump administration. In contrast, some governments of low income
countries have decided to support health development far above the average of countries at their income level and have
made great progress with their programs.

1.1.2 Poverty Scenario—Essential Healthcare Not Available or Unaffordable

Many of those health compromising factors discussed above still persist in the poorest areas of the world, in Sub-Saharan
Africa, some parts of Southern Asia, in countries that are or have recently been facing war or civil war, and in some rural
areas of countries that have only during the last 20–50 years left wide spread extreme poverty behind them.
The World Bank states on its website: “There has been marked progress on reducing poverty over the past two decades
(most notably in East Asia). The world attained the first Millennium Development Goal target—to cut the 1990 poverty
rate in half by 2015—5 years ahead of schedule, in 2010. Despite this progress, the number of people living in poverty
globally remains unacceptably high” [3]. We are looking at approximately 1 billion people who are today still living in
“extreme poverty” (income < $1.25/day) and an estimated 2.2 billion under what is defined as an “average poverty” line
in the developing world (income < $2.00/day). In 2011, over 80% of the extremely poor lived, almost equally distributed,
in South Asia and Sub-Saharan Africa [3].
 Disease and Healthcare Priorities Chapter | 1 5

TABLE 1.2 Top Priority—Fundamental Conditions for a Healthy Life

Condition Effect
Clean water Prevention of infectious disease

Access to Sanitation Prevention of infectious disease

Sufficient nutrition Improved chances for normal healthy life

Air pollution (in-house and out-house) Prevention of respirational tract disease

Access to medical services Prevention of mother/child disease or death

Vaccination of children Long-term reduction of communicable disease burden

Safety standards in traffic, at work, at home Reduction of injuries and disease

Absence of violence (war or interpersonal) Reduction of injuries, more productive society

For the poverty scenario, creating and stabilizing the fundamental conditions for health constitute the highest priority for improve-
ments, paired with key preventive medical interventions, including healthcare access for pregnant women and newborn babies,
and basic vaccination of all children. Therapeutic products covering the basic needs must be available at affordable, usually very
low cost in these areas. Advanced, more recently developed therapeutics such as many biopharmaceuticals will not be afford-
able for this group if they remain in poverty or unless the nations they live in create subsidized insurance programs making more
advanced medical interventions a possibility, even for those who can't afford them out of pocket.

1.1.3 Prosperity Scenario—Essential Healthcare Available and Affordable

“In 1900, the leading causes of death in the United States were pneumonia, influenza and tuberculosis. A century later, they
are heart disease and cancer” [4]. Elementary health challenges have been overcome. This has been the case for almost a
century for most of the population, the “middle class,” in Western Europe (apart from periods of war) and North America
(apart from the uninsured population), and several other countries that enjoy similar standards of living (e.g., Australia,
New Zealand, Singapore, Israel, South Korea, and Japan). Now, during the two decades from 2010 to 2030 three billion new
“members” will join the world's middle class, almost 90% of them in the Asia Pacific region. Middle class can be defined
by daily purchasing power parity1 between $10 and $100 per person [5]. People in this group generally have access to and
can afford a good level of healthcare provided they are members of an insurance program.
Once in this fortunate position, a new dynamic sets in: this group begins to demand improved quality and is no longer satisfied
with “just access” and status quo (Fig 1.1). According to a 2008 poll conducted by Harris Interactive [6], the US healthcare system
may be the least popular, with more than 33% demanding a complete rebuild of the system, another 50% voting that fundamental
changes are required and only 12% thinking the system is generally working well. Italy on the other side of the Atlantic is not
considered much better. The Dutch and the French systems are a lot more popular at 42% and 29% positive ratings, respectively.
At the World Bank MIC2 forum in April 2013 it was argued that in order to stabilize the positive economic development
coming along with a growing middle class in the long term, countries would need to put in place policies that motivate the
middle class to opt into a “social contract” [7]. The participants did not take this “opt-in” as a given: it would require good
social services, namely insurances, high quality of education, avoidance of unbalanced burden on the young generation
from the needs of the ageing population, and a positive outlook of stability for the large group who initially, after leav-
ing poverty, would still be vulnerable. Without these measures, even the established middle class would face the risk of
eventually dropping into poverty again. The implementation of policy maintaining the “social contract” is viewed to be a
prerequisite for achieving a stability of prosperity and health like the levels that the Western World has enjoyed for quite a
long time now. There are multiple conditions and behaviors promoting health or preventing disease that need to be in place
before the health status of a human being, a society, or a geographical region can reach satisfactory levels.

1. Purchasing Power Parity (PPP) is used worldwide to compare the income levels in different countries. It adjusts the exchange rates of two currencies to
make them at par with the purchasing power of each other. PPP thus makes it possible to understand and interpret income data of each country even with
very different local economic conditions.
2. MIC, middle income countries.
6 SECTION | I Healthcare and Biopharma Industry, Products and Processes

The Netherlands

France

Canada

Australia
Complete rebuild required
Spain Fundamental changes needed
Not sure
Great Britain
Minor changes required
Germany

United States

Italy

0% 20% 40% 60% 80% 100%

FIG. 1.1 Survey of satisfaction with national healthcare systems: popular demand for improvement of the good [6].

The prosperity scenario of healthcare has its priorities in providing advanced health services to everyone at the time needed, in
providing social insurances covering state-of-the art of medicine and healthcare for everyone, and in supporting further progress
via developments in medical and pharmaceutical research that lead to closing the gaps in prevention or treatment of disease. In
stating the latter, we assume that such research will only be affordable in economies that have achieved the essential health priori-
ties for their population and can focus part of their wealth on further improving the medical toolkit.

Historically, it has hardly ever happened that an improvement of living standards or of essential knowledge and tools has
become available everywhere immediately once introduced at the place of invention. Today's digital networks, global trade,
and travel may provide knowledge and products everywhere on the globe with minor delays, but that should not be confused
with the affordability issue and the prioritization of national budgets, i.e., the implementation of structures and services
providing the healthcare and medication access for whole populations, which is still a different, often very complex matter.

1.2 STATUS AND MEASUREMENT OF GLOBAL HEALTH

With poverty and prosperity resulting in very different scenarios in different parts of the world, one may expect similar dif-
ferences for the health situation of populations in these areas. The Institute for Health Metrics and Evaluation (IHME) has
coordinated work on the Global Burden of Disease Reports 2010 [8–10] and 2013 [11,12], with the intention to provide policy
makers with comprehensive and standardized data on diseases, injuries, and risk factors across age groups, gender and geog-
raphy. These reports that are now regularly updated and amended and additional studies, e.g., from the United Nations, the
World Health Organization (WHO), and the World Bank confirm the conclusion. The following discussion attempts to provide
a summary of the findings and give guidance useful for the later review of medical research priorities (Section 1.6).

1.2.1 Life Expectancy and Mortality

In reports looking at disease and health status of populations, the consequences of disease are often summarized using
­parameters such as life expectancy3 or mortality [13]4, i.e., condensing the individual human situation into a fact based
health management terminology.
At this lowest level of granularity, the status of health in a population may be described using life expectancy at birth,
i.e., considering the conditions in the area that a child is born into but not analyzing underlying factors or future trends on

3. Life expectancy (at birth) equals the average number of years a person born in each area would live if mortality rates (see next footnote) at each age
were to remain constant in the future. The number reflects the quality and safety of life in the region, including impacts from healthcare quality, disease
burden, specific health risk factors, and violence.
4. Mortality is another term for death. The “crude death rate” (CDR) is the mortality due to a disease divided by the total population and provides input on
the size of the challenge for a healthcare system. CDR is influenced by the age composition of a population. For meaningful comparisons between regions
or over time the crude rate thus needs to be adjusted for demographic differences between populations using the “age standardized death rate” (ASDR).
Life expectancy can be calculated from ASDR using so called Life Tables.
 Disease and Healthcare Priorities Chapter | 1 7

48 84
50 55 60 65 70 75 80
FIG. 1.2 Life expectancy at birth 2013 (both sexes), Sub-Saharan Africa populations and parts of South Asia have up to 36 years lower life expectancy
than people in the most developed economies. Reproduced with permission from Institute for Health Metrics and Evaluation (IHME), GBD Compare,
Seattle, WA, IHME, University of Washington, 2016, http://vizhub.healthdata.org/gbd-compare/ (accessed January 2017).

factors impacting health (Fig. 1.2). Life expectancy at birth has increased by more than 6 years since 1990, to 71.5 years
in 2013 (global average, both sexes) and the mean age of death has been increasing to 59.3 years in 2013 [12]. In Western
Europe, Israel, Canada, Japan, South Korea, Australia, New Zealand, and Chile a child born in 2012 is estimated to have a
life expectancy of >80 years (range 80–84). The United States is not too far behind at 79 years. On average, children born
in Eastern Europe, Central and South America, Northern Africa and the Middle East, as well as China and South East Asia
have a 5–10 years lower life expectancy. Russia, Central Asia, India, and some African countries have 10–20 years lower
life expectancy. In most Sub-Saharan countries of Africa, the average life expectancy at birth is between 20 and 30 years
lower than in the industrialized parts of the world. In the poorest countries of Sub-Saharan Africa, 10%–20% of all children
are still expected to die below the age of 5 [14] (see details in Fig. 1.3).
The WHO estimated for 2013 that, on average, disease burden and health risks resulted in a loss of nearly 9 years of healthy
life overall per individual. Global Health Adjusted Life Expectancy (HALE) at birth, a measure estimating the time lived at
“full health” [15], was 62 years versus the actual life expectancy of 71 years averaged for males and females. Due to successful
improvement of the health situation for children, the number of deaths for the youngest (< age 5) has declined by ~50%. Child
mortality up to age 5, however, is 12-fold higher in low income compared to high income countries. However, the countries
with lowest child mortality (Luxembourg, Iceland) versus the highest in this comparison (Sub-Saharan region) differ by a fac-
tor of 60–80 (2 deaths before age 5 per 1000 live births versus 167 at the maximum), see Fig. 1.3. Significant differences exist
also between sexes at adult age. Correcting for change of age composition of the population, it becomes obvious that the world
has made significant progress on health status: the age standardized death rate (ASDR) fell by almost 25%.
While equal opportunity between genders is a key feature of an advanced health promotion program, men and women
are not the same and today still do not share the same daily environment seen from the mortality risk point of view. The
differences by gender and their social, cultural, and biological context need to be considered for the improvement programs
to be acceptable to the target groups and to be overall successful.

1.2.2 Morbidity and Disease Burden

Disease results in various stages of loss of well-being ranging from a mere disturbance, without much else than a small
individual impact, through temporary inability to perform daily tasks, e.g., with short term sick-leave from work, up to
longer term disability, paired with dependency on others and significant economic implications both for an individual, any
dependent family, and the society. Ultimately, disease may lead to death, but the clear majority of disease incidents will only
involve varying degrees of inability or disability. Any serious attempt to objectively measure the burden of disease on a pop-
ulation and to recommend health program priorities therefore needs to take everything into account, at all levels of impact.
With improvements made in disease related interventions the survival rates have significantly improved, and mortal-
ity alone is not any more sufficient to indicate a populations' health status. Indicators of morbidity5 such as the prevalence

5. Morbidity refers to the diseases and illness, injuries and disabilities in a population. Prevalence, the total number of cases (newly diagnosed incidences
plus previously diagnosed patients still alive) in a population may be used to determine the level of morbidity in a population, which then forms guidance
complementary to mortality information.
 8
SECTION | I Healthcare and Biopharma Industry, Products and Processes
FIG. 1.3 Healthcare priority measured as “under-five mortality;” countries in Sub-Saharan Africa and South/South East Asia face a much more fundamental health challenge than countries in
the upper-middle or high income group of countries. Data from H. Wang, M. Naghavi, C. Allen, R.M. Barber, Z.A. Bhutta, A. Carter…C.J.L. Murray, Global, regional, and national life expectancy,
all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2013;2015: a systematic analysis for the Global Burden of Disease Study 2015, The Lancet 388 (10053) (2016)
1459–1544; Population: data from CIA, World Factbook (https://www.cia.gov/library/publications/resources/the-world-factbook/index.html Central Intelligence Agency (CIA), The World Factbook,
2015); Income data source World Bank, World Development Indicators 2016, World Bank, Washington, DC, 2016, https://doi.org/10.1596/978–1-4648–0683–4; Adopted and updated from K. Owen,
Bill and Medina Gates Foundation, Presented at DCVMAnnual Meeting 2016, Buenos Aires, Argentina.
 Disease and Healthcare Priorities Chapter | 1 9

DALY = YLD + YLL

Overall disease burden Years with disability Years Lost

Highest
Birth Periods of Premature life
illness/injury death expectancy

Heathy life Years with

Years of
Disease/Disability
(accumulated)
life lost

0 Years 80
FIG. 1.4 Disability-adjusted life years (DALY), a concept to accommodate disease burden including both fatal and nonfatal outcomes. This concept has
been developed by Christopher J.L. Murray and his team at the Institute for Health Metrics and Evaluation (IHME), Seattle, WA and was initially highly
controversial in a global system of mortality focused activity funding and priority setting (based on: J.N. Smith, Epic Measures: One Doctor. Seven Billion
Patients, HarperCollins, https://books.google.hu/books?id=EvqEoAEACAAJ, 2015.)

of chronic diseases and injuries, as well as disease burden descriptors such as “Disability-Adjusted Life Year (DALY)”6
become important in order to cover other consequences of disease (sequelae7) than death in a meaningful way [16]. “Years
Lived with Disability (YLD)”8, and “years of life lost (YLL)”9 are the parameters that sum up to DALYs in that they cover
the time with disease impact before death and the lost time due to premature death (Fig 1.4). A high YLL value means first
that death occurs prematurely before reaching the maximum life expectancy, but also significant mortality for the disease or
condition. A low YLL means that death typically occurs closer to the maximum life expectancy. A high YLD value means
high prevalence (many people are living with the disease or condition) and a high burden from the consequences measured
by the “weight factor” for each sequela. As the sum of YLL and YLD, DALYs are being used as a powerful tool for priority
setting as they measure disease burden from nonfatal as well as fatal conditions.
Table 1.3 shows a summary of the top 30 diseases, conditions, and injuries by global disease burden (DALY), years
lived with disability (YLD), years of life lost (YLL), prevalence, and mortality. In 2013 global mortality was at 55 million,
an increase of ~16% over 1990 in absolute numbers but a decline of 24% when standardized for ageing and normalized for
population growth. The overall disease burden decreased slightly (−3.6%) between 1990 and 2013. This trend is composed
of a 33% increase in years lived with disability indicating an improved survival of disease and a 16% decline in years of life
lost, which mainly is caused by the successful reduction in child mortality [12].

1.3 THE WORLD IS CHANGING—DEMOGRAPHIC TRANSITION

The human population on our planet has passed 7 billion people in 2011 and by the end of the century, it is expected to
increase to 11.2 billion [20]. UNICEF estimates that ~4 babies are born each second, which equals 130 M births per year.
Largely correlating to income levels, population growth is, however, very unevenly distributed across geographic regions
and nations. The same is true for the age structure of different populations (see Fig. 1.7). For prioritization of disease re-
search and healthcare planning it is important to understand how the age structure of a population will develop as this is one
of the main drivers for the portfolio of diseases to be dealt with in the future.

1.3.1 Factors Driving Population Growth and Ageing

Population growth is driven by four major components: fertility, mortality, the current age structure of a population, and
migration to or from an area [21]. Ageing, i.e., change of the age structure, is driven by an increase in life expectancy
and declining fertility. Assuming improvements of prosperity and health, populations move from high mortality and high

6. One DALY can be viewed as a lost year of healthy life. In a population, the sum of DALYs represents the gap between the current health status and the
ideal health situation. DALYs are calculated as the sum of years of life lost (YLL) due to premature death and years lived with disability (YLD). DALY
is the key parameter used to quantify morbidity.
7. Sequela is a consequence of disease or injury such as the degree of disability or inability.
8. YLD is a cause specific parameter. It is calculated as the number of prevalent cases times the “disability weight,” which reflects the severity of the
disease on a scale from 0 (perfect health) to 1 (equivalent to death).
9. YLL is the number of life years lost due to premature death. It is accumulated from the differences between highest known life expectancy (in any
population) and the age at premature death for each incident.
TABLE 1.3 Health Losses From Global Top 30 Disorders by Disease Burden (DALY), Years Lived with Disability (YLD), Years of Life Lost (YLL), Prevalence,
and Mortality: Shift From Communicable to Noncommunicable Disease and to Longer Survival With Disease/Disability
Rank
DALY [12] (× 1000) = YLD Δ DALY % Prevalence Mortality Δ Mortality comp.
Disorder [13] + YLL [17] vs 1990 [13] (× 1000) [14] (× 1000) % vs 1990 (D/M) Comments
Ischemic [15] heart disease 150,239 = 5804 + 144,434 32.3 92,937 8140 41.7 1/1

Lower respiratory infections 113,363 = 461 + 112,902 −48.2 4473 2653 −22.2 2/4 High (59%) mortality relative to prevalence;
influenza and pneumonia

Cerebrovascular [15] disease (stroke) 112,879 = 3744 + 109,135 25.1 25,669 6447 40.2 3/2 High (25%) mortality relative to prevalence,
long convalescence process

Low back pain and neck pain 106,666 = 106,666 + 0 55.8 1,000,314 0 n.a. 4/– Slipped disc et al.

Road injuries 73,251 = 8593 + 64,658 13.6 171,494 1396 32.4 5/7 Traffic safety in developing world

Diarrheal [15] diseases 72,797 = 6854 + 65,942 −59.5 42,410 1264 −51.0 6/10 Major progress with young children
a
COPD 71,901 = 26,131 + 45,769 16.8 328,504 2931 21.0 7/3

Preterm birth complications 70,843 = 6690 + 64,153 −49.0 56,272 742 −52.9 8/15 Major progress on maternal health

HIV [15]/AIDS [15] 69,363 = 4064 + 65,300 341.5 29,331 1341 374.2 9/8 Peak 2005–06, slow decline since then

Malaria 65,493 = 3171 + 62,323 −3.9 351,051 dia854 −4.4 10/13 Peak in 2004, malaria estimates are
considered highly uncertain

Depressive disorders 61,632 = 61,633 + 0 53.6 355,724 0 n.a. 11/–

Neonatal encephalopathy [15] 58,013 = 2388 + 55,625 −24.5 22,859 644 −26.1 12/16

Congenital anomalies [15] 57,173 = 8023 + 49,151 −11.7 88,095 632 −13.5 13/17

Diabetes 55,832 = 29,518 + 26,315 97.5 409,967 1299 89.7 14/9

Sense organ diseases(eyes and ears) 54,428 = 54,428 + 0 47.4 2,049,200 0 n.a. 15/– Main condition: hearing loss

Tuberculosis 49,816 = 3670 + 46,147 −28.4 12,112 129 −27.7 16/25

Iron-deficiency anemia [15] 43,748 = 36,664 + 7084 −14.7 1,208,216 183 −13.8 17/23

Skin/subcutaneous diseases 41,598 = 39,051 + 2545 36.5 24,80,527 99 68.3 18/26 Highest: fungal skin diseases, Acne
vulgaris, and dermatitis

Cirrhosisbof the liver 36,858 = 545 + 36,313 35.6 3297 1221 45.6 19/11 High (37%) mortality relative to prevalence

Self-harm 35,170 = 232 + 34,939 9.3 5941 842 17.8 20/14 Suicides
c
Cancer of trachea, bronchus, lung 34,733 = 467 + 34,266 39.6 3227 1634 56.5 21/6 High (50%) mortality relative to prevalence
Chronic kidney disease 33,187 = 12,347 + 20,840 72.4 471,916 956 134.2 22/12

Neonatal sepsis [18]/infections 31,632 = 7 + 31,625 6.2 50 366 6.1 23/22

Migraine 28,898 = 28,898 + 0 46.1 848,367 0 n.a. 24/–

Protein-energy malnutrition 27,771 = 2574 + 25,136 −27.1 20,757 469 −7.3 25/20

Falls 27,491 = 12,818 + 14,673 21.1 250,044 556 66.7 26/18

Neglected tropical diseases (NTDs) 25,184 = 16,939 + 8253 −27.5 2,185,979 143 −30.0 27/24 17 different diseases, very high prevalence

Alzheimer's disease/dementias 22,239 = 7774 + 14,465 90.3 53,051 1655 107.2 28/5 Alzheimer's disease higher on years lost to
disease than all cancers, fastest growing

Asthma 22,183 = 10,596 + 11,587 −2.6 241,695 489 −2.9 29/19

Interpers violence 21,404 = 822 + 20,582 9.4 20,082 405 18.4 30/21 Murder, violent crime (highest in Latin
America, #1 YLL cause in some countries)

Total (all causes) 24,49,810 = 764,804 + −3.6 26,526,084 54,863 15.6 Age standardized mortality: −21.5%
16,85,006

Communicable 486,826 = 45,739 + 441,087 −27.1 4,947,734 8787 −24.1 88% of all deaths in low and lower middle
income countries, NTDs in top ten YLD list

Maternal, neonatal, nutrition 282,463 = 55,756 + 226,707 −25.5 1,440,139 3023 −33.8 Progress in developing regions

Noncommunicable diseases (NCDs) 14,32,939 = 626,478 + 32.6 19,398,748 38,267 41.7

806,461

Neoplasms [19] 197,094 = 6764 + 190,330 29.1 94,518 8236 45.6 1/1 Breast, prostate, colon and rectum leading
by prevalences, lung et al leading by
mortality

Neurol. disordersd 84,048 = 59,360 + 24,688 55.2 2,555,646 1977 94.0 5/5 Fastest growing category of fatal disease
(Alzheimer's disease doubles in 20 years)

Injuries 278,665 = 36,832 + 210,751 −8.4 739,463 4787 10.4

a
Ischaemia, restriction in blood supply to tissues; cerebrovascular, disease of the cerebral circulation; diarrhea, condition of having at least three loose or liquid bowel movements each day; COPD, chronic obstructive
pulmonary disease; HIV, human immunodeficiency virus; AIDS, acquired immune deficiency syndrome; encephalopathy, disease of the brain; congenital anomalies, birth defects; anemia, deficiency of red cells or of
hemoglobin in the blood.
b
Cirrhosis, condition with long-term liver damage; sepsis, presence in tissues of harmful bacteria and their toxins, typically through infection of a wound.
c
More than 100 cancers are collectively referred to as Neoplasms. The global morbidity and mortality from all Neoplasms puts cancer at rank #1 in DALY and mortality
d
Neurological disorders include Alzheimer's (AD), Parkinson's, and Multiple sclerosis (MS).
12 SECTION | I Healthcare and Biopharma Industry, Products and Processes

f­ ertility to low mortality and low fertility, a development referred to as “demographic transition” (Fig. 1.5). Fertility reaches
replacement level (stable population size), when women have one surviving daughter on average, i.e., at two children
per woman10. Fertility below replacement rate for a longer period is observed in many high and some medium income
countries. If migration and policy that supports families including equal opportunity for women cannot mitigate this, the
potential consequence may be a partial collapse of social security systems (the “social contract”) as the burden for the
young population from current forms of insurances and other support activities to care for the aged majority becomes first
unpopular and then unbearable.
On the other hand, long term fertility rates above replacement are expected for Africa: the continent is expected to
increase population by 3.2 billion people almost reaching the population size of Asia by the end of the 21st century. The
challenges to provide a stable economic climate of opportunity, a long-term sustainable basis of natural resources, and thus
the prerequisites for health for this growing population may be considered dramatic without success in reduction of fertil-
ity rates, which in turn is known to be strongly dependent on just that: economic improvement, a potentially vicious cycle!
Fig. 1.6 shows the population development across age groups. In Africa, all age groups are growing strongly, but most
notably the appearance of a significant old age group indicates a change in disease and health challenges: the number of

Older Fertility below replacement level

and Mortality low, stable
shrinking Old age population structure
population
Family Pr
os
planning pe
im rity a
pro nd
ve
me hea
nt lth Family
planning
Younger
Fertility above replacement level and
Mortality high, declining growing
Young age population structure population

FIG. 1.5 Demographic transition: the factors influencing population growth and age structure. In demographic models, the key assumption is that all
populations sooner or later will experience improvements in prosperity and health that trigger the transition.

Age 0–14 Age 14–24 Age 25–29 Age 60 plus Age 80 plus

1400
Population growth 2013–2100 [millions]

1200

1000

800

600

400

200

–200

–400
Oceania Northern America Latin America & Caribbean Europe Asia Africa
FIG. 1.6 Demographic transition in the 21st century: Africa and Asia drive global population growth and age structure changes; the world is ageing!

10. In practice, the ratio children/woman is slightly higher as the survival rate for babies is not 100% and because more boys than girls are born.
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et je dis à un policeman de me conduire à la direction générale du service
des détectives.
«Par bonheur, j’arrivai à temps, quoique le chef de la sûreté, le fameux
inspecteur Blunt, fût précisément sur le point de s’en aller chez lui. C’était
un homme de taille moyenne et d’une charpente ramassée, et, quand il
réfléchissait profondément, il avait une manière à lui de froncer les sourcils
et de se taper le front avec les doigts qui vous donnait tout de suite la
conviction que vous vous trouviez en présence d’un personnage comme il y
en a peu. Du premier coup d’œil il m’inspira de la confiance et me donna de
l’espoir.
«Je lui exposai l’objet de ma visite. Ma déclaration ne l’émut en aucune
façon, elle n’eut pas plus d’effet apparent sur son sang-froid de fer, que si
j’étais venu lui dire simplement qu’on m’avait volé mon chien; il m’offrit
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—«Permettez-moi de réfléchir un moment, je vous prie.»
«Cela dit, il s’assit à son bureau et resta la tête appuyée sur la main. Des
commis écrivaient à l’autre bout de la pièce: le grattement de leurs plumes
fut le seul bruit que j’entendis pendant les six ou sept minutes qui suivirent.
Entre temps l’inspecteur était enseveli dans ses pensées. Enfin il leva la
tête, et la fermeté des lignes de son visage me prouva que dans son cerveau
il avait achevé son travail, que son plan était arrêté. Alors, d’une voix basse
mais impressive:
—«Ce n’est pas un cas ordinaire. Chaque pas que nous allons faire doit
être fait avec prudence et il ne faut pas risquer un second pas avant d’être
sûr du premier. Il faut garder le secret, un secret profond et absolu. Ne
parlez à personne de cette affaire, pas même aux reporters. Je me charge
d’eux et j’aurai soin de ne leur laisser connaître que juste ce qu’il entre dans
mes vues de leur faire savoir.»
«Il toucha un timbre. Un garçon entra:
—«Alaric, dites aux reporters d’attendre.»
«Le garçon se retira.
—«Maintenant, en besogne et méthodiquement. On ne fait rien dans
notre métier sans une méthode stricte et minutieuse.»
«Il prit une plume et du papier.
—«Voyons. Le nom de l’éléphant?»
 —«Hassan-ben-Ali-ben-Sélim-Abdalah-Mohamed-Moïse-Alhallmall-
Jamset-Jejeeboy-Dhuleep-Sultan-Ebou-Bhoudpour.»
—«Très bien. Surnom?»
—«Jumbo.»
—«Très bien. Lieu de naissance?»
—«Capitale du Siam.»
—«Les parents, vivants?»
—«Non, morts.»
—«Ont-ils eu d’autres enfants que celui-ci?»
—«Non. Il est fils unique.»
—«Parfait. Cela suffit sur ce point. Maintenant ayez l’obligeance de me
faire la description de l’éléphant et n’omettez aucun détail, pas même le
plus insignifiant, je veux dire le plus insignifiant à votre point de vue, car
dans notre profession il n’y a pas de détails insignifiants; il n’en existe pas.»
«Je fis la description, il écrivit. Quand j’eus fini, il dit:
—«Écoutez, maintenant. Si j’ai commis des erreurs, veuillez les
corriger.»
«Il lut ce qui suit:
«Hauteur, dix-neuf pieds.
«Longueur, du sommet de la tête à l’insertion de la queue, vingt-six
pieds.
«Longueur de la trompe, seize pieds.
«Longueur de la queue, six pieds.
«Longueur totale, y compris la trompe et la queue, quarante-huit pieds.
«Longueur des défenses, neuf pieds et demi.
«Oreilles en rapport avec ces dimensions.
«Empreinte du pied: semblable à celle qu’on laisse dans la neige quand
on culbute un tonneau.
«Couleur de l’éléphant: blanc terne.
«Un trou de la grandeur d’une assiette dans chaque oreille pour
l’insertion des bijoux.
«A l’habitude, à un remarquable degré, de lancer de l’eau sur les
spectateurs et de maltraiter avec sa trompe, non seulement les personnes
qu’il connaît, mais celles qui lui sont absolument étrangères.
«Boite légèrement du pied droit de derrière.
«A une petite cicatrice sous l’aisselle gauche, provenant d’un ancien
furoncle.
«Portait au moment du vol une tour renfermant des sièges pour quinze
personnes et une couverture en drap d’or de la grandeur d’un tapis
ordinaire.»
«Il n’y avait pas d’erreur. L’inspecteur sonna, donna le signalement à
Alaric et dit:
—«Cinquante mille exemplaires à faire imprimer à la minute et à
envoyer par la malle-poste à tous les bureaux de mont-de-piété du
continent.»
«Alaric se retira.
—«Voilà pour le moment. Maintenant il nous faut une photographie de
l’objet volé.»
«Je la lui donnai. Il l’examina en connaisseur et dit:
—«On s’en contentera puisque nous ne pouvons faire mieux; mais il a la
trompe rentrée dans la bouche. Cela est fâcheux et pourra causer des
erreurs, car, évidemment, il n’est pas toujours dans cette position.»
«Il toucha le timbre.
—«Alaric, cinquante mille exemplaires de cette photographie, demain, à
la première heure, et expédiez par la malle avec les signalements.»
«Alaric se retira pour exécuter les ordres. L’inspecteur dit:
—«Il faudra offrir une récompense, naturellement. Voyons, quelle
somme?»
—«Combien croyez-vous?»
—«Pour commencer, je crois que... Disons vingt-cinq mille dollars.
C’est une affaire embrouillée et difficile. Il y a mille moyens d’échapper et
mille facilités de recel. Ces voleurs ont des amis et des complices partout.»
—«Dieu me bénisse! vous les connaissez donc!»
«La physionomie prudente, habile à ne laisser transparaître ni les
pensées ni les sentiments, ne me fournit aucun indice, pas plus que les mots
suivants, placidement prononcés:
 —«Ne vous occupez pas de cela. Je les connais ou je ne les connais pas.
Généralement nous avons vite une idée assez nette de l’auteur par la
manière dont le délit a été commis, et l’importance du profit possible pour
lui. Il ne s’agit pas d’un pickpocket ou d’un voleur de foires, mettez-vous
cela dans la tête. L’objet n’a pas été escamoté par un novice. Mais, comme
je le disais, considérant le voyage qu’il faudra accomplir, la diligence que
les voleurs mettront à faire disparaître leurs traces à mesure qu’ils
avanceront, vingt-cinq mille dollars me paraissent une faible somme, à quoi
nous pouvons cependant nous en tenir, pour commencer.»
«Nous partîmes donc de ce chiffre. Puis cet homme, qui n’oubliait rien
de ce qui pouvait fournir une indication, me dit:
—«Il y a des cas dans les annales de la police qui démontrent que parfois
des criminels ont été retrouvés par des singularités dans leur façon de se
nourrir. Pouvez-vous me dire ce que mange l’éléphant, et en quelle
quantité?»
—«Bon! ce qu’il mange? Il mange de tout. Il mangera un homme, il
mangera une bible. Il mangera n’importe quoi compris entre un homme et
une bible.»
—«C’est parfait. Un peu trop général toutefois. Il me faut quelques
détails. Les détails sont la seule chose utile dans notre métier. Très bien,
pour les hommes. Mais, voyons. A un repas, ou si vous préférez, en un jour,
combien d’hommes mangera-t-il, viande fraîche?»
—«Il lui importera peu qu’ils soient frais ou non. En un seul repas, il
pourra manger cinq hommes ordinaires.»
—«Parfait.—Cinq hommes.—C’est noté. Quelles nationalités préfère-t-
il?»
—«Il est tout à fait indifférent à la nationalité. Il préfère les gens qu’il
connaît, mais il n’a pas de parti pris contre les étrangers.»
—«Très bien! Maintenant, les bibles. Combien de bibles peut-il manger
à un repas?»
—«Il en mangera une édition tout entière.»
—«Ce n’est pas assez explicite. Parlez-vous de l’édition ordinaire, in-
octavo, ou de l’édition de famille, illustrée?»
—«Je ne crois pas qu’il se préoccupe des illustrations. C’est-à-dire je ne
pense pas qu’il fasse plus de cas des éditions illustrées que des autres.»
 —«Vous ne saisissez pas ma pensée. Je parle du volume. L’édition
ordinaire in-octavo pèse environ deux livres et demie, tandis que la grande
édition in-quarto, avec les illustrations, pèse dix ou douze livres. Combien
de bibles de Doré mangerait-il à un repas?»
—«Si vous connaissiez l’animal, vous ne demanderiez pas. Il prendrait
tout ce qu’on lui donnerait.»
—«Eh bien, calculez alors en dollars et en centimes. Il nous faut arriver
à nous fixer. Le Gustave Doré coûte cent dollars l’exemplaire, en cuir de
Russie, reliure à biseaux.»
—«Il lui faudrait une valeur d’environ cinquante mille dollars; mettons
une édition de cinq cents exemplaires.»
—«Bon, c’est plus exact. J’écris. Très bien: il aime les hommes et les
bibles. Ça va, qu’aime-t-il encore? Voyons... des détails...»
—«Il laissera les bibles pour des briques, il laissera les briques pour des
bouteilles, il laissera les bouteilles pour du drap, il laissera le drap pour des
chats, il laissera les chats pour des huîtres, il laissera les huîtres pour du
jambon, il laissera le jambon pour du sucre, il laissera le sucre pour des
pâtés, il laissera les pâtés pour des pommes de terre, il laissera les pommes
de terre pour du son, il laissera le son pour du foin, il laissera le foin pour de
l’avoine, il laissera l’avoine pour du riz qui a toujours formé sa principale
alimentation; il n’y a du reste rien qu’il ne mange si ce n’est du beurre
d’Europe; mais il en mangerait s’il l’aimait.»
—«Très bien, et quelle quantité en moyenne par repas?»
—«Nous disons environ... Eh bien! environ un quart de tonne à une
demi-tonne.»
—«Il boit?»
—«Tout ce qui est liquide: du lait, de l’eau, du whisky, de la mélasse, de
l’huile de ricin, de la térébenthine, de l’acide phénique... inutile d’insister
sur les détails; indiquez tous les liquides qui vous viennent à l’esprit;
d’ailleurs il boira n’importe quoi, excepté du café d’Europe.»
—«Très bien. Et quelle quantité?»
—«Mettons de cinq à quinze barriques, cela dépend de sa soif, qui varie,
mais son appétit ne varie pas.»
—«Ce sont des habitudes peu ordinaires; elles serviront à nous mettre
sur la piste.»
 «Il sonna.
—«Alaric, faites venir le capitaine Burns.»
«Burns arriva. L’inspecteur Blunt lui expliqua l’affaire, en entrant dans
tous les détails, puis il dit de ce ton clair et décisif d’un homme qui a son
plan nettement arrêté dans son esprit et qui est accoutumé à commander:
—«Capitaine Burns, vous chargerez les détectives Jones, Davis, Halsey,
Bates et Hackett de suivre l’éléphant comme une ombre.»
—«Oui, Monsieur.»
—«Vous chargerez les détectives Moses, Dakin, Murphy, Rogers,
Tupper, Higgins et Barthélemy de suivre les voleurs comme une ombre.»
—«Oui, Monsieur.»
—«Vous placerez un poste de trente hommes, trente hommes d’élite
avec un renfort de trente à l’endroit où l’éléphant a été volé, avec ordre de
faire faction nuit et jour, et de ne laisser approcher personne, excepté les
reporters, sans un ordre écrit de moi.»
—«Oui, Monsieur.»
—«Des détectives en bourgeois sur le chemin de fer, les bateaux à
vapeur et sur les bacs et bateaux de passeurs, et sur toutes les routes et tous
les chemins qui partent de Jersey-City, avec ordre de fouiller toutes les
personnes suspectes.»
—«Oui, Monsieur.»
—«Vous leur donnerez à chacun des photographies avec le signalement
de l’éléphant, et vous leur enjoindrez de fouiller tous les trains et tous les
bateaux et navires qui sortent du port.»
—«Oui, Monsieur.»
—«Si on trouve l’éléphant, vous le ferez arrêter et vous m’avertirez
immédiatement par télégraphe.»
—«Oui, Monsieur.»
—«Vous m’avertirez immédiatement si on trouve des empreintes de pied
d’animal ou toute autre chose de même nature.»
—«Oui, Monsieur.»
—«Vous vous ferez donner l’ordre enjoignant à la police du port de faire
des patrouilles vigilantes devant les façades des maisons.»
—«Oui, Monsieur.»
 —«Vous ferez partir des détectives en bourgeois, par les chemins de fer,
et ils iront au nord jusqu’au Canada, à l’ouest jusqu’à l’Ohio, au sud jusqu’à
Washington.»
—«Oui, Monsieur.»
—«Vous aurez des hommes sûrs et capables dans tous les bureaux de
télégraphes pour lire les dépêches, avec ordre de se faire interpréter toutes
les dépêches chiffrées.»
—«Oui, Monsieur.»
—«Que tout cela soit exécuté dans le plus profond secret, dans le plus
impénétrable secret.»
—«Oui, Monsieur.»
—«Vous viendrez sans faute me faire votre rapport à l’heure habituelle.»
—«Oui, Monsieur.»
—«Allez maintenant.»
—«Oui, Monsieur.»
«Il était parti. L’inspecteur Blunt demeura silencieux et pensif un
moment; le feu de son regard s’éteignit et disparut. Il se tourna vers moi et
me dit d’une voix calme:
—«Je n’aime pas à me vanter. Ce n’est pas mon habitude, mais je crois
pouvoir dire que nous trouverons l’éléphant.»
«Je lui pris les mains chaleureusement et le remerciai. J’étais sincère,
tout ce que je voyais de cet homme me le faisait aimer davantage, et me
faisait émerveiller sur les étonnants mystères de sa profession. Il était tard.
Nous nous séparâmes, et je retournai chez moi le cœur autrement joyeux
qu’à mon arrivée à son bureau.

II
«Le lendemain matin, les détails complets étaient dans tous les journaux.
Il y avait même, en supplément, l’exposé des théories de l’agent un tel, ou
un tel, sur la manière dont le coup avait été fait, sur les auteurs présumés du
vol, et la direction qu’ils avaient dû prendre avec leur butin. Il y avait onze
théories, embrassant toutes les possibilités. Et ce simple fait montra quels
gens indépendants sont les détectives. Il n’y avait pas deux théories
semblables, ou se rapprochant en quoi que ce fût, excepté sur un certain
point, sur lequel les onze étaient absolument d’accord. C’était que,
quoiqu’on eût bouleversé et démoli l’arrière de ma maison, et que la porte
seule fût restée fermée à clef, l’éléphant n’avait pu passer par la brèche
pratiquée, mais par quelque autre issue encore inconnue. Tous s’accordaient
à dire que les voleurs n’avaient pratiqué cette brèche que pour induire la
police en erreur. Cela ne me serait pas venu à l’idée, non plus qu’à tout
homme ordinaire, mais les détectives ne s’y laissèrent pas prendre un seul
instant.
«Ainsi la chose qui me paraissait la seule claire était celle où je m’étais
le plus lourdement trompé. Les onze théories mentionnaient toutes le nom
des voleurs supposés, mais pas deux ne donnaient les mêmes noms. Le
nombre total des personnes soupçonnées était de trente-sept. Les divers
comptes-rendus des journaux se terminaient par l’énoncé de l’opinion la
plus importante de toutes, celle de l’inspecteur en chef Blunt. Voici un
extrait de ce qu’on lisait:
«L’inspecteur en chef connaît les deux principaux coupables. Ils se
nomment «Brick Duffy» et «Rouge Mac Fadden». Dix jours avant que le
vol fût accompli, il en avait eu connaissance, et avait sans bruit pris les
mesures pour mettre à l’ombre ces deux coquins notoires. Malheureusement
on perdit leurs traces juste la nuit du rapt, et avant qu’on les eût retrouvées,
l’oiseau, c’est-à-dire l’éléphant, s’était envolé.
«Duffy et Mac Fadden sont les deux plus insolents vauriens de leur
profession. Le chef a des raisons de croire que ce sont les mêmes qui
dérobèrent, l’hiver dernier, par une nuit glaciale, le poêle du poste de police,
ce qui eut pour conséquence de mettre le chef et les hommes de police entre
les mains des médecins avant l’aube, les uns avec des doigts gelés, d’autres,
les oreilles, ou d’autres membres.»
«Après avoir lu la moitié de ce passage, je fus plus étonné que jamais de
la merveilleuse sagacité de cet homme. Non seulement il voyait d’un œil
clair tous les détails présents, mais l’avenir même ne lui était pas caché!
J’allai aussitôt à son bureau, et lui dis que je ne pouvais m’empêcher de
regretter qu’il n’eût pas fait tout d’abord arrêter ces gens et empêché ainsi le
mal et le dommage. Sa réponse fut simple et sans réplique:
—«Ce n’est point notre affaire de prévenir le crime, mais de le punir.
Nous ne pouvons pas le punir tant qu’il n’a pas été commis.»
 «Je lui fis remarquer en outre que le secret dont nous avions enveloppé
nos premières recherches avait été divulgué par les journaux; que non
seulement tous nos actes, mais même tous nos plans et projets avaient été
dévoilés, que l’on avait donné le nom de toutes les personnes soupçonnées;
elles n’auraient maintenant rien de plus pressé que de se déguiser ou de se
cacher.
—«Laissez faire. Ils éprouveront que, quand je serai prêt, ma main
s’appesantira sur eux, dans leurs retraites, avec autant de sûreté que la main
du destin. Pour les journaux, nous devons marcher avec eux. La renommée,
la réputation, l’attention constante du public sont le pain quotidien du
policier. Il doit rendre manifeste ce qu’il fait, pour qu’on ne suppose pas
qu’il ne fait rien; il faut bien qu’il fasse connaître d’avance ses théories, car
il n’y a rien d’aussi curieux et d’aussi frappant que les théories d’un
détective, et rien qui lui vaille plus de respect et d’admiration. Si les
journaux publient nos projets et nos plans, c’est qu’ils insistent pour les
avoir, et nous ne pouvons leur refuser sans leur faire injure; nous devons
constamment mettre nos agissements sous les yeux du public, sinon le
public croira que nous n’agissons pas. Il est d’ailleurs plus agréable de lire
dans un journal: «Voici l’ingénieuse et remarquable théorie de l’inspecteur
Blunt», que d’y trouver quelque boutade de mauvaise humeur, ou pis
encore, quelque sarcasme.»
—«Je vois la force de votre raisonnement, mais j’ai remarqué qu’en un
passage de vos observations dans les journaux de ce matin, vous aviez
refusé de faire connaître votre opinion sur un point accessoire.»
—«Oui, c’est ce que nous faisons toujours, cela fait bon effet. D’ailleurs,
je n’avais pas d’opinion du tout sur ce point.»
«Je déposai une somme d’argent considérable entre les mains de
l’inspecteur, pour couvrir les dépenses courantes; et je m’assis pour attendre
des nouvelles: nous pouvions espérer avoir des télégrammes à chaque
minute. Entre temps, je relus les journaux et notre circulaire, et je constatai
que les 25,000 dollars de récompense semblaient n’être offerts qu’aux
détectives seulement; je dis qu’il aurait fallu les offrir à quiconque
trouverait l’éléphant, mais l’inspecteur me répondit:
—«Ce sont les détectives qui trouveront l’éléphant, par conséquent la
récompense ira à qui de droit. Si la trouvaille est faite par quelque autre
personne, ce ne sera jamais que parce qu’on aura épié les détectives, et
qu’on aura mis à profit les indications qu’ils se seront laissé voler, et ils
auront droit, de toute façon, à la récompense. Le but d’une prime de cette
nature est de stimuler le zèle des hommes qui consacrent leur temps et leurs
talents acquis à ces sortes de recherches, et non pas de favoriser des
citoyens quelconques qui ont la chance de faire une capture sans avoir
mérité la récompense par des mérites et des efforts spéciaux.»
«Cela me parut assez raisonnable. A ce moment, l’appareil télégraphique
qui était dans un coin de la pièce commença à cliqueter et la dépêche
suivante se déroula:
 «Flower Station, New-York, 7 h. 30 matin.
«Suis sur une piste. Trouvé série de profonds sillons traversant ferme
près d’ici, les ai suivis pendant deux milles direction est. Sans résultat.
Crois éléphant a pris direction ouest. Je filerai de ce côté.
«Darley, détective.»
—«Darley est un des meilleurs hommes de la division, dit l’inspecteur;
nous aurons bientôt d’autres nouvelles de lui.»
«Le télégramme nº2 arriva.
«Barker’s, N. J., 7 h. 30 matin.
«Arrive à l’instant. Effraction dans verrerie ici nuit dernière, huit cents
bouteilles enlevées. Eau en grande quantité ne se trouve qu’à cinq milles
d’ici; me transporte de ce côté. Éléphant probablement altéré, bouteilles
vides trouvées.
«Baker, détective.»
—«Cela promet, dit l’inspecteur, je vous avais bien dit que le régime de
l’animal nous mettrait sur la trace.»
«Télégramme nº3.
«Taylorville, L. I., 8 h. 15 matin.
«Une meule de foin près d’ici disparue pendant la nuit. Probablement
dévorée. Relevé et suivi la piste.
«Hubard, détective.»
—«Quel chemin il fait! dit l’inspecteur. Je savais d’ailleurs que nous
aurions du mal, mais nous l’attraperons.»
«Flower Station, N. Y., 9 h. matin.
«Relevé les traces à trois milles vers l’ouest. Larges, profondes,
déchiquetées. Nous venons de rencontrer un fermier qui dit que ce ne sont
pas des traces d’éléphant. Il prétend que ce sont des traces de trous où il mit
des plants d’arbres lors des gelées de l’hiver dernier. Donnez-moi des
indications sur la marche à suivre.
«Darley, détective.»
—«Ah! ah! un complice des voleurs! Nous brûlons», dit l’inspecteur.
 «Il télégraphia à Darley:
«Arrêtez l’homme et forcez-le à nommer ses complices. Continuez à
suivre les traces... jusqu’au Pacifique, s’il le faut.
«Blunt, chef détective.»
«Autre télégramme.
«Coney-Point, Pa., 8 h. 45 matin.
«Effraction à l’usine à gaz pendant la nuit. Quittances trimestrielles non
payées disparues. Relevé et suivi la piste.»
—«Ciel! s’exclama l’inspecteur. Mange-t-il aussi des quittances?»
—«Par inadvertance, sans doute, répondis-je. Des quittances ne peuvent
être une nourriture suffisante. Du moins, prises seules.»
«Puis arriva ce télégramme émouvant:
«Ironville, N. Y., 9 h. 30 matin.
«J’arrive. Ce village est dans la consternation. Éléphant passé ici à cinq
heures du matin. Les uns disent qu’il se dirige vers l’ouest; d’autres, vers le
nord; quelques-uns, vers le sud. Mais personne n’est resté pour faire au
moment une observation précise. Il a tué un cheval. J’en ai mis un morceau
de côté comme indice. Il l’a tué avec la trompe. D’après la nature du coup,
je crois qu’il a été porté à gauche. D’après la position où on a trouvé le
cheval, je crois que l’éléphant se dirige au nord, suivant la ligne du chemin
de fer de Berkley. Il a une avance de quatre heures et demie. Mais nous le
suivons de près.
«Harves, détective.»
«Je poussai une exclamation de joie. L’inspecteur était calme comme une
image. Il toucha posément son timbre.
—«Alaric, envoyez-moi le capitaine Burns.»
«Burns entra.
—«Combien d’hommes disponibles avez-vous?»
—«Quatre-vingt-seize, Monsieur.»
—«Envoyez-les dans le nord, immédiatement. Concentration sur la ligne
de Berkley, au nord d’Ironville.»
—«Oui, Monsieur.»
 —«Que tous les mouvements se fassent dans le plus grand secret. Dès
que vous aurez d’autres hommes disponibles, prévenez-moi.»
—«Oui, Monsieur.»
—«Allez.»
—«Oui, Monsieur.»
«A ce moment arrivait un autre télégramme.
«Sage Corners, N. Y., 10 h. 30 matin.
«J’arrive. L’éléphant passé ici à 8 h. 15. Tous les habitants de la ville ont
pris la fuite, sauf un policeman. Il semble que l’éléphant ait attaqué non pas
le policeman, mais un réverbère. Tué tous les deux. J’ai ai mis de côté un
morceau du policeman comme indice.
«Stumm, détective.»
—«Ainsi l’éléphant a tourné à l’ouest, dit l’inspecteur. D’ailleurs il ne
peut échapper. J’ai des hommes partout.»
«Le télégramme suivant disait:
«Glovers, 11 h. 15 matin.
«J’arrive. Le village est abandonné. Restent les malades et les vieillards.
Éléphant passé ici il y a trois quarts d’heure. La société de protestation
contre les buveurs d’eau était réunie en séance, il a passé sa trompe par la
fenêtre et l’a vidée dans la salle; la trompe était pleine d’eau de puits,
quelques assistants l’ont avalée et sont morts, d’autres ont été noyés. Les
détectives Cross et O’Shaughnessy ont traversé la ville, mais allant au sud,
ont manqué l’éléphant. Tout le pays à plusieurs milles à la ronde saisi de
terreur. Les gens désertent leurs maisons, fuyant partout, mais partout ils
rencontrent l’éléphant. Beaucoup de tués.
«Brant, détective.»
«J’aurais voulu répandre des larmes, tant ces ravages me consternaient,
mais l’inspecteur se contenta de dire:
—«Vous voyez que nous nous rapprochons; il sent notre présence, le
voilà de nouveau à l’est.»
«Mais d’autres nouvelles sinistres nous étaient préparées. Le télégraphe
apporta ceci:
«Hoganport, 12 h. 19.
 «Arrive à l’instant. Éléphant passé ici il y a une demi-heure. Semé
partout terreur et désolation. Course furieuse à travers les rues. Deux
plombiers passant, un tué, l’autre blessé, regrets unanimes.
«O’Flaherty, détective.»
—«Enfin, le voilà au milieu de mes hommes, dit l’inspecteur, rien ne
peut le sauver.»
«Alors ce fut une série de télégrammes expédiés par des détectives
disséminés entre New-Jersey et la Pensylvanie et qui suivaient des traces,
granges ravagées, usines détruites, bibliothèques scolaires dévorées, avec
grand espoir, espoir valant certitude.
—«Je voudrais, dit l’inspecteur, pouvoir être en communication avec eux
et leur donner l’ordre de prendre le nord, mais c’est impossible. Un
détective ne va au bureau du télégraphe que pour envoyer son rapport, puis
il repart et vous ne savez jamais où mettre la main sur lui.»
«Alors arriva une dépêche ainsi conçue:
«Bridge-port, Ct., 12 h. 15.
«Barnum offre 4,000 dollars par an pour le privilège exclusif de se servir
de l’éléphant comme moyen d’annonce ambulante, à partir d’aujourd’hui
jusqu’au moment où les détectives le trouveront. Voudrait le couvrir
d’affiches de son cirque. Demande réponse immédiate.
«Boggs, détective.»
—«C’est absurde!» m’écriai-je.
—«Sans doute, dit l’inspecteur. Évidemment M. Barnum, qui se croit
très fin, ne me connaît pas. Mais je le connais.»
«Et il dicta la réponse à la dépêche:
«Offre de M. Barnum refusée. 7,000 dollars ou rien.
«Inspect. chef, Blunt.»
—«Voilà, nous n’aurons pas à attendre longtemps la réponse. M.
Barnum n’est pas chez lui, il est dans le bureau du télégraphe, c’est son
habitude quand il traite une affaire. Dans trois...»
«Affaire faite. P.-T. Barnum...» interrompit l’appareil télégraphique en
cliquetant.
 «Avant que j’eusse le temps de commenter cet extraordinaire épisode, la
dépêche suivante changea désastreusement le cours de mes idées:
«Bolivia, N. Y., 12 h. 50.
«Éléphant arrivé ici, venant du sud, a passé se dirigeant vers la forêt à 11
h. 50, dispersant un enterrement et diminuant de deux le nombre des
suiveurs. Des citoyens lui ont tiré quelques balles, puis ont pris la fuite. Le
détective Burke et moi sommes arrivés dix minutes trop tard, venant du
nord. Mais des traces fausses nous ont égarés, et nous avons perdu du
temps. A la fin, nous avons trouvé la vraie trace et l’avons suivie jusqu’à la
forêt. A ce moment nous nous sommes mis à quatre pattes, et avons relevé
les empreintes attentivement. Nous avons aperçu l’animal dans les
broussailles. Burke était devant moi. Malheureusement l’éléphant s’est
arrêté pour se reposer. Burke, qui allait la tête penchée, les yeux sur la piste,
buta contre les jambes postérieures de l’animal avant de l’avoir vu. Il se
leva aussitôt, saisit la queue, et s’écria joyeusement: «Je réclame la pri...»
Mais avant qu’il eût achevé, un simple mouvement de la trompe jeta le
brave garçon à bas, mort et en pièces. Je fis retraite, l’éléphant se retourna
et me poursuivit de près jusqu’à la lisière du bois, à une allure effrayante.
J’aurais été pris infailliblement, si les débris de l’enterrement n’étaient
miraculeusement survenus pour détourner son attention. On m’apprend
qu’il ne reste rien de l’enterrement. Ce n’est pas une perte sérieuse. Il y a ici
plus de matériaux qu’il n’en faut pour un autre. L’éléphant a disparu.
«Mulrooney, détective.»
«Nous n’eûmes plus de nouvelles, sinon des diligents et habiles
détectives dispersés, dans le New-Jersey, la Pensylvanie, le Delaware, la
Virginie, qui, tous, suivaient des pistes fraîches et sûres. Un peu après deux
heures, vint ce télégramme:
«Baxter centre, 2 h. 15 soir.
«Éléphant passé ici, tout couvert d’affiches de cirque. A dispersé une
conférence religieuse, frappant et blessant un grand nombre de ceux qui
étaient venus là pour le bien de leurs âmes. Les citoyens ont pu le saisir et
l’ont mis sous bonne garde. Quand le détective Brown et moi arrivâmes,
peu après, nous entrâmes dans l’enclos, et commençâmes à identifier
l’animal avec les photographies et descriptions. Toutes les marques
concordantes étaient reconnues, sauf une, que nous ne pouvions pas voir, la
marque à feu sous l’aisselle. Pour la voir, Brown se glissa sous l’animal, et
eut aussitôt la tête broyée; il n’en resta pas même les débris. Tous prirent la
fuite, et aussi l’éléphant, portant à droite et à gauche des coups meurtriers.
—Il s’est sauvé, mais a laissé des traces de sang, provenant des boulets de
canon. Nous sommes sûrs de le retrouver. Traverse dans la direction du sud
une forêt épaisse.»
«Ce fut le dernier télégramme. A la tombée du soir, il y eut un brouillard
si opaque que l’on ne pouvait distinguer les objets à trois pas. Il dura toute
la nuit. La circulation des bateaux et des omnibus fut interrompue.

III
Le lendemain matin, les journaux étaient pleins d’opinions de détectives.
Comme auparavant on racontait toutes les péripéties de la tragédie par le
menu et l’on ajoutait beaucoup d’autres détails reçus des correspondants
télégraphiques particuliers. Il y en avait des colonnes et des colonnes, un
bon tiers du journal avec des titres flamboyants en vedette et mon cœur
saignait à les lire. Voici le ton général:
l’éléphant blanc en liberté! il poursuit sa marche fatale! des
villages entiers abandonnés par leurs habitants frappés d’épouvante!
la pâle terreur le précède! la dévastation et la mort le suivent! puis
viennent les détectives! granges détruites! usines saccagées!
moissons dévorées! assemblées publiques dispersées! scènes de carnage
impossibles a décrire! opinion de trente-quatre détectives les plus
éminents de la division de sûreté. opinion de l’inspecteur en chef
blunt.
—«Voilà, dit l’inspecteur Blunt, trahissant presque son enthousiasme;
voilà qui est magnifique! La plus splendide aubaine qu’ait jamais eue une
administration de la sûreté. La renommée portera le bruit de nos exploits
jusqu’aux confins de la terre. Le souvenir s’en perpétuera jusqu’aux
dernières limites du temps et mon nom avec lui.»
«Mais, personnellement, je n’avais aucune raison de me réjouir; il me
semblait que c’était moi qui avais commis tous ces crimes sanglants et que
l’éléphant n’était que mon agent irresponsable. Et comme la liste s’était
accrue! Dans un endroit il était tombé au milieu d’une élection et avait tué
cinq scrutateurs. Acte de violence manifeste suivi du massacre de deux
pauvres diables nommés O’Donohue et Mac Flannigan, qui avaient «trouvé
un refuge dans l’asile des opprimés de tous les pays la veille seulement et
exerçaient pour la première fois le droit sacré des citoyens américains en se
présentant aux urnes, quand ils avaient été frappés par la main impitoyable
du fléau du Siam». Dans un autre endroit, il avait attaqué un vieux fou
prêcheur qui préparait pour la prochaine campagne son attaque héroïque
contre la danse, le théâtre et autres choses immorales, et il avait marché
dessus. Dans un autre endroit encore il avait tué un agent préposé au
paratonnerre, et la liste continuait de plus en plus sanglante, de plus en plus
navrante: il y avait soixante tués et deux cent quarante blessés. Tous les
rapports rendaient hommage à la vigilance et au dévouement des détectives
et tous se terminaient par cette remarque que le monstre avait été vu par
trois cent mille hommes et quatre détectives, et que deux de ces derniers
avaient péri.
«Je redoutais d’entendre de nouveau cliqueter l’appareil télégraphique.
Bientôt la pluie de dépêches recommença; mais je fus heureusement déçu:
on ne tarda pas à avoir la certitude que toute trace de l’éléphant avait
disparu.
«Le brouillard lui avait permis de se trouver une bonne cachette où il
restait à l’abri des investigations. Les télégrammes de localités les plus
absurdement éloignées les unes des autres annonçaient qu’une vaste masse
sombre avait été vaguement aperçue à travers le brouillard, à telle ou telle
heure, et que c’était indubitablement «l’éléphant». Cette vaste masse
sombre aurait été aperçue vaguement à New-Haven et New-Jersey, en
Pensylvanie, dans l’intérieur de l’État de New-York, à Brooklyn et même
dans la ville de New-York; mais chaque fois la vaste masse sombre s’était
évanouie et n’avait pas laissé de traces. Chacun des détectives de la
nombreuse division répandue sur cette immense étendue de pays envoyait
son rapport d’heure en heure; et chacun d’eux avait relevé une piste sûre,
épiait quelque chose et le talonnait.
«Le jour se passa néanmoins sans résultat.
«De même, le jour suivant.
«Et le troisième.
«On commençait à se lasser de lire dans les journaux des renseignements
sans issue, d’entendre parler de pistes qui ne menaient à rien, et de théories
dont l’intérêt, l’amusement et la surprise s’étaient épuisés.
 «Sur le conseil de l’inspecteur, je doublai la prime.
«Suivirent quatre jours encore de morne attente. Le coup le plus cruel
frappa alors les pauvres détectives harassés. Les journalistes refusèrent de
publier plus longtemps leurs théories, et demandèrent froidement quelque
répit.
«Quinze jours après le vol, j’élevai la prime à 75,000 dollars, sur le
conseil de l’inspecteur. C’était une somme importante, mais je compris
qu’il valait mieux sacrifier toute ma fortune personnelle que perdre mon
crédit auprès de mon gouvernement. Maintenant que les détectives étaient
en mauvaise posture, les journaux se tournèrent contre eux, et se mirent à
leur décocher les traits les plus acérés. Le théâtre s’empara de l’histoire. On
vit sur la scène des acteurs déguisés en détectives, chassant l’éléphant de la
plus amusante façon. On fit des caricatures de détectives parcourant le pays
avec des longues-vues, tandis que l’éléphant, derrière eux, mangeait des
pommes dans leurs poches. Enfin on ridiculisa de cent façons les insignes
des détectives.
«Vous avez vu l’insigne imprimé en or au dos des romans sur la police.
C’est un œil grand ouvert avec la légende: «Nous ne dormons jamais.»
Quand un agent entrait dans un bar, le patron facétieux renouvelait une
vieille plaisanterie: «Voulez-vous qu’on vous ouvre un œil?» Il y avait
partout des sarcasmes dans l’air.
«Mais un homme demeurait calme, immuable, insensible à toutes les
moqueries. C’était ce cœur de chêne, l’inspecteur. Pas une fois son regard
limpide ne se troubla, pas une fois sa confiance ne fut ébranlée. Il disait:
—«Laissez-les faire et dire. Rira bien qui rira le dernier.»
«Mon admiration pour cet homme devint un véritable culte. Je ne quittai
plus sa société. Son bureau m’était devenu un séjour de moins en moins
agréable. Cependant, puisqu’il se montrait si héroïque, je me faisais un
devoir de l’imiter, aussi longtemps du moins que je le pourrais. Je venais
régulièrement et m’installais. J’étais le seul visiteur qui parût capable de
cela. Tout le monde m’admirait. Parfois il me semblait que j’aurais dû
renoncer. Mais alors je contemplais cette face calme et apparemment
insoucieuse, et je demeurais.
«Trois semaines environ après le vol de l’éléphant, je fus un matin sur le
point de dire que j’allais donner ma démission et me retirer. A ce moment
même, pour me retenir, le grand détective me soumit un nouveau plan
génial.
«C’était une transaction avec les voleurs. La fertilité de ce génie inventif
surpassait tout ce que j’avais jamais vu, et pourtant j’ai été en relations avec
les esprits les plus distingués. Il me dit qu’il était sûr de pouvoir transiger
pour cent mille dollars, et de me faire avoir l’éléphant. Je répondis que je
croyais pouvoir réunir cette somme, mais je demandai ce que deviendraient
ces pauvres détectives qui avaient montré tant de zèle.
—«Dans les transactions, m’assura-t-il, ils ont toujours la moitié.»
«Cela écartait ma seule objection. L’inspecteur écrivit deux billets ainsi
conçus:
«Chère Madame,
«Votre mari peut gagner une forte somme d’argent (et compter
absolument sur la protection de la loi) en venant me voir immédiatement.
«Blunt, chef inspecteur.»
«Il envoya un de ces billets à la femme supposée de Brick Duffy, l’autre
à celle de Rouge Mac Fadden.
«Une heure après arrivèrent ces deux réponses insolentes:
«Vieux hibou, Brick Mac Duffy est mort depuis deux ans.»
«Bridget Mahoney.»
«Vieille chauve-souris, Rouge Mac Fadden a été pendu il y a dix-huit
mois. Tout autre âne qu’un détective sait cela.
«Mary O’Hooligan.»
—«Je m’en doutais depuis longtemps, dit l’inspecteur. Ce témoignage
prouve que mon flair ne m’a pas trompé.»
«Dès qu’une ressource lui échappait, il en trouvait une autre toute prête.
Il envoya aussitôt aux journaux du matin une annonce dont je gardai la
copie.
«A—XWBLV, 242, N, Tjd—Fz, 328 wmlg. Ozpo—2m!
«Ogw Mum.»
«Il me dit que si le voleur était encore vivant, cela le déciderait à venir
au rendez-vous habituel; il m’expliqua que ce rendez-vous était dans un
endroit où se traitaient tous les compromis entre détectives et criminels.
L’heure fixée était minuit sonnant.
«Nous ne pouvions rien faire jusque-là. Je quittai le bureau sans retard,
heureux d’un moment de liberté.
«A onze heures du soir, j’apportai les 100,000 dollars en billets de
banque et les remis entre les mains du chef détective. Peu après, il me
quitta, avec dans le regard la lueur d’espérance et de confiance que je
connaissais bien. Une heure s’écoula, presque intolérable. Puis j’entendis
son pas béni. Je me levai tout ému et chancelant de joie, et j’allai vers lui.
Quelle flamme de triomphe dans ses yeux! Il dit:
—«Nous avons transigé. Les rieurs déchanteront demain. Suivez-moi.»
«Il prit une bougie et descendit dans la vaste crypte qui s’étendait sous la
maison, et où dormaient continuellement soixante détectives, tandis qu’un
renfort de vingt autres jouaient aux cartes pour tuer le temps. Je marchais
sur ses pas. Il alla légèrement jusqu’au bout de la pièce sombre, et au
moment précis où je succombais à la suffocation et me préparais à
m’évanouir, je le vis trébucher et s’étaler sur les membres étendus d’un
objet gigantesque. Je l’entendis crier en tombant:
—«Notre noble profession est vengée. Voici l’éléphant!»
«On me transporta dans le bureau. Je repris mes sens en respirant de
l’éther.
«Tous les détectives accoururent. Je vis une scène de triomphe comme je
n’en avais jamais vu encore. On appela les reporters. On éventra des paniers
de champagne. On porta des toasts. Il y eut des serrements de mains, des
congratulations, un enthousiasme indicible et infini. Naturellement le chef
fut le héros du moment et son bonheur était si complet, il avait si
patiemment, si légitimement, si bravement remporté la victoire que j’étais
heureux moi-même de le voir ainsi, quoique je ne fusse plus pour ce qui me
concernait qu’un mendiant sans feu ni lieu: le trésor inappréciable qu’on
m’avait confié était perdu et ma position officielle m’échappait par suite de
ce que l’on considérait toujours comme une négligence coupable dans
l’accomplissement de ma grande mission. Bien des regards éloquents
témoignèrent leur profonde admiration pour le chef, et plus d’un détective
murmurait à voix basse:
—«Voyez-le, c’est le roi de la profession; il ne lui faut qu’un indice et il
n’y a rien de caché qu’il ne puisse retrouver.»