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Biotechnology
Biotechnology
Second Edition
Wallace Woon-Fong Leung

Elsevier
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The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
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experience broaden our understanding, changes in research methods, professional practices,
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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.
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Contents
In God, I trust xvii

Preface to Second Edition (2019) xix
Preface to First Edition (2007) xxiii
1 Introduction 1
1.1 Introduction 1
1.1.1 Common Host Cells for Secreting Recombinant
Protein 6
1.1.2 Platform for Protein Expression 8
1.1.3 Extracellular Protein 9
1.1.4 Intracellular Protein—Liquid 9
1.1.5 Intracellular Protein—Inclusion Body 10
1.2 Centrifugal Separation and Filtration 12
1.2.1 Sedimenting Centrifuge 14
1.2.2 Filtering Centrifuges 15
1.3 Pros and Cons of Filtration Versus Centrifugation 16
1.4 Generic Flow Sheet for Biopharmaceutical Process 18
1.5 Other Centrifugal Separations 19
1.6 Inputs and Outputs of Centrifuge 19
1.7 Separation Metrics 20
1.7.1 Protein Yield 20
1.7.2 Centrate Suspended Solids 20
1.7.3 Throughput Rate 21
1.7.4 Cell Viability 21
1.8 Text Organization 22
1.9 Summary 23
References 23
Problems 25
v
vi CONTENTS
2 Principles of Centrifugal Sedimentation 27

2.1 Introduction 27
2.2 Nonintuitive Phenomena 27
2.2.1 Pressure Gradient in a Fluid Under Centrifugal
Acceleration 27
2.2.2 Combined Centrifugal and Gravitational
Accelerations 28
2.2.3 Coriolis Effect 32
2.3 Intuitive Phenomena 35
2.3.1 Centrifugal Acceleration 35
2.3.2 Fluid in a Centrifuge Bowl Not at Solid-Body
Rotation 38
2.3.3 Regimes of Sedimentation 40
2.3.4 Stokes’ Law 42
2.3.5 Settling With Concentrated Solids 43
2.4 Process Functions 44
2.5 Summary 46
References 46
Problems 47
3 Batch and Semibatch Centrifuges 49

3.1 Spintube 49
3.2 Centrifugal Filter 53
3.3 Ultracentrifuges 54
3.3.1 Analytical Ultracentrifuge 55
3.3.2 Preparative Ultracentrifuge 56
3.3.3 Centrifugal Elutriation 58
3.4 Tubular Centrifuge 60
3.4.1 General Tubular Bowl Geometry 60
3.4.2 Ribs and Solids Scraper 63
3.4.3 Automatic Plunger Cake Discharge 67
3.5 Summary 69
References 70
Problems 70
4 Disk Centrifuge 73
4.1 Lamella/Inclined Plate Settler 73
4.1.1 Inclined Plate Settler Principle 73
4.1.2 Complications in Inclined Plate Settler 74
CONTENTS vii
4.2 Disk-Stack Centrifuge 75

4.2.1 General Disk Geometry 75
4.2.2 Disk Angle 77
4.2.3 Disk Spacing 77
4.2.4 Process Functions of Disk Centrifuge 79
4.2.5 Feed Solids 80
4.2.6 Manual Disk Centrifuge 80
4.2.7 Intermittent Discharge 82
4.2.8 Chamber Bowl 87
4.2.9 Continuous Concentrate Discharge 87
4.2.10 Liquid Discharge 95
4.2.11 Solution to Adverse Heating Effect 100
4.3 Feed Inlet and Accelerator 100
4.3.1 Introduction to Low Shear 101
4.3.2 Hydro-Hermetic Feed Design 101
4.3.3 Power Loss 102
4.3.4 Feed Acceleration Visual and Quantitative
Testing 104
4.3.5 Improved Feed Accelerator 108
4.4 Other Considerations 111
4.4.1 Materials of Construction 111
4.4.2 Clean-in-Place 112
4.4.3 Sterilization-in-Place 113
4.4.4 Containment 114
4.4.5 Surface Finish 114
4.4.6 Temperature Control 115
4.4.7 Water Requirements 115
4.4.8 Noise Level 115
4.4.9 Explosion Proof Design 115
4.5 Examples of Commercial Disk-Stack Centrifuge 115
4.6 Summary 119
References 119
Problems 120
5 Decanter Centrifuge 121

5.1 Solid Bowl or Decanter Centrifuge 121
5.2 Feed Rate 122
5.3 Pool Depth 122
5.4 Rotation Speed and G-Force 123
viii CONTENTS
5.5 Differential Speed 124

5.6 Sedimentation Enhancement Using Chemical 127
5.7 Three-Phase Separation 127
5.8 Cake Conveyance 129
5.8.1 Dry Beach 129
5.8.2 Hydraulic Assist 130
5.9 Summary 132
References 132
Problems 133
6 Commercial Applications of Centrifugation in

Biotechnology 135
6.1 Generic Flow Sheet of Biopharmaceutical 136
6.2 Mammalian Cell 138
6.3 Yeast Processing 141
6.4 Hormones Processing 144
6.5 Insulin Production 145
6.6 Biotech Separation of Inclusion Bodies 145
6.7 Vaccines Processing 147
6.7.1 Concentrated Cell-Based Product 147
6.7.2 Serum Product 148
6.8 Enzymes Processing 148
6.8.1 Extracellular Enzymes 148
6.8.2 Intracellular Enzymes 149
6.9 Probiotic Processing 150
6.10 Aquaculture 151
6.11 Alternative Meat 152
6.12 Baker Yeast Processing 153
6.13 Omega-3 From Microalgae 154
6.14 Ethanol Production 155
6.15 Other Biotech Processing 157
6.15.1 Recovery of Coagulation Factors From
Blood Plasma 157
6.15.2 Tissue From Animal Cells 157
6.15.3 Laboratory Concentration and Buffer
Exchange Using Centrifugal Filter 158
6.16 Summary 159
References 159
Problems 160
CONTENTS ix
7 Concentrating Solids by Centrifugation 161

7.1 Introduction 161
7.2 Concentrating Underflow 161
7.3 Compaction 163
7.4 Expression or Percolation 164
7.5 Compaction Testing 166
7.6 Compaction Pressure 167
7.6.1 Test-Tube Compaction 168
7.6.2 Decanter Compaction 168
7.6.3 Considerations of Cake Compaction 170
7.7 Recommendations for Increasing Solid Concentration
in Underflow 170
7.8 Summary 171
References 171
Problems 172
8 Laboratory and Pilot Testing 173

8.1 Process Objectives 173
8.2 Solid, Liquid, and Suspension Properties 174
8.2.1 Solids Properties 174
8.2.2 Mother Liquid Properties 175
8.2.3 Feed Slurry Properties 175
8.3 Bench-Scale Testing 175
8.3.1 Separability 175
8.3.2 Flocculant and Coagulant in Bench Tests 176
8.3.3 Test Variables 177
8.3.4 Material Balance 177
8.3.5 Acceleration and Deceleration Time Duration 180
8.3.6 Settling Velocity 180
8.4 Centrifugal Filter Testing 185
8.4.1 Steady-State Membrane Centrifugal Filtration
to Determine Protein Diffusivity and Solubility 185
8.4.2 Transient Membrane Centrifugal Filtration to
Determine Protein Osmotic Pressure and
Membrane Resistance 186
8.5 Pilot Testing 187
8.5.1 Material Balance Consideration for Pilot/
Production Scale 188
8.5.2 Product (Protein) Yield 190
8.5.3 Pilot Test Factors 192
8.6 Summary 199
References 199
Problems 200
x CONTENTS
9 Selection and Sizing of Centrifuges 203

9.1 Selection 203
9.1.1 Introduction 203
9.1.2 Tubular Centrifuge Selection 204
9.1.3 Disk Centrifuge Selection 204
9.1.4 Centrifuge Comparison 205
9.2 Centrifuge Sizing 207
9.2.1 Sizes and Rates 207
9.2.2 Dimensionless Le Number 208
9.2.3 Spintube (Bottle) Centrifuge 210
9.2.4 Sizing for Disk Centrifuge 213
9.2.5 Sizing for Tubular, Chamber, and Decanter
Centrifuge 219
9.3 Feed Particle Size Distribution 222
9.4 Performance of Tubular Centrifuge 223
9.5 Summary 224
References 224
Further Reading 225
Problems 225
10 Troubleshoot and Optimization 229

10.1 Troubleshooting 229
10.1.1 Timescale of Occurrence 229
10.1.2 Mechanical or Process Problem 230
10.1.3 Process Problems 230
10.1.4 Mechanical Problem 233
10.2 Optimization 235
10.2.1 Separation Metrics 235
10.2.2 Monitored Variables 236
10.2.3 Controlled Variables 237
10.2.4 Simple Optimization Scheme 237
10.3 Summary 240
Problems 240
11 Visualization and Modeling of Flow and Separation in

Tubular Centrifuge 243
11.1 Flow Visualization 243
11.2 Improved Moving Layer Flow Model 248
11.3 Effect of Velocity Profile 251
11.4 Effect of Friction Within the Flow Layer 252
CONTENTS xi
11.5 Dimensionless Le Parameter 252

11.6 Quantitative Prediction 253
11.6.1 Total Solids Recovery in Cake 253
11.6.2 Total Solids Recovery in the Centrate 253
11.6.3 Particle Size Distribution of Supernatant/
Overflow 254
11.6.4 Cumulative Size Recovery 254
11.7 Sedimentation Tests 255
11.7.1 Experiments on Sedimentation in Rotating
Bowl Centrifuge 255
11.8 Summary 258
References 259
Problems 259
12 Disk-Stack Modeling 261

12.1 Disk Model 261
12.1.1 Continuum Phase 264
12.1.2 Dispersed Phase 264
12.2 Model Validation 268
12.3 Complications 270
12.4 Summary 271
References 271
Problems 272
13 Performance Projection of Centrifuges in Bioseparation 273

13.1 Disk Centrifuge 273
13.1.1 Baseline Case (400-mm Disk) 275
13.1.2 Effect of Fine Size Distribution
(400-mm Disk) 276
13.1.3 Effect of G-Force (580-mm disk) 277
13.1.4 Efficiency η in Le Number (580-mm Disk) 280
13.1.5 Disk Centrifuge for Yeast Processing
(500-mm Disk) 282
13.1.6 Disk Centrifuge for Inclusion Body
Separation (260-mm Disk) 284
13.1.7 Enzymes (580-mm Disk) 286
13.2 Tubular Centrifuge 288
13.2.1 High-G Tubular (150- and 300-mm Tubular) 289
13.2.2 Low-G Tubular (150- and 300-mm Tubular) 290
13.3 Decanter 292
xii CONTENTS
13.4 Spintube 294

13.5 Further Discussion on Numerical Simulations 296
13.6 Summary 297
References 297
Problems 297
14 Rotating Membrane in Bioseparation 299

14.1 Membrane 299
14.1.1 Osmotic Pressure Resistance 300
14.1.2 Gel Resistance 301
14.1.3 Membrane Fouling and Cake Formation 302
14.1.4 Two Scenarios on Rotation 302
14.2 Rotating Disk Membrane With Surface Parallel to
the G-Force 303
14.2.1 Dimensionless Numbers 304
14.2.2 Governing Equations and Solutions 306
14.2.3 Gel Concentration 310
14.2.4 Determining Diffusivity 311
14.2.5 Parametric Effects 313
14.3 Rotating Membrane With Membrane Perpendicular
to the G-Force 315
14.3.1 Spintube Equipped With Membrane
Module—Centrifugal Filter 316
14.3.2 Model on Swinging Bucket Equipped With
Ultrafiltration Membrane 320
14.3.3 Comparing Test Results With Predictions 323
14.4 Summary 328
References 329
Problems 329
15 Flocculation With Decanter Centrifuges 331

15.1.1 Coagulation and Flocculation 331
15.1.2 Decanter Centrifuge 332
15.1.3 Problems 332
15.2 Monotonic Size Distribution Model 333
15.2.1 Moving Layer 334
15.2.2 Floc Model 336
15.2.3 Exponential Floc Size Distribution 336
15.2.4 Leung Number Calculation 338
15.2.5 Model Solution 339
CONTENTS xiii
15.3 Field Test 340

15.3.1 Two Decanter Tests at Wastewater
Treatment 340
15.3.2 Determining In Situ Floc Size 341
15.4 Prediction 346
15.5 Scale-Up 346
15.5.1 Le scale-Up 346
15.5.2 Sigma Scale-Up 348
15.5.3 G/g-Volume Scale-Up 348
15.5.4 Surface Area Scale-Up 348
15.6 Summary 350
References 350
Further Reading 351
Problems 351
16 Case Studies of Monotonic and Unimodal Size

Distribution Models 353
16.2 Monotonic Model Equivalent for Disk-Stack and
Tubular Centrifuges 354
16.3 Disk-Stack Centrifuge for Processing Protein From
Mammalian Cell Culture 356
16.3.1 Low Cell Density Cell Culture 358
16.3.2 High Cell Density Cell Culture 361
16.3.3 Centrate Solids and Turbidity 363
16.4 Tubular Centrifuge for Separating E. coli Lysate 364
16.5 Tubular Centrifuge for Separating S. pneumoniae
Flocculate 366
16.6 Unimodal Size Distribution Model 367
16.6.1 Unflocculated Suspension 369
16.6.2 Flocculation in Disk-Stack Centrifuge 370
16.7 Comparing the Solids Recovery Between Monotonic
and Unimodal Size Distributions 375
16.8 Summary 377
References 378
Problems 378
17 Classifying Bimodal Particle Size Distribution and Case

Study of Inclusion Body Classification 381
17.2 Mammalian Cells With Cell Debris 383
xiv CONTENTS
17.3 Processing Hybridoma Cell Broth 384

17.4 Bimodal Size Model 387
17.5 Application of Bimodal Model on Hybridoma Cell
Separation 388
17.6 Variation in Fine Fractions From Debris 390
17.7 Size of Whole Cells 391
17.7.1 Whole Cells Average Size 392
17.7.2 Size Range on Whole Cells 392
17.8 Effect of Smaller Size (The Debris) 394
17.9 Size Recoveries 398
17.9.1 Size Recovery of Small (S) Particles in
Centrate 399
17.9.2 Size Recovery of Large (L) Particles in
Centrate 400
17.9.3 Example on Classification 401
17.9.4 Smaller Size Fraction Further Apart From
Larger Size Fraction in Bimodal Feed 405
17.9.5 Smaller Size Fraction Closer to the Larger
Size Fraction in Bimodal Feed 407
17.10 Classification of Inclusion Bodies 408
17.10.1 Conventional Inclusion Bodies Processing 409
17.10.2 New Inclusion Bodies Processing 411
17.11 Centrifuges for Inclusion Bodies processing 413
17.11.1 Disk-Stack Centrifuge 413
17.11.2 Tubular Centrifuge 415
17.11.3 Spintube Centrifuge 417
17.12 Separation by Size and Density Difference 418
17.13 Summary 422
References 422
Problems 423
18 Integration of Unified Modeling With Practice in

Centrifugal Separations 425
18.2 Unified Modeling to Centrifugal Separation 425
18.3 Applications of the Unified Separation Models 428
18.3.1 Analysis of Test Data 428
18.3.2 Prediction/Forecast 429
18.3.3 Guiding Testing 429
18.3.4 Optimization 429
CONTENTS xv
18.3.5 Troubleshooting 429

18.3.6 Scale-Up/Scale-Down 430
18.4 Integration of Unified Separation Models With
Practice 431
18.5 Summary 433
Appendix A: Nomenclature 435

Appendix B: Buckingham-π Analysis for Decanter and
Tubular, Disk-Stack, and Spintube Centrifuges 439
Appendix C: Centrate or Concentrate Discharge Through
Rotating Impeller 445
Appendix D: Answers to Problems in Chapters 2 17 449
Index 457
In God, I trust
xvii
Preface to Second Edition (2019)
Since the Centrifugal Separation in Biotechnology (CSB) has been

published in 2007, it has well served the community of biotechnology
separation. I have been contacted by practitioners, researchers, engineers,
students, users, and equipment manufacturers in the biotechnology and
biopharmaceutical community, who told me that they have recommended
the text whole-heartedly to others as they find the contents in it are informa-
tive and helpful. They said CSB is the most comprehensive treatise in deal-
ing with centrifugal separation in biotechnology. There is a good balance of
practice and fundamentals on the subject matter in the text. The text is well
illustrated with tables, figures, sketches, and photographs, and is written in
layman’s language that is easy to understand. The readers also found the
questions at the end of each chapter both challenging and stimulating,
which help them to think and digest the contents that have been discussed.
Over a decade since the first publication of CSB, there has been an
exponential growth in biotech activities. This is especially for the world
of biopharma. As an example, there are much more therapeutic mono-
clonal antibodies that have been under various stages of testing than
before and they have been approved, in record numbers, by the
European Medicines Agencies and the United States Federal Drug
Administration. There is a popular demand from the biotechnology and
biopharmaceutical community for a revised edition of the text. After a
year and a half of preparation we finally come up with the second edi-
tion of the book. We have kept the same approach as our first edition,
again with good “balance” between practice and fundamentals, together
with the author’s 44 years of experience in separation and filtration from
both industry and academia injecting into this delicate balance. We have
updated the text with new technologies and new designs for both the
disk-stack and tubular centrifuges, which are the workhorse of the indus-
try. A decade ago, flocculation was unheard of for disk-stack centrifuge.
Given the bottom feed design which is available today, flocculation can
be implemented for disk-stack centrifuge, when process permits. In
some applications processing flowable concentrate containing biological
cells or bacteria, after separation the concentrate is discharged through
external nozzles. The impact of the concentrate discharge stream at high
speed from the nozzle disk-stack centrifuge results in destroying the
cells or bacteria and losing their probiotic function. Today this probiotic
xix
xx PREFACE TO SECOND EDITION (2019)
flowable concentrate stream can be routed to a small diameter near the

axis of the centrifuge, where the concentrate stream can be gently dis-
charged under pressure without being exposed to air causing oxidation
and being destroyed from high-speed impact. These are only a few of the
new technologies that are presented in the second edition. We have
expanded the discussion on clean-in-place (CIP) and sterilization-in-place
(SIP), which are very important practice for biotechnology and biophar-
maceutical to avoid cross-contamination of their products. Despite there is
no satisfactory standard to execute CIP and SIP, the text has discussed
good practice that can be used as a general guideline for practitioners.
Chapter 6, Commercial Applications of Centrifugation in Biotechnology,
discusses several typical flow sheets for biotech and biopharma processing,
and this has received very favorable response from the readers. We have
included a few more typical flow sheets in biotech and biopharma proces-
sings, and they are by no means exhaustive but serve as good examples.
Some of the flow sheets are related to separation of recombinant protein,
while others are for different applications. Not only have recombinant pro-
tein being used for diagnostic, therapeutics, and disease prevention, they
have been expanded into human food (beef, pork, and chicken made from
recombinant protein) and aquaculture. As medical scientists discover new
antigens that are specific to different types of cancer cells, more monoclo-
nal antibodies are also discovered for identifying the antigens associated
with these cancer cells so that human immune system can launch self-
defense against them. Despite each type of protein being secreted by the
common hosts (mammalian cells, microbial, yeast, virus infected insect
cells, etc.) are different, there are certain commonalities in the separation
and purification steps. CSB continues to use case studies throughout to
illustrate these commonalities so that biotech practitioner not only can use
them in generic form but tailor-make for their own process.
In the first edition, the author has developed basic models of separa-
tion for tubular/decanter and disk-stack centrifuges (Chapter 11:
Visualization and Modeling of Flow and Separation in Tubular
Centrifuge, and Chapter 12: Disk-Stack Modeling). These models have
been used to complement with case studies to discuss separation
(Chapter 13: Performance Projection of Centrifuges in Bioseparation).
One shortcoming is that the particle size distributions, which were used
in Chapter 13, Performance Projection of Centrifuges in Bioseparation,
were often lacking. To that end, some of the models being developed
may be handicapped in short of that measurement. In the second edition,
we have come up with three analytical forms of particle size distribution
for the feed to the separating centrifuge, which depend on two to five
PREFACE TO SECOND EDITION (2019)
parameters. These analytical forms include monotonic size distribution,

unimodal size distribution, and bimodal size distribution. By modifying
the parameters, we can easily change the particle size of the feed and
investigate the sensitivity of the separation outcome from the centrifuge.
We have used these size distributions together with a unified approach
on separation using spintube, disk-stack, tubular, and decanter centrifuges
to study a lot more cases. This includes a very important application on
inferring the flocculated size in centrifuge (Chapter 15: Flocculation With
Decanter Centrifuges), which has been a culprit on scale-up/scale-down
problems. We have also used the monotonic size distribution to interpret
tests being conducted on disk-stack and tubular centrifuges (Chapter 16:
Case Studies of Monotonic and Unimodal Size Distribution Models) with
both dispersed and flocculated feeds, respectively, and to demonstrate the
benefits of flocculation with disk-stack centrifuge (Chapter 16: Case
Studies of Monotonic and Unimodal Size Distribution Models).
Next, we have adopted the bimodal model to classify biologics with
both a smaller and a larger size fraction (Chapter 17: Classifying Bimodal
Particle Size Distribution and Case Study of Inclusion Body Classification)
in the feed to the centrifuge. The size recovery of the smaller fraction, as
well as the larger fraction, in centrate and concentrate, respectively, have
been developed. One can fine-tune the classification in accordance to the
S-shaped size recovery curves to maximize recovery of smaller/larger size
fraction with minimum cross contamination. This can speed-up the devel-
opment or optimization process improving the purity of the products.
Finally, in Chapter 18, Integration of Unified Modeling With Practice
in Centrifugal Separation, we propose an integrated approach, irrespec-
tive of the type of centrifuges, to separation by sedimentation in using
known/measured, or estimated analytical representation of, particle size
distribution to tackle separation problem. The author has suggested inte-
grating the models into practices at all stages from laboratory testing, pilot
testing, and clinical manufacturing to full-scale production. This reassures
the reliability of the separation process despite frequently we have only
limited test results as a basis to make important decisionon the process.
Overall, the second edition not only provides an update to various
centrifugal technologies for biotechnology, but fills in a number of miss-
ing gaps. I believe this new edition of CSB will provide a more power-
ful resource for readers to tackle their centrifugal separation problem.
I sincerely hope that the text can help to push the biotech and biopharma
forward with good practice and advanced technologies in centrifugal
separation. Consequently, drugs substances and intermediates, diagnostic
and therapeutic protein-based drugs, and health supplements, based on
xxii PREFACE TO SECOND EDITION (2019)
recombinant protein and other biotech processes, becomes available in

shorter development time and become more affordable to the general
public.
Finally, I give thanks to all those who have provided valuable inputs
into this new edition. I also thank my wife, Stella, in giving me the
extra-time and space to devote in preparation of the new edition of CSB
that has taken much longer time than I originally estimated. The support
from the rest of my family, my mom, Jessica, Daniel, Jeffrey, and
Sandar has been tremendous. I am grateful to God for His keeping and
strengthening of me, without which I cannot complete this revision.
In Him, I trust.

July 2019
Preface to First Edition (2007)
In processing biological materials to produce high value-added

intermediates or finished products, no matter whether it is liquid or
solid, often involves a separation step, especially after the fermenter or
bioreactor. In one instance, the high-value solid products in a dilute con-
centration have to be separated from the waste liquid with spent cells
and debris; therefore it is essential to prevent loss of valuable solids in
the liquid stream. A further requirement is that contaminants have to be
washed from the solids. Alternatively, the high-value liquid containing
dissolved protein needs to be separated from the biomass and that the
liquid product should be free of solid particles to avoid downstream sepa-
ration and contamination of purification equipment, such as chromatogra-
phy column. The difficulty in carrying out separation step is that biosolids
do not filter well and often foul and blind the filter, such as microfiltration
and ultrafiltration membranes. Instead of filtration, separation by sedimen-
tation utilizing the density difference between the solid and the suspending
liquid can be employed. However, the density difference between bioso-
lids and liquid (typically water based) is very small, rendering the separa-
tion very slow and ineffective, especially under the Earth’s gravity. In
addition, if RNA and other protein materials are dissolved in the liquid,
the liquid phase can be very viscous, which further slows down sedimenta-
tion. Another difficulty is that the solids concentration in suspension for
processing is relatively dilute and requires equipment that has large volu-
metric capacity for handling the flow and process.
Centrifugation has proven to be a rather robust process for enhancing
settling by using thousands to almost millions of times the Earth’s gravita-
tional acceleration. In biopharmaceutical processing for producing a
recombinant therapeutic protein for antibiotics and drug substances from
yeast, microbial, and mammalian cells, such as the Chinese Hamster
Ovary cells, centrifuges have been widely used to perform separation,
classification of cell debris, concentration of suspension, and separation
and washing of solids, such as inclusion body or crystalline protein. No
doubt, given the escalating research activities in biotechnology, many new
sources of therapeutic proteins and other valuable biological materials
will be discovered and developed, and more stringent requirements are
demanded from separation/recovery and purification. There will be more
xxiii
xxiv PREFACE TO FIRST EDITION (2007)
growing needs of centrifugation in combination with other separations and

filtrations to perform the often overlooked, yet important, duty.
While there are many texts and reference books on bioseparation,
there is very little coverage on centrifugation. This book is the first ref-
erence book of its kind devoted to centrifugal separation in biotechnol-
ogy. It is an outgrowth of a series of seminars, short courses and
presentations that the author has delivered to biopharmaceutical compa-
nies all over the world. This new and challenging topic has received
excellent global reception, which is quite comforting and rewarding. The
contents of this book are also based on the author’s research and exten-
sive experiences, respectively, in practice, mentoring and lecturing on
the subject for over 20 years.
The book starts out with an introduction on the topic (Chapter 1) fol-
lowed by Chapter 2 on sedimentation, which is the key step of separation.
Subsequently, various batch (spintube centrifuge, ultracentrifuge) and
semi-batch (tubular centrifuge) centrifuges are discussed in Chapter 3. The
workhorse of the industrial separation process, disk-stack centrifuge, is
presented and discussed in detail in Chapter 4. Also, decanter centrifuge,
which is more applicable to high-solids feed under relatively lower centrif-
ugal acceleration, has also been included in Chapter 5. However, the dis-
cussion will be brief in favor of giving room to various other topics.
Commercial applications of centrifuges in biotechnology are discussed in
Chapter 6. This is perhaps one of the most interesting topics for practi-
tioners who are more concerned about where proven processes are and
how their new process may build on what is already known and practiced.
Despite there being lot of applications discussed in this chapter, unfortu-
nately there might be applications that have been inadvertently omitted,
given that the biotech applications are very diverse. Subsequently, we dis-
cuss in Chapter 7 the importance and practice of increasing, or at least
maintaining, high-solids concentration in the underflow stream of the cen-
trifuge. Laboratory and pilot testing and selection and sizing are essential
functions for establishing and implementing the biotech process and they
are discussed in Chapters 8 and 9, respectively. A new unified approach in
scale-up and prediction with use of a dimensionless Leung (Le) number is
introduced. The Le number works for all types of centrifuges, including
spintube, tubular, chamber, disk-stack, and decanter centrifuges. This pro-
vides a solid foundation for practitioners to scale-up equipment and analyze
test results. Troubleshooting and optimization are two important topics of
general interest, especially for installed machines, and these are discussed in
Chapter 10. Subsequently, modeling of tubular and disk-stack centrifuges
are covered in Chapters 11 and 12, respectively, for researchers who are
PREFACE TO FIRST EDITION (2007)
interested. Readers who are not interested in modeling can jump directly
to Chapter 13. The Le number provides a basis for the scale-up and per-
formance prediction covered in Chapter 13. Here, numerous examples
are used to demonstrate the versatility of the numerical simulator built on
the Le-approach to forecast performance in parallel with concurrent test-
ing, which is often limited for various reasons. Numerical simulation can
also be used to analyze laboratory, pilot, and production test results to
validate machine and process performance. Therefore numerical simula-
tion can be used for laboratory screening, pilot testing, clinical
manufacturing testing, full-scale production testing, and even for small-
scale testing in the laboratory to investigate alternatives and improve-
ments of the existing process under production. Membrane processes,
such as microfiltration, ultrafiltration, and diafiltration, are frequently
used in bioseparation. Lastly, Chapter 14 is devoted to combining two
separation processes: centrifugation and membrane separation. Two
examples, respectively, on centrifugal filter in spintube and large rotating
membrane systems are discussed. The general approach can be extended
to other rotating membrane geometry.
Centrifugation has been treated as a black box in the past, as the sub-
ject is quite complex and nonintuitive. The subject involves multiple dis-
ciplines, such as fluid dynamics, mechanics and vibration, design,
material science, rheology, chemical and process engineering, chemistry,
biology, and physics. I hope this text will fulfill the quest of knowledge
rendering centrifuge a lot more “transparent” to biologists, biotechnolo-
gists, chemists, physicists, scientists, researchers, and practicing engi-
neers. The more they know the better they can deploy, comfortably and
without reservation, centrifuge as handy process equipment.
Problems are listed at the end of each chapter in the text, and they
complement and supplement the contents in the chapter. They are also
meant to reinforce the concept for the readers through practices, chal-
lenging their thoughts and understanding on the topic. Apart from practi-
tioners and researchers, this book is written primarily for 4th year
university student in the 4-year undergraduate study and research gradu-
ates in their MS or PhD research program in university taking biosepara-
tion, bioprocessing, advanced unit-operation/process engineering, or
similar courses.
I am grateful to Stella, Jessica, Jeffrey, my mother, and my late father
for putting up with me while I was devoted to preparing this book. Both
my late father and my dear friend and mentor, the late Professor Ascher
H. Shapiro, demonstrated dedication and perseverance in their lives,
which inspired me all along, especially during the trying times when I
xxvi PREFACE TO FIRST EDITION (2007)
was working on the manuscript among other responsibilities that also

demanded my undivided attention. I also thank Alice Tang for skillfully
helping out with the manuscript work and meeting the publisher’s
deadline.

2007
1
Introduction
1.1 Introduction
Biotechnology has revolutionized our life in the 20th century [1]. Its
impact is only now being felt from engineering food [2 4], engineering
and delivering drugs [5,6], to engineering consumable products. Without
doubt it will continue to influence our daily lives for years to come. One
of the many successful examples is that drugs, such as monoclonal anti-
bodies (mAbs) and some basic drug substances (i.e., the building block
for various drugs), can now be manufactured and formulated from bior-
eaction. One of the commonly used methods in biotechnology is the
recombinant DNA technique [7 9]. A desired gene is isolated from one
organism, and this is inserted into a small piece of carrier DNA called a
vector. It is highly desirable that the recombined DNA (vector plus
gene) can propagate in a similar or unrelated host/recipient cell.
The mammalian cell, such as the Chinese Hamster Ovary cell, is a
popular host cell. Fig. 1.1 shows a schematic of an animal cell which is
very similar to that of a mammalian cell. A characteristic size of the
mammalian cell is about 10 20 µm. Unlike a plant cell, there is no cell
wall for animal and mammalian cells, so they rely on a plasma mem-
brane to keep the intracellular contents intact. High shear stress acting
on the cell can rupture the fragile membrane releasing the intracellular
material.
Yeast (see schematic in Fig. 1.2), in eukaryotic single-celled microor-
ganisms classified as a member of fungus kingdom, has been commonly
used as a host cell in the recombinant DNA process, the knowledge and
experience of which we have gained from the brewery industry. Unlike
a mammalian cell, the yeast cell has a strong cell wall. Yeast cells are
smaller than mammalian cells and are typically between 7 and 10 µm.
Some common yeast hosts include, Saccharomyces cerevisiae (referred
commonly as baker yeast) and Pichia pastoris.
Bacteria, such as Escherichia coli (hereafter abbreviated as E. coli)
and Bacillus subtilis, have been used as host cells for the recombinant
Centrifugal Separations in Biotechnology. DOI: https://doi.org/10.1016/B978-0-08-102634-2.00001-5

© 2020 Elsevier Ltd. All rights reserved. 1
Figure 1.1 Animal cell schematic showing plasma membrane.
Figure 1.2 Yeast cell schematic showing both cell wall and membrane.
Introduction 3
Figure 1.3 Escherichia coli cell schematic.
DNA technique. A schematic of E. coli bacteria cell is shown in

Fig. 1.3. Again, E. coli has a sturdy cell wall with both an outer mem-
brane and an inner membrane. E. coli is typically elongated with a
dimension of 3 µm long by 1 µm width. Therapeutic protein can be
“expressed” by these host cells or organisms with the recombinant
DNA. The protein of interest may remain in the cell (intracellular) or be
secreted to the exterior of the cell (extracellular). The aforementioned
biosynthesis provides more engineering flexibility, specificity, versatil-
ity, reliability, and cost-effectiveness.
Therapeutic proteins are quite diverse in the application treatments,
such as human insulin for diabetes, erythropoietin for anemia and chronic
renal failure, interferon-beta and gamma for cancer, DNase for pulmonary
treatment, vaccines for hepatitis B, interleukin-2 for AIDS, prourokinase
for heart attacks, and tissue plasminogen activator (enzyme) for strokes.
Therapeutic proteins are present in many different kinds of mAbs.
mAbs are antibodies that are identical, because they are produced by
one type of immune cells, and they are all copies or clones of a single
parent cell. mAbs are first produced by Kohler and Milstein in 1975 [10],
for which they were awarded the Nobel Prize in Physiology or Medicine
in 1984. By virtue of the mAb being identical copies produced by one
type of immune cells, they have a high specificity for their targets. mAb
has been used in diagnosis. There are over 100 different diagnostic pro-
ducts available in the world that are mAb [11]. mAb is also used for
4 Centrifugal Separations in Biotechnology
therapeutics. For example, mAb has been a popular antibody made in the
laboratory used for cancer treatment where the antibody is designed to
attach as a label to their counterpart protein (antigen) on a specific cancer
cell so that immune cells can spot and attack the cancer cells. As an
example, the mAb known commercially as alemtuzumab drug (note all
mAb drugs have the last three alphabets labeled as “mab,” which distin-
guishes them being mAb-based drugs) can target at the antigen CD52
found on the cancer cells that causes chronic lymphocytic leukemia [12].
As antigens are discovered to be linked to more specific cancers, more
mAbs have also been developed for cancer treatment. Some mAbs work
better on certain cancers than others. mAb can also attach to the antigen
on breast cancer cells blocking the growth of breast cancer cells.
Cancer cells can “turn off the switch” of immune cells to avoid being
attacked by the immune system in our bodies. Inhibitors, or commonly
known as checkpoints, are mAb produced by the recombinant protein
process, that inhibit the protein secreted from the cancer cells in “fool-
ing” the immune cells, thereby allowing the immune cells to carry out
their normal functions. As an example, PD-1 is a protein on the immune
T-cells. The immune T-cells are normally in a switch-off condition
because PD-1 has been attached by their counterpart PD-L1, another
protein that both normal cells and cancer cells have. In other words,
they have been switched off. Some cancer cells have an abundant PD-
L1 that is used to attach to the PD-1 of the immune T-cells thereby
evading being attacked. On the other hand, mAb can target at either PD-
1 or PD-L1 and inhibit their binding, thereby allowing the immune cells
to attack the cancer cells. For example, pembrolizumab is a PD-1 inhibi-
tor that can treat skin melanoma, non-small-cell lung cancer, kidney can-
cer, bladder cancer, head and neck cancer, and Hodhkin lymphoma [12].
As another example, atezolizumab is a PD-L1 inhibitor that can treat
bladder cancer, non-small-cell lung cancer, and Merkel cell carcinoma
[12]. New inhibitors are being developed rapidly over time as more
knowledge is being gained on the specifics of different cancers and their
behavior. The examples mentioned in the forgoing are just a few under
the broad umbrella of immunotherapy for which mAb plays an impor-
tant role. The main objective of immunotherapy is to enable the immune
system of patients to recognize or target specific cancer cells and destroy
them [13]. In 2005 the total mAb therapeutics entering first-in-human
studies per year is 35, 16 out of which are for cancer treatment. In 2017
the total mAb therapeutics rose to 105, and nearly 80 were for cancer
treatment. Indeed, the antibody therapeutics entering clinical study and
being approved are in record numbers [14].
Introduction 5
Extracellular proteins secreted from yeasts are produced for making

insulin, human serum albumin, and hepatitis vaccines. Insulin drug has
reached over USD 24 billion market in 2018 according to a market study
in 2019. The fast-growing biopharmaceutical business in producing ther-
apeutic proteins is getting so popular that all major drug manufacturers
also carry a parallel line of this business.
Unfortunately, the protein expressed from the bioprocess is in very
small amounts in a large volume of suspension, that is, low concentration.
The two key hurdles in recombinant DNA techniques to produce therapeu-
tic protein [15] are (a) to recover this small concentration of protein after
fermentation by separation and (2) to provide high purity of the protein
product through separation and purification. It is prudent that both separa-
tion and purification processes should be robust and cost-effective for the
biopharmaceutical technology to be viable and competitive. Although this
text is focused on separation, one should bear in mind that given these two
steps are sequential, poor separation can adversely affect purification
downstream. Therefore it is prudent to have an integrated approach for
downstream processing. To say the least, if there is an upset from the fer-
menter upstream producing, say, off-spec finer feed, the centrifuge should
take on the upset feed and try to produce a consistent output downstream
to the filter, membrane and chromatography column downstream in the
interim, while the upset condition is being fixed. Otherwise, the entire
chain of downstream processes can be seriously affected.
Other biotechnology involves synthesis and/or modification of inter-
mediates or final products. Frequently, this is in a suspension form so
that mechanical mixing, separation, spray or thermal drying, and other
allied processes are required.
Given separation is an important task [16 20] in biotechnology in
lieu of the above, it can be a very difficult task due to the low concen-
tration of the protein present and the large volume of liquid to handle,
the fragility of the cells, the presence of cell debris, fine particulates and
colloids, and the high viscosity due to dissolution of intracellular sub-
stances, such as RNA. Typically, separation can be achieved by filtration
and sedimentation. There are some specific problems relating to each as
discussed in the following.
Filtering a suspension containing biomass is quite tricky as the mate-
rial can foul the filter surface, reducing permeate or filtrate flow regard-
less whether the media is a microfiltration or an ultrafiltration
membrane. It is equally challenging to settle biomass as the density of
the biomass material is just slightly greater than that of the liquid phase,
which often is aqueous based. Given that settling rate is proportional to
the difference in the two densities, it takes a very long time to separate,
translating in simple terms—to an impractically low-capacity, high-cost
operation. On the other hand, separation by sedimentation can be much
enhanced under centrifugal acceleration. This is possible by introducing
the suspension with biomass in a centrifuge rotating at high speed where
centrifugal acceleration can be hundreds to millions of times that of the
Earth’s gravitational acceleration.
1.1.1 Common Host Cells for Secreting Recombinant Protein

Fig. 1.4A shows the use of centrifugation in biopharmaceutical produc-
tion of therapeutic protein using the commonly employed mammalian
cells, bacteria and other microbial hosts [21], and yeasts. Centrifugation
(A)
Fermentation
and bioreaction
Mammalian
cell Bacteria Yeast
Extracellular Intracellular Extracellular
Centrifuge Centrifuge
Lysing
separation clarification
Solid product Liquid product
Depth filter Centrifuge Centrifuge lysate Centrifuge

polishing IBClassification clarification polishing
Centrifuge/filter
IB washing
polishing
Downstream processing
Drug substance and monoclonal antibodies
Figure 1.4A Drug substances produced from fermentation and down-

stream processes where centrifugation has been widely employed for
various duties.
Introduction 7
can be used for separation, clarification/polishing, thickening, classifica-

tion, and washing-and-separation.
With reference to the left bioprocess in Fig. 1.4A, after harvesting
from the bioreactor, the cell culture suspension containing mammalian
cells is sent to a centrifuge wherein the cells are separated from the
liquid product which contains the extracellular protein secreted from the
mammalian cells. The separated liquid is then sent to a depth filter for
further polishing, removing any solid particulates before sending it
downstream for processing. Some mammalian cells also secrete
soluble protein intracellularly, despite majority secrete liquid protein
extracellularly.
With reference to the middle bioprocess in Fig. 1.4A, the protein is
expressed intracellular in the bacteria. After harvesting and homogeniz-
ing, the protein is released from the lysed bacteria in the inclusion
bodies (IBs) that need to be isolated before additional downstream pro-
cessing. Centrifugation is used to sediment the IBs while the cellular
contents, cell debris, and finer materials leave with the liquid phase to
wasting. The IB is further washed and separated several times until it
reaches the desired purity for downstream processing. Alternatively, the
protein from the bacteria may be expressed in the intracellular liquid,
and upon homogenizing, this protein is released in the liquid. The task is
to remove all solid materials and to recover the liquid bearing the solu-
ble protein. The biomass may have to be washed to ensure all the pro-
tein that is adhered to the biomass has been recovered, otherwise this
represents a loss or lower yield for the process.
With reference to the right bioprocess in Fig. 1.4A, after harvesting
from the fermenter, the yeast suspension is sent to centrifugation where
the liquid containing extracellular protein is separated from the yeast
solids. The liquid leaving the centrifuge may have to be centrifuged
again (i.e., clarification or polishing) to remove any particulates and tur-
bidity before downstream processing. Some yeast cells may also secrete
protein intracellularly as liquid protein.
The above three paths are central to biopharmaceutical production of
therapeutic proteins using host cells. These will be discussed in much
greater detail throughout the text.
Besides mammalian cells, microbials, and yeast, infected baculovirus
insect cells can also secrete protein. For example, baculovirus-infected
insect cells can express mAb, such as antibody C017-1A that recognizes
antigen GA733 on colorectal cancer cell. Once the mAb C017-1A is
docked onto the antigen GA733, immune cells can attack the colorectal
cancer cells [22]. They are being used for multiple gene delivery
platforms with subsequent cellular expression of protein for labeling

cells that can serve as sensors for monitoring cellular architecture or
metabolism, and other functions [23].
1.1.2 Platform for Protein Expression

Despite we have used as an introduction in Section 1.1.1 the three most
common host cells to discuss the downstream separation and recovery of
protein, it is more prudent to examine how protein is being secreted irre-
spective of the host cells, from which the downstream protein separation
and recovery steps will typically follow.
In general, there are three platforms for recombinant protein expres-
sions from cells and microorganisms. The protein can be secreted extra-
cellularly into the broth of the cell culture in the bioreactor or fermenter.
It can also be secreted inside the cells in a bioreactor or fermenter.
There are two types of intracellular expressions. In one type, the protein
is secreted inside the cell as liquid form. In another type, the protein is
secreted as a dense mass, commonly referred as IB, which contains mis-
folded and biological inactive protein. This is illustrated in Fig. 1.4B. It
is possible that some cells or microorganisms secrete mostly intracellular
protein but also a small amount of extracellular protein and vice versa.
Mammalian cells have been thought of secreting only extracellular pro-
tein, but some new processes and host cells have shown that they can
also secrete intracellular protein. They can release the protein in the
(B) Recombinant
protein expression
Extracellular Intracellular Intracellular

protein (liquid) protein protein
(liquid) (inclusion
Protein bodies)
IB
Protein
Cell Cell
Cell
Figure 1.4B Three types of secreted recombinant protein by cells and

microorganisms.
Introduction 9
broth during maturity without the need of cell lysis. With the advent of
new research and technology, it is expected that more new recombinant
processes and new host cells will be discovered over time. Regardless,
we can look at these protein expression processes being the “model” for
downstream processes, including separation, which is a key step
upstream of purification, whether it be extracellular or intracellular pro-
tein or even a hybrid of both. Without achieving the objective in separa-
tion, the steps downstream of separation will only become more difficult
leading to poor protein recovery and low yield. Below, we will briefly
describe each of these expression processes and the downstream flow
sheet in general terms.
1.1.3 Extracellular Protein

Protein is secreted by the mammalian cells in cell culture or yeast in
fermenter extracellularly. When protein mixed with the broth is har-
vested from the bioreactor or fermenter, the objective is to remove any
cells or debris from the broth as the liquid contains the liquid protein. If
centrifugation is used to carry out the separation, the liquid product
should be clarified (free of suspended cells and cell debris) while all the
solids should settle in the concentrate stream. The centrate product will
be subsequently sent to the depth filter or microfiltration for polishing,
that is, to remove any residual fine solids not being removed by centrifu-
gation. Downstream, the liquid product is washed (reslurry with buffer
solution) by diafiltration to dissolve any dissolved impurities, including
salts, and subsequently sent to ultrafiltration for concentration. Finally,
the protein is purified in the chromatography column. The process flow
sheet is shown in Fig. 1.4C.
1.1.4 Intracellular Protein—Liquid

Protein can be secreted inside the cells using mammalian cells, yeast,
and bacteria in the bioreactor or fermenter. For yeast and bacteria, at
harvest, the cells are lysed breaking the cell walls releasing the protein
liquid that will be mixed with the broth. The objective of the separation
by centrifugation subsequent to lysis is to remove the cell debris and
some unlysed cells from the protein liquid. The liquid lysate is clarified
using centrifuge in this step. Subsequently, the liquid protein will go
through fine filtration, washing, and concentration, the steps of which
are analogous to the case of extracellular protein. Finally, the protein is
purified by the chromatography. It is perceived that given the size of the
cell debris is very small on the order of submicrons, centrifugation at
(C)
Cell culture/
fermentation
Clarification and
Cell cell separation
(centrifugation)
Protein
For example Fine filtration

mAb, (depth filtration/
enzymes, microfiltration)
antibiotics,
amino acids
Washing and
concentration
(diafiltration,
ultrafiltration)
Purification
(chromatography)
Figure 1.4C Extracellular protein secreted into broth.
high speed is necessary to generate large centrifugal force to carry out

separation. It is indeed a very important step in the process. Otherwise,
any unseparated cell debris will be carried downstream overloading the
depth filter/microfilter and even to the diafilter and ultrafilter and possi-
bly fouling the chromatography column. The process flow sheet is
shown in Fig. 1.4D.
For mammalian cells, there are exceptions for which the intracellu-
lar protein secreted can be released into the broth. Therefore periodi-
cally the broth is removed from the bioreactor, and separation is made
for which the separated whole cells are returned back to the bioreactor.
The product centrate with the cell debris will undergo another separa-
tion, where debris are removed by high-speed centrifugation. The cen-
trate liquid containing protein is sent to the depth filter for further
polishing.
1.1.5 Intracellular Protein—Inclusion Body

Extracellular protein can be secreted quite prolifically from the micro-
bials, commonly bacterial cells, in the production of recombinant pro-
tein. Unfortunately, the protein is misfolded and inactive and requires
Introduction 11
(D)
Fermentation/
cell culture
Cell harvest
Cell lysis
Protein
Cell
Lysate
clarification
For example (centrifugation)
enzymes,
growth,
factors,
vaccines Fine
separation,
washing,
concentration
Purification
(chromatography)
Figure 1.4D Intracellular protein secreted inside the cells.
subsequent processing to restore its bioactivity before use. At harvest, the

cells are lysed releasing the protein in the dense IBs. Subsequently, the IB
cells are homogenized releasing the IB cells. The IB cells have to be sepa-
rated from the cell debris and in order to have effective separation, the
properties for which separation is based should be distinctly different.
Typically, the IB cells are in the size range of 0.2 1.4 µm [24]. The den-
sity of the solids is about 1.3 g/cm3, but the IB cells have large void
fraction filled with liquid; therefore the effective bulk density may still be
about 1.1 g/cm3 or smaller depending on the void fraction. On the other
hand, the cell debris in the lysate is also quite small, 0.5 µm and below,
and has bulk density in the same range as the cell debris. In the separation
step, the IB cells are separated from the cell debris. High-speed centrifu-
gation is used to settle the IB cells from the liquid leaving the fine cell
debris to leave with the liquid centrate from the centrifuge. This fraction-
ation or classification step is not perfect. Given the IB cell size and the
cell debris are not too far from each other, there is always some cell debris
mixed with the IB cells in the sediment. This interesting case will be taken
up in Chapter 17, Classifying Bimodal Particle Size Distribution and Case
Study of Inclusion Body Classification, when we discuss classification.
(E)
Fermentation
IB
Cell harvest
Cell
Cell lysis
Classification
Inclusion bodies and
cell debris
(centrifugation)
Solubilization,
Solubilization Refolding refolding
Purification
(chromatography)
Figure 1.4E Intracellular protein secreted into the inclusion bodies.
The IB cells are washed and centrifuged a few times before sending
to the next process step. There the IB cells are solubilized moderately
by alkaline-based chemicals to dissolve the protein IB. Subsequently the
misfolded protein is refolded to restore the bioactivity of the protein.
The protein in liquid is subsequently purified in chromatography. If clas-
sification of the IB cells and cell debris is not carried out properly by
centrifugation, cell debris will be carried downstream causing contami-
nation and fouling of the chromatography column. On the other hand,
IB cells which have not been separated by centrifugation and leave the
centrate also represent a loss of yield for the entire process. The process
flow sheet is summarized in Fig. 1.4E.
1.2 Centrifugal Separation and Filtration

Industrial centrifugal separation [25,26] can be divided generally into
two classes: sedimenting and filtering. It should be noted that straight
centrifugation can only separate suspended solids and not dissolved
solids with the exception of a centrifugal filter, to be discussed later,
which can separate soluble solids. Heavier solids settle to the solid wall
of a sedimenting centrifuge under centrifugal acceleration that is much
Introduction 13
greater than the Earth’s gravity. A density difference between the solid
and the liquid phase is required to affect separation. A schematic of a
sedimenting solid-wall centrifuge is shown in Fig. 1.5. Similarly, a ligh-
ter dispersed solid phase, like fat or solids with attached air bubbles, can
also float (instead of sink) in a continuous liquid phase, and separation
by flotation can be enhanced in a centrifugal field.
On the other hand, density difference between the two phases is not
required to separate the solid from liquid phase in a filtering centrifuge.
Both phases are driven under the centrifugal body force to the perforated
wall lined with a filter medium. Liquid permeates (see Fig. 1.6) through
the filter medium while solids, comparable or larger than the openings
of the filter medium, are retained. Sometimes even smaller solids can be
retained as they “bridge across” or “jam” the medium openings preclud-
ing them from filtering through. As such, openings in the filter medium
can be selected normally two to three times larger than the particle size
to be retained. Once a “cake” layer of particles forms on the medium
despite some smaller particles that may still percolate through during
initial filtration, the cake layer further acts as a frontline filter medium
in series with the original medium to retain the incoming particles.
Slurry pool
Axis
Solid wall
Cake
Figure 1.5 Solid-wall sedimenting centrifuge.
Cake
Axis Filtrate
Slurry pool Filter medium
Figure 1.6 Filtering perforate-wall centrifuge.

It can be seen that filtering centrifuges can separate particles and liquid
regardless of their density difference; this is very much different from a
sedimenting centrifuge that relies on the density difference of the two
phases to drive separation.
1.2.1 Sedimenting Centrifuge

Sedimenting centrifuge can be divided into, respectively, batch and con-
tinuous sediment discharge as represented in Fig. 1.7. For batch dis-
charge, this can be further divided to batch and continuous feed.
Spintube, ultracentrifuge, zonal centrifuge are all classified as batch feed
centrifuges despite some zonal centrifuges can have continuous feed and
continuous removal features. In batch feed, a fixed amount of feed slurry
is introduced to the centrifuge. Upon separation, heavier solids settle to
the bowl wall or tube bottom at a large radius. Here they accumulate
temporarily until separation stops.
Tubular, manual disk, chamber, multibowl, and solid basket are con-
sidered as semicontinuous as they take continuous feed of suspension.
Heavier and larger solids from suspension get settled under centrifugal
Sedimenting
centrifuge
Batch Continuous
discharge discharge
Continuous
Batch feed Disk Decanter
feed
Dropping- Conventional
Spintube Tubular
bottom disk decanter
Ultra- Manual Nozzle Dual-cone

centrifuge disk disk decanter
Zonal Chamber and Compound-

centrifuge multibowl beach decanter
Solid Nozzle
basket decanter
Figure 1.7 Batch and continuous centrifuge classification.

Introduction 15
acceleration and the sediment is stored temporarily in the bowl until the
quality of the separated liquid gets affected by the growing sediment in
the bowl. At that point, feeding stops, the centrifuge is allowed to coast
down, the liquid pool is drained, and the sediment is removed. The cen-
trifuge needs to be cleaned before the next cycle.
For continuous or semicontinuous discharge of sediment, both disk
and decanter centrifuges fall in this category. Under disk centrifuge,
there are two types depending on how the concentrated solids are dis-
charged—dropping bottom with intermittent solid discharge, and the
nozzle disk with continuous solid discharge. On the other hand, there
are four types under decanter, a conventional decanter, dual-cone
decanter for classifying two solids and a liquid phase, compound beach
decanter for dewatering fine solids producing a paste-like cake [27], and
nozzle decanter for classifying kaolin suspension with 1 2 µm particles.
1.2.2 Filtering Centrifuges

Filtering centrifuges are also divided into two types: batch and continu-
ous feed (see Fig. 1.8). Under batch discharge, there are two categories.
First, a small-batch feed under which perforated spintube and basket and
centrifugal filter both belong. Second, a large-batch feed under which
conventional basket, peelers, siphon, and inverting bag centrifuges all
belong. Regardless of the large- or small-batch feed, they take a batch
of feed suspension or “charge” and perform various operations to pro-
cess the suspension, including filtering, washing, deliquoring/dewatering
with or without drying, unloading and decelerating (for peeler) or decel-
erating and unloading (for regular baskets), and cleaning.
Pusher, conical screen, and screenbowl are continuous centrifuges.
Feed is continuously introduced in these centrifuges, and cake retained
by the filter media is continuously being removed. Filtrate liquid, with
minimal suspended solids, is also removed separately.
Suspension containing biological solids filter very slowly and they
often clog up the filter media, forming an impermeable cake. As a con-
sequence, filtration is often affected by a thin cake filtration in a con-
trolled batch mode under moderate centrifugal gravity so that the cake
does not compact to an impermeable “skin” layer adjacent to the filter
medium. In addition, solids should be at least greater than 10 µm, other-
wise filtration is slow and impractical. If the valuable product is the liq-
uid, it is possible to use a filter aid, such as diatomaceous earth, to
enhance the filtration rate provided the filter aid does not interact with
the product. In essence, this increases the permeability of the filter cake.
Filtering
centrifuge
Batch Continuous
discharge discharge
Small-batch Large-batch Conical

Pusher Screenbowl
feed feed Screen
Perforated Conventional Single-stage Conventional Scroll

spimtube basket pusher screenbowl screen
Peeler and
Centrifugal Multistage Insitu cake Vibrating
siphon
filter pusher washing screen
basket
Inverting-
Tumbling
bag
screen
basket
Wide-angle
screen
Figure 1.8 Batch and continuous filtering centrifuge classification.
When the product protein is in the solids, such as in the biological cells,
filter aid should not be used as it is almost impossible to separate the fil-
ter aid from the valuable biological material.
1.3 Pros and Cons of Filtration Versus

Centrifugation
Centrifugation followed by depth filtration, two-stage depth filtration, or
microfiltration diafiltration can all be used alone for solid liquid sepa-
ration when the valuable protein is in liquid phase. With reference to
Fig. 1.4A, centrifugation is frequently used for lysed cells involving
release of protein solids to be separated from the cell debris when bacte-
ria are used as host cells. Table 1.1 compares the pros and cons of cen-
trifugation and microfiltration. It is quite interesting that centrifugation
Introduction 17
Table 1.1 Comparing centrifugation and microfiltration (MF).
Pros Less shear compared with that generated from tangential

flow filtration or crossflow filtration
Cell debris does not foul or clog up pores leading to blinding;
advantageous for higher feed solids (future trend)
Remove solids down to 0.2 µm at high G
Compact
Less downtime (from fouling)
Single pass or multiple passes
Robust
Cons Slightly higher capital costs
Comparable operating costs (power and maintenance) as MF, which requires

periodic replacement of membranes from fouling.
actually generates less shear stress than tangential flow filtration con-
trary to conventional wisdom, provided a good feed acceleration is
adopted in the centrifuge, see Chapter 4, Disk Centrifuge. Also, centrifu-
gation does not suffer from the fouling of membrane that leads to costly
membrane replacement and downtime. Also, it is a very robust and reli-
able system.
The solids in suspension for the process of interest are very small, in
the domain of 10 µm and below, and sometimes even in the 1 2 2 µm
range. While the small density difference affects sedimentation and not
as much for filtration, as discussed, the fine biosolids affect depth filtra-
tion, clogging flow path of the filter, leading to rapid large pressure drop
across the depth filter. For microfiltration, the membrane needs to be
stayed “unclogged” or “unfouled” by the use of a crossflow using a high
shear rate to scour the membrane surface (see Chapter 14: Rotating
Membrane in Bioseparation) and to use cleaning agent to wash the
membrane during downtime. Also, diafiltration helps to maintain a
lower solid concentration, to prevent fouling. Nevertheless, fouling lead-
ing to the replacement of membrane and an associated downtime are the
key drawbacks of microfiltration in this application. In addition, a much
larger volume of liquid product (two to four times the original volume)
results from washing or diafiltration to recover the protein. This implies
a higher cost for the process as it involves more concentration by ultra-
filtration and other processes downstream for treating the spent liquid.
A fourth possibility is to combine centrifugation with depth
filtration as an integrated approach to separate mammalian cells.
Centrifugation takes up the solids loading from the feed stream leaving
the fermenter/bioreactor, while the depth filter works best in removing

the submicron particles (separated liquid from the centrifuge) from a
low-solid stream leaving the centrifuge. This will be discussed in more
detail in Chapter 6, Commercial Applications of Centrifugation in
Biotechnology.
1.4 Generic Flow Sheet for Biopharmaceutical

Process
The recombinant protein process has become very popular for engineer-
ing a protein to have specific configurations and functions [2 9].
Various recombinant proteins are commonly expressed through an engi-
neering culture such as yeast, bacteria (e.g., E. coli), and living cells
(e.g., mammalian and plant cells). The extracellular (exterior of cells)
protein expressed by yeast and mammalian cells are in the liquid phase,
while protein expressed by the engineered bacteria is inside the bacteria
(i.e., intracellular), which subsequently needs to be lysed to release the
protein. The process condition of a cell culture is carried out under
specific range of temperature, pressure, and agitation/mixing, with fer-
mentation usually subject to shorter time and more intensive condition
(higher process temperature, pressure, and with more rigorous mixing),
while bioreaction takes place under longer reaction time and milder con-
ditions (lower process temperature, pressure, and only moderate mixing).
Fig. 1.9 shows a generic flow sheet for processing recombinant pro-
tein in which protein is expressed extracellularly wherein liquid is the
product. The immediate first step—the separation step—is also referred
to as primary recovery of protein. After solid liquid separation, by any
of the aforementioned four different possible separation methods, the
protein buffer liquid may be replaced or diluted with a more appropriate
buffer with a different pH and ionic strength followed by a concentration
using an ultrafiltration and diafiltration combination. The end product
is a concentrated protein solution in a suitable buffer liquid. At
this stage, the protein can be purified using ion-exchange or affinity
Bioreactor/
cell culture
Concentration,
Purification
Coarse and buffer Sterile Drug
Centrifugation (chromatography)
fine filtration exchange filtration substance
(UF/DF)
Fermenter
Figure 1.9 Generic flow sheet of biopharmaceutical drug substance.

Introduction 19
chromatography to remove any impurities and contaminants. A final

sterile filtration involves the use of a 0.2-µm-sized microfilter to remove
bacteria that are incurred during processing. The final product is typi-
cally a drug substance or an antibiotic.
1.5 Other Centrifugal Separations

Other than for primary recovery in the downstream process of recombinant
protein, centrifugal separation is also used in many biotechnology
solid liquid separations in manufacturing of drugs/hormones such as insu-
lin and many others. In the process of manufacturing, drugs (in solid form)
frequently contain salt and other impurities. In such cases, the drugs need
to be washed by reslurrying followed by centrifugal separation. Also, crys-
tallization and precipitation in the purification step require solid liquid
separation by centrifugation. The objective in these processes is to fully
capture or recover the valuable suspended solids, unlike the recovery of
soluble protein expressed extracellularly by yeast and mammalian cells
wherein the product is the liquid. Another equally important objective is to
reduce the impurity level of the crystals to an acceptable level for down-
stream formulation, such as washing followed by centrifugal separation.
1.6 Inputs and Outputs of Centrifuge

Centrifuge has often been considered as a black box, as the mechanics
and fluid dynamics are quite complex. Here we will discuss the scien-
tific basis and understanding of centrifugation and operation of various
types of available centrifuges as commonly used in separation in bio-
technology. In the simplest terms, for a centrifuge processing, a wet feed
suspension after centrifuging, a centrate, supernatant, or overflow con-
taining a small amount of suspended solids leave the centrifuge together
with a moist concentrate, wet cake, or underflow. This is depicted in
Fig. 1.10. There could be also another input such as chemicals (coagu-
lants and flocculants) added to flocculate the feed suspension (not shown
in Fig. 1.10). Based on the previous discussion, the valuable protein
Centrate/effluent/
Feed Centrifuge overflow liquid
Concentrate/cake/
underflow solids
Figure 1.10 Typical input and output streams of centrifugation.

product can be in the fine suspended solids, such as the IBs, crystals or
precipitants containing protein, or in the centrate liquid phase, as in the
extracellular protein expression (yeast and mammalian cells). The centri-
fuge needs to be tuned to separate the product from the rest (waste or
recycle stream). Depending on the specific process, as discussed below,
some metrics or measures are commonly used to assess the centrifugal
separation.
1.7 Separation Metrics

Several measures of centrifuge performance are common—protein yield,
suspended solids in the centrate or solid recovery, throughput or capacity,
and cell viability.
1.7.1 Protein Yield

For soluble protein expressed from extracellular process, one important
measure of the separation performance of the centrifuge is the protein
yield Y. Yield is defined as the ratio of the amount (e.g., kg/min or
g/min) of protein recovered in the liquid product to the amount (kg/min
or g/min) of protein in the feed to the centrifuge. A complete recovery
of protein without loss is 100%. Usually, the yield should be very high
before the separation process can be considered viable. A 90% or higher
in yield is typical. The specific yield depends on how difficult is the sep-
aration. An example on protein yield is given, respectively, in
Chapter 7, Concentrating Solids by Centrifugation, and Chapter 8,
Laboratory and Pilot Testing.
For continuous feed centrifuge, the volumetric rate (L/min) and protein
concentration of both feed and centrate need to be measured, respectively,
for the calculation of yield. For batch feed centrifuge, the volume and
protein concentration of both feed and supernatant (i.e., centrate) should
be measured, respectively, for the yield calculation. It is evident that liq-
uid loss in the concentrate or cake affects the yield as the protein is dis-
solved in liquid; therefore the amount of liquid in the concentrate should
be minimized (see Chapter 7: Concentrating Solids by Centrifugation).
1.7.2 Centrate Suspended Solids

The centrate suspended solids should be minimized unless this is for
classification, wherein finer-sized solids in the centrate are separated
from larger solids in the concentrate as found for separating cell debris
Introduction 21
from IBs. A measure of clarity of the liquid centrate is the amount of

suspended solids by weight or by bulk volume after the centrate is spun
in a spintube centrifuge for a prescribed time. For cell culture, only fine
solids in the submicron range escape, with the centrate or supernatant to
be ultimately captured by the downstream filter.
An indirect method on assessing the centrate suspended solid is to
measure the optical opacity (or turbidity) of the centrate liquid. The tur-
bidity measurement should be calibrated against a standard on a frequent
basis. A consequence of good clarification is that the solids recovered
by sedimentation (kg/h, dry basis) compared with the feed solids (kg/h,
dry basis) should be very high. The ratio is referred to the solid recovery
Rs. When Rs is at 100%, this implies perfect separation, and there is no
solid in the product centrate or supernatant. For cell culture, we may
achieve, say, 99.9% recovery of cells by centrifugation, leaving minimal
cells escaped in the centrate or supernatant.
1.7.3 Throughput Rate

The volumetric rate or capacity of centrifuge (in L/min) is an important
measure of the volumetric liquid throughput capacity that a centrifuge
can attain. Once the total capacity (size and number) of fermenter or
bioreactor is fixed, the rate of the centrifuge(s) is determined, and this in
turn bears out the total capacity (size and number) of centrifuges. High-
rate larger centrifuges require fewer centrifuges when compared with
low-rate smaller centrifuges. On the other hand, a spare centrifuge needs
to be furnished to cover the operating centrifuges when one of these cen-
trifuges is rotated out for maintenance. Again, the operation planning
requires the information on centrifuge throughput rate.
1.7.4 Cell Viability

Mammalian cells are gaining popularity in expressing protein as there is
more flexibility in pursuing this route. However, unlike plant cells or
yeast, mammalian cells are very shear sensitive as they do not have a
cell wall. They are highly susceptible to shear, such as during accelera-
tion of the feed stream. Cells can be destroyed in the process, releasing
an intracellular protein substance that can be harmful for downstream
purification (cross-contamination of product protein) and finer debris
which renders the separation problem more difficult, resulting in
increased suspended solids loading up the depth filter. As a minimum,
cell viability of mammalian cell lines should be maintained at a high
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I wasn’t certain of it. That gives me a great deal of pleasure. It
doesn’t surprise me, he’s highly intelligent. It’s a great thing, that is.”
Dreyfusism had brought to Swann an extraordinary simplicity of
mind, and had imparted to his way of looking at things an
impulsiveness, an inconsistency more noticeable even than had
been the similar effects of his marriage to Odette; this new loss of
caste would have been better described as a recasting, and was
entirely to his credit, since it made him return to the ways in which
his forebears had trodden and from which he had turned aside to mix
with the aristocracy. But Swann, just at the very moment when with
such lucidity it had been granted to him, thanks to the gifts he had
inherited from his race, to perceive a truth that was still hidden from
people of fashion, shewed himself nevertheless quite comically blind.
He subjected afresh all his admirations and all his contempts to the
test of a new criterion, Dreyfusism. That the anti-Dreyfusism of Mme.
Bontemps should have made him think her a fool was no more
astonishing than that, when he was first married, he should have
thought her intelligent. It was not very serious either that the new
wave reached also his political judgments and made him lose all
memory of having treated as a man with a price, a British spy (this
latter was an absurdity of the Guermantes set), Clemenceau, whom
he declared now to have always stood up for conscience, to be a
man of iron, like Cornély. “No, no, I never told you anything of the
sort. You’re thinking of some one else.” But, sweeping past his
political judgments, the wave overthrew in Swann his literary
judgments also, and even affected his way of pronouncing them.
Barrès had lost all his talent, and even the books of his early days
were feeble, one could hardly read them again. “You try, you’ll find
you can’t struggle to the end. What a difference from Clemenceau!
Personally, I am not anti-clerical, but when you compare them
together you must see that Barrès is invertebrate. He’s a very great
fellow, is old Clemenceau. How he knows the language!” However,
the anti-Dreyfusards were in no position to criticise these follies.
They explained that one was a Dreyfusard by one’s being of Jewish
origin. If a practising Catholic like Saniette stood out also for a fresh
trial, that was because he was buttonholed by Mme. Verdurin, who
behaved like a wild Radical. She was out above all things against the
“frocks”. Saniette was more fool than knave, and had no idea of the
harm that the Mistress was doing him. If you pointed out that Brichot
was equally a friend of Mme. Verdurin and was a member of the
Patrie Française, that was because he was more intelligent. “You
see him occasionally?” I asked Swann, referring to Saint-Loup. “No,
never. He wrote to me the other day hoping that I would ask the Duc
de Mouchy and various other people to vote for him at the Jockey,
where for that matter he got through like a letter through the post.”
“In spite of the Case!” “The question was never raised. However I
must tell you that since all this business began I never set foot in the
place.”
M. de Guermantes returned, and was presently joined by his wife,
all ready now for the evening, tall and proud in a gown of red satin
the skirt of which was bordered with spangles. She had in her hair a
long ostrich feather dyed purple, and over her shoulders a tulle scarf
of the same red as her dress. “How nice it is to have one’s hat lined
with leather,” said the Duchess, whom nothing escaped. “However,
with you, Charles, everything is always charming, whether it’s what
you wear or what you say, what you read or what you do.” Swann
meanwhile, without apparently listening, was considering the
Duchess as he would have studied the canvas of a master, and then
sought her gaze, making with his lips the grimace which implies:
“The devil!” Mme. de Guermantes rippled with laughter. “So my
clothes please you? I’m delighted. But I must say that they don’t
please me much,” she went on with a sulking air. “Good Lord, what a
bore it is to have to dress up and go out when one would ever so
much rather stay at home!” “What magnificent rubies!” “Ah! my dear
Charles, at least one can see that you know what you’re talking
about, you’re not like that brute Monserfeuil who asked me if they
were real. I must say that I’ve never seen anything quite like them.
They were a present from the Grand Duchess. They’re a little too
large for my liking, a little too like claret glasses filled to the brim, but
I’ve put them on because we shall be seeing the Grand Duchess this
evening at Marie-Gilbert’s,” added Mme. de Guermantes, never
suspecting that this assertion destroyed the force of those previously
made by the Duke. “What’s on at the Princess’s?” inquired Swann.
“Practically nothing,” the Duke hastened to reply, the question having
made him think that Swann was not invited. “What’s that, Basin?
When all the highways and hedgerows have been scoured? It will be
a deathly crush. What will be pretty, though,” she went on, looking
wistfully at Swann, “if the storm I can feel in the air now doesn’t
break, will be those marvellous gardens. You know them, of course. I
was there a month ago, at the time when the lilacs were in flower,
you can’t have any idea how lovely they were. And then the fountain,
really, it’s Versailles in Paris.” “What sort of person is the Princess?” I
asked. “Why, you know quite well, you’ve seen her here, she’s as
beautiful as the day, also rather an idiot. Very nice, in spite of all her
Germanic high-and-mightiness, full of good nature and stupid
mistakes.” Swann was too subtle not to perceive that the Duchess, in
this speech, was trying to shew the “Guermantes wit”, and at no
great cost to herself, for she was only serving up in a less perfect
form an old saying of her own. Nevertheless, to prove to the
Duchess that he appreciated her intention to be, and as though she
had really succeeded in being funny, he smiled with a slightly forced
air, causing me by this particular form of insincerity the same feeling
of awkwardness that used to disturb me long ago when I heard my
parents discussing with M. Vinteuil the corruption of certain sections
of society (when they knew very well that a corruption far greater sat
enthroned at Montjouvain), Legrandin colouring his utterances for
the benefit of fools, choosing delicate epithets which he knew
perfectly well would not be understood by a rich or smart but illiterate
public. “Come now, Oriane, what on earth are you saying?” broke in
M. de Guermantes. “Marie a fool? Why, she has read everything,
she’s as musical as a fiddle.” “But, my poor little Basin, you’re as
innocent as a new-born babe. As if one could not be all that, and
rather an idiot as well. Idiot is too strong a word; no, she’s in the
clouds, she’s Hesse-Darmstadt, Holy Roman Empire, and wa-wa-
wa. Her pronunciation alone makes me tired. But I quite admit that
she’s a charming looney. Simply the idea of stepping down from her
German throne to go and marry, in the most middle-class way, a
private citizen. It is true that she chose him! Yes, it’s quite true,” she
went on, turning to me, “you don’t know Gilbert. Let me give you an
idea of him, he took to his bed once because I had left a card on
Mme. Carnot.... But, my little Charles,” said the Duchess, changing
the conversation when she saw that the story of the card left on the
Carnots appeared to irritate M. de Guermantes, “you know, you’ve
never sent me that photograph of our Knights of Rhodes, whom I’ve
learned to love through you, and I am so anxious to make their
acquaintance.” The Duke meanwhile had not taken his eyes from his
wife’s face. “Oriane, you might at least tell the story properly and not
cut out half. I ought to explain,” he corrected, addressing Swann,
“that the British Ambassadress at that time, who was a very worthy
woman, but lived rather in the moon and was in the habit of making
up these odd combinations, conceived the distinctly quaint idea of
inviting us with the President and his wife. We were—Oriane herself
was rather surprised, especially as the Ambassadress knew quite
enough of the people we knew not to invite us, of all things, to so ill-
assorted a gathering. There was a Minister there who is a swindler,
however I pass over all that, we had not been warned in time, were
caught in the trap, and, I’m bound to admit, all these people behaved
most civilly to us. Only, once was enough. Mme. de Guermantes,
who does not often do me the honour of consulting me, felt it
incumbent upon her to leave a card in the course of the following
week at the Elysée. Gilbert may perhaps have gone rather far in
regarding it as a stain upon our name. But it must not be forgotten
that, politics apart, M. Carnot, who for that matter filled his post quite
adequately, was the grandson of a member of the Revolutionary
Tribunal which caused the death of eleven of our people in a single
day.” “In that case, Basin, why did you go every week to dine at
Chantilly? The Duc d’Aumale was just as much the grandson of a
member of the Revolutionary Tribunal, with this difference, that
Carnot was a brave man and Philippe Egalité a wretched scoundrel.”
“Excuse my interrupting you to explain that I did send the
photograph,” said Swann. “I can’t understand how it hasn’t reached
you.” “It doesn’t altogether surprise me,” said the Duchess, “my
servants tell me only what they think fit. They probably do not
approve of the Order of Saint John.” And she rang the bell. “You
know, Oriane, that when I used to go to Chantilly it was without
enthusiasm.” “Without enthusiasm, but with a nightshirt in a bag, in
case the Prince asked you to stay, which for that matter he very
rarely did, being a perfect cad like all the Orléans lot. Do you know
who else are to be dining at Mme. de Saint-Euverte’s?” Mme. de
Guermantes asked her husband. “Besides the people you know
already, she’s asked at the last moment King Theodosius’s brother.”
At these tidings the Duchess’s features breathed contentment and
her speech boredom. “Oh, good heavens, more princes!” “But that
one is well-mannered and intelligent,” Swann suggested. “Not
altogether, though,” replied the Duchess, apparently seeking for
words that would give more novelty to the thought expressed. “Have
you ever noticed with princes that the best-mannered among them
are not really well-mannered? They must always have an opinion
about everything. Then, as they have none of their own, they spend
the first half of their lives asking us ours and the other half serving it
up to us second-hand. They positively must be able to say that one
piece has been well played and the next not so well. When there is
no difference. Listen, this little Theodosius junior (I forget his name)
asked me what one called an orchestral motif. I replied,” said the
Duchess, her eyes sparkling while a laugh broke from her beautiful
red lips: “‘One calls it an orchestral motif.’ I don’t think he was any
too well pleased, really. Oh, my dear Charles,” she went on, “what a
bore it can be, dining out. There are evenings when one would
sooner die! It is true that dying may be perhaps just as great a bore,
because we don’t know what it’s like.” A servant appeared. It was the
young lover who used to have trouble with the porter, until the
Duchess, in her kindness of heart, brought about an apparent peace
between them. “Am I to go up this evening to inquire for M. le
Marquis d’Osmond?” he asked. “Most certainly not, nothing before
to-morrow morning. In fact I don’t want you to remain in the house
to-night. The only thing that will happen will be that his footman, who
knows you, will come to you with the latest report and send you out
after us. Get off, go anywhere you like, have a woman, sleep out, but
I don’t want to see you here before to-morrow morning.” An immense
joy overflowed from the footman’s face. He would at last be able to
spend long hours with his lady-love, whom he had practically ceased
to see ever since, after a final scene with the porter, the Duchess
had considerately explained to him that it would be better, to avoid
further conflicts, if he did not go out at all. He floated, at the thought
of having an evening free at last, in a happiness which the Duchess
saw and guessed its reason. She felt, so to speak, a tightening of the
heart and an itching in all her limbs at the sight of this happiness
which an amorous couple were snatching behind her back,
concealing themselves from her, which left her irritated and jealous.
“No, Basin, let him stay here; I say, he’s not to stir out of the house.”
“But, Oriane, that’s absurd, the house is crammed with servants, and
you have the costumier’s people coming as well at twelve to dress
us for this show. There’s absolutely nothing for him to do, and he’s
the only one who’s a friend of Mama’s footman; I would a thousand
times rather get him right away from the house.” “Listen, Basin, let
me do what I want, I shall have a message for him to take in the
evening, as it happens, I can’t tell yet at what time. In any case
you’re not to go out of the house for a single instant, do you hear?”
she said to the despairing footman. If there were continual quarrels,
and if servants did not stay long with the Duchess, the person to
whose charge this guerrilla warfare was to be laid was indeed
irremovable, but it was not the porter; no doubt for the rougher tasks,
for the martyrdoms that it was more tiring to inflict, for the quarrels
which ended in blows, the Duchess entrusted the heavier
instruments to him; but even then he played his part without the least
suspicion that he had been cast for it. Like the household servants,
he admired the Duchess for her kindness of heart; and footmen of
little discernment who came back, after leaving her service, to visit
Françoise used to say that the Duke’s house would have been the
finest “place” in Paris if it had not been for the porter’s lodge. The
Duchess “played” the lodge on them, just as at different times
clericalism, freemasonry, the Jewish peril have been played on the
public. Another footman came into the room. “Why have not they
brought up the package that M. Swann sent here? And, by the way
(you’ve heard, Charles, that Mama is seriously ill?), Jules went up to
inquire for news of M. le Marquis d’Osmond: has he come back yet?”
“He’s just come this instant, M. le Duc. They’re waiting from one
moment to the next for M. le Marquis to pass away.” “Ah! He’s alive!”
exclaimed the Duke with a sigh of relief. “That’s all right, that’s all
right: sold again, Satan! While there’s life there’s hope,” the Duke
announced to us with a joyful air. “They’ve been talking about him as
though he were dead and buried. In a week from now he’ll be fitter
than I am.” “It’s the Doctors who said that he wouldn’t last out the
evening. One of them wanted to call again during the night. The
head one said it was no use. M. le Marquis would be dead by then;
they’ve only kept him alive by injecting him with camphorated oil.”
“Hold your tongue, you damned fool,” cried the Duke in a paroxysm
of rage. “Who the devil asked you to say all that? You haven’t
understood a word of what they told you.” “It wasn’t me they told, it
was Jules.” “Will you hold your tongue!” roared the Duke, and,
turning to Swann, “What a blessing he’s still alive! He will regain his
strength gradually, don’t you know. Still alive, after being in such a
critical state, that in itself is an excellent sign. One mustn’t expect
everything at once. It can’t be at all unpleasant, a little injection of
camphorated oil.” He rubbed his hands. “He’s alive; what more could
anyone want? After going through all that he’s gone through, it’s a
great step forward. Upon my word, I envy him having such a
temperament. Ah! these invalids, you know, people do all sorts of
little things for them that they don’t do for us. Now to-day there was a
devil of a cook who sent me up a leg of mutton with béarnaise sauce
—it was done to a turn, I must admit, but just for that very reason I
took so much of it that it’s still lying on my stomach. However, that
doesn’t make people come to inquire for me as they do for dear
Amanien. We do too much inquiring. It only tires him. We must let
him have room to breathe. They’re killing the poor fellow by sending
round to him all the time.” “Well,” said the Duchess to the footman as
he was leaving the room, “I gave orders for the envelope containing
a photograph which M. Swann sent me to be brought up here.”
“Madame la Duchesse, it is so large that I didn’t know if I could get it
through the door. We have left it in the hall. Does Madame la
Duchesse wish me to bring it up?” “Oh, in that case, no; they ought
to have told me, but if it’s so big I shall see it in a moment when I
come downstairs.” “I forgot to tell Mme. la Duchesse that Mme. la
Comtesse Molé left a card this morning for Mme. la Duchesse.”
“What, this morning?” said the Duchess with an air of disapproval,
feeling that so young a woman ought not to take the liberty of leaving
cards in the morning. “About ten o’clock, Madame la Duchesse.”
“Shew me the cards.” “In any case, Oriane, when you say that it was
a funny idea on Marie’s part to marry Gilbert,” went on the Duke,
reverting to the original topic of conversation, “it is you who have an
odd way of writing history. If either of them was a fool, it was Gilbert,
for having married of all people a woman so closely related to the
King of the Belgians, who has usurped the name of Brabant which
belongs to us. To put it briefly, we are of the same blood as the
Hesses, and of the elder branch. It is always stupid to talk about
oneself,” he apologised to me, “but after all, whenever we have been
not only at Darmstadt, but even at Cassel and all over Electoral
Hesse, the Landgraves have always, all of them, been most
courteous in giving us precedence as being of the elder branch.”
“But really, Basin, you don’t mean to tell me that a person who was a
Major in every regiment in her country, who had been engaged to the
King of Sweden.” “Oriane, that is too much; anyone would think that
you didn’t know that the King of Sweden’s grandfather was tilling the
soil at Pau when we had been ruling the roost for nine hundred years
throughout the whole of Europe.” “That doesn’t alter the fact that if
somebody were to say in the street: ‘Hallo, there’s the King of
Sweden,’ everyone would at once rush to see him as far as the
Place de la Concorde, and if he said: ‘There’s M. de Guermantes,’
nobody would know who M. de Guermantes was.” “What an
argument!” “Besides, I never can understand how, once the title of
Duke of Brabant has passed to the Belgian Royal Family, you can
continue to claim it.”
The footman returned with the Comtesse Molé’s card, or rather
what she had left in place of a card. Alleging that she had none on
her, she had taken from her pocket a letter addressed to herself, and
keeping the contents had handed in the envelope which bore the
inscription: “La Comtesse Molé.” As the envelope was rather large,
following the fashion in notepaper which prevailed that year, this
manuscript “card” was almost twice the size of an ordinary visiting
card. “That is what people call Mme. Molé’s ‘simplicity’,” said the
Duchess ironically. “She wants to make us think that she had no
cards on her, and to shew her originality. But we know all about that,
don’t we, my little Charles, we are quite old enough and quite original
enough ourselves to see through the tricks of a little lady who has
only been going about for four years. She is charming, but she
doesn’t seem to me, all the same, to be quite ‘big’ enough to imagine
that she can take the world by surprise with so little effort as merely
leaving an envelope instead of a card and leaving it at ten o’clock in
the morning. Her old mother mouse will shew her that she knows a
thing or two about that.” Swann could not help smiling at the thought
that the Duchess, who was, incidentally, a trifle jealous of Mme. de
Molé’s success, would find it quite in accordance with the
“Guermantes wit” to make some impertinent retort to her visitor. “So
far as the title of Duc de Brabant is concerned, I’ve told you a
hundred times, Oriane...” the Duke continued, but the Duchess,
without listening, cut him short. “But, my little Charles, I’m longing to
see your photograph.” “Ah! Extinctor draconis latrator Anubis,” said
Swann. “Yes, it was so charming what you said about that when you
were comparing the Saint George at Venice. But I don’t understand:
why Anubis?” “What’s the one like who was an ancestor of Babal?”
asked M. de Guermantes. “You want to see his bauble?” retorted his
wife, dryly, to shew that she herself scorned the pun. “I want to see
them all,” she added. “Listen, Charles, let us wait downstairs till the
carriage comes,” said the Duke; “you can pay your call on us in the
hall, because my wife won’t let us have any peace until she’s seen
your photograph. I am less impatient, I must say,” he added with a
satisfied air. “I am not easily moved myself, but she would see us all
dead rather than miss it.” “I am entirely of your opinion, Basin,” said
the Duchess, “let us go into the hall; we shall at least know why we
have come down from your study, while we shall never know how we
have come down from the Counts of Brabant.” “I’ve told you a
hundred times how the title came into the House of Hesse,” said the
Duke (while we were going downstairs to look at the photograph,
and I thought of those that Swann used to bring me at Combray),
“through the marriage of a Brabant in 1241 with the daughter of the
last Landgrave of Thuringia and Hesse, so that really it is the title of
Prince of Hesse that came to the House of Brabant rather than that
of Duke of Brabant to the House of Hesse. You will remember that
our battle-cry was that of the Dukes of Brabant: ‘Limbourg to her
conqueror!’ until we exchanged the arms of Brabant for those of
Guermantes, in which I think myself that we were wrong, and the
example of the Gramonts will not make me change my opinion.”
“But,” replied Mme. de Guermantes, “as it is the King of the Belgians
who is the conqueror.... Besides the Belgian Crown Prince calls
himself Duc de Brabant.” “But, my dear child, your argument will not
hold water for a moment. You know as well as I do that there are
titles of pretension which can perfectly well exist even if the territory
is occupied by usurpers. For instance, the King of Spain describes
himself equally as Duke of Brabant, claiming in virtue of a
possession less ancient than ours, but more ancient than that of the
King of the Belgians. He calls himself also Duke of Burgundy, King of
the Indies Occidental and Oriental, and Duke of Milan. Well, he is no
more in possession of Burgundy, the Indies or Brabant than I
possess Brabant myself, or the Prince of Hesse either, for that
matter. The King of Spain likewise proclaims himself King of
Jerusalem, as does the Austrian Emperor, and Jerusalem belongs to
neither one nor the other.” He stopped for a moment with an
awkward feeling that the mention of Jerusalem might have
embarrassed Swann, in view of “current events”, but only went on
more rapidly: “What you said just now might be said of anyone. We
were at one time Dukes of Aumale, a duchy that has passed as
regularly to the House of France as Joinville and Chevreuse have to
the House of Albert. We make no more claim to those titles than to
that of Marquis de Noirmoutiers, which was at one time ours, and
became perfectly regularly the appanage of the House of La
Trémoïlle, but because certain cessions are valid, it does not follow
that they all are. For instance,” he went on, turning to me, “my sister-
in-law’s son bears the title of Prince d’Agrigente, which comes to us
from Joan the Mad, as that of Prince de Tarente comes to the La
Trémoïlles. Well, Napoleon went and gave this title of Tarente to a
soldier, who may have been admirable in the ranks, but in doing so
the Emperor was disposing of what belonged to him even less than
Napoleon III when he created a Duc de Montmorency, since
Périgord had at least a mother who was a Montmorency, while the
Tarente of Napoléon I had no more Tarente about him than
Napoleon’s wish that he should become so. That did not prevent
Chaix d’Est-Ange, alluding to our uncle Condé, from asking the
Procureur Impérial if he had picked up the title of Duc de
Montmorency in the moat of Vincennes.”
“Listen, Basin, I ask for nothing better than to follow you to the
ditches of Vincennes, or even to Taranto. And that reminds me,
Charles, of what I was going to say to you when you were telling me
about your Saint George at Venice. We have an idea, Basin and I, of
spending next spring in Italy and Sicily. If you were to come with us,
just think what a difference it would make! I’m not thinking only of the
pleasure of seeing you, but imagine, after all you’ve told me so often
about the remains of the Norman Conquest and of ancient history,
imagine what a trip like that would become if you came with us! I
mean to say that even Basin—what am I saying, Gilbert—would
benefit by it, because I feel that even his claims to the throne of
Naples and all that sort of thing would interest me if they were
explained by you in old romanesque churches in little villages
perched on hills like primitive paintings. But now we’re going to look
at your photograph. Open the envelope,” said the Duchess to a
footman. “Please, Oriane, not this evening; you can look at it to-
morrow,” implored the Duke, who had already been making signs of
alarm to me on seeing the huge size of the photograph. “But I like to
look at it with Charles,” said the Duchess, with a smile at once
artificially concupiscent and psychologically subtle, for in her desire
to be friendly to Swann she spoke of the pleasure which she would
have in looking at the photograph as though it were the pleasure an
invalid feels he would find in eating an orange, or as though she had
managed to combine an escapade with her friends with giving
information to a biographer as to some of her favourite pursuits. “All
right, he will come again to see you, on purpose,” declared the Duke,
to whom his wife was obliged to yield. “You can spend three hours in
front of it, if that amuses you,” he added ironically. “But where are
you going to stick a toy of those dimensions?” “Why, in my room, of
course. I like to have it before my eyes.” “Oh, just as you please; if
it’s in your room, probably I shall never see it,” said the Duke, without
thinking of the revelation he was thus blindly making of the negative
character of his conjugal relations. “Very well, you will undo it with
the greatest care,” Mme. de Guermantes told the servant, multiplying
her instructions out of politeness to Swann. “And see that you don’t
crumple the envelope, either.” “So even the envelope has got to be
respected!” the Duke murmured to me, raising his eyes to the ceiling.
“But, Swann,” he added, “I, who am only a poor married man and
thoroughly prosaic, what I wonder at is how on earth you managed
to find an envelope that size. Where did you pick it up?” “Oh, at the
photographer’s; they’re always sending out things like that. But the
man is a fool, for I see he’s written on it ‘The Duchesse de
Guermantes,’ without putting ‘Madame’.” “I’ll forgive him for that,”
said the Duchesse carelessly; then, seeming to be struck by a
sudden idea which enlivened her, checked a faint smile; but at once
returning to Swann: “Well, you don’t say whether you’re coming to
Italy with us?” “Madame, I am really afraid that it will not be
possible.” “Indeed! Mme. de Montmorency is more fortunate. You
went with her to Venice and Vicenza. She told me that with you one
saw things one would never see otherwise, things no one had ever
thought of mentioning before, that you shewed her things she had
never dreamed of, and that even in the well-known things she had
been able to appreciate details which without you she might have
passed by a dozen times without ever noticing. Obviously, she has
been more highly favoured than we are to be.... You will take the big
envelope from M. Swann’s photograph,” she said to the servant,
“and you will hand it in, from me, this evening at half past ten at
Mme. la Comtesse Molé’s.” Swann laughed. “I should like to know,
all the same,” Mme. de Guermantes asked him, “how, ten months
before the time, you can tell that a thing will be impossible.” “My dear
Duchess, I will tell you if you insist upon it, but, first of all, you can
see that I am very ill.” “Yes, my little Charles, I don’t think you look at
all well. I’m not pleased with your colour, but I’m not asking you to
come with me next week, I ask you to come in ten months. In ten
months one has time to get oneself cured, you know.” At this point a
footman came in to say that the carriage was at the door. “Come,
Oriane, to horse,” said the Duke, already pawing the ground with
impatience as though he were himself one of the horses that stood
waiting outside. “Very well, give me in one word the reason why you
can’t come to Italy,” the Duchess put it to Swann as she rose to say
good-bye to us. “But, my dear friend, it’s because I shall then have
been dead for several months. According to the doctors I consulted
last winter, the thing I’ve got—which may, for that matter, carry me off
at any moment—won’t in any case leave me more than three or four
months to live, and even that is a generous estimate,” replied Swann
with a smile, while the footman opened the glazed door of the hall to
let the Duchess out. “What’s that you say?” cried the Duchess,
stopping for a moment on her way to the carriage, and raising her
fine eyes, their melancholy blue clouded by uncertainty. Placed for
the first time in her life between two duties as incompatible as getting
into her carriage to go out to dinner and shewing pity for a man who
was about to die, she could find nothing in the code of conventions
that indicated the right line to follow, and, not knowing which to
choose, felt it better to make a show of not believing that the latter
alternative need be seriously considered, so as to follow the first,
which demanded of her at the moment less effort, and thought that
the best way of settling the conflict would be to deny that any
existed. “You’re joking,” she said to Swann. “It would be a joke in
charming taste,” replied he ironically. “I don’t know why I am telling
you this; I have never said a word to you before about my illness. But
as you asked me, and as now I may die at any moment.... But
whatever I do I mustn’t make you late; you’re dining out, remember,”
he added, because he knew that for other people their own social
obligations took precedence of the death of a friend, and could put
himself in her place by dint of his instinctive politeness. But that of
the Duchess enabled her also to perceive in a vague way that the
dinner to which she was going must count for less to Swann than his
own death. And so, while continuing on her way towards the
carriage, she let her shoulders droop, saying: “Don’t worry about our
dinner. It’s not of any importance!” But this put the Duke in a bad
humour, who exclaimed: “Come, Oriane, don’t stop there chattering
like that and exchanging your jeremiads with Swann; you know very
well that Mme. de Saint-Euverte insists on sitting down to table at
eight o’clock sharp. We must know what you propose to do; the
horses have been waiting for a good five minutes. I beg your pardon,
Charles,” he went on, turning to Swann, “but it’s ten minutes to eight
already. Oriane is always late, and it will take us more than five
minutes to get to old Saint-Euverte’s.”
Mme. de Guermantes advanced resolutely towards the carriage
and uttered a last farewell to Swann. “You know, we can talk about
that another time; I don’t believe a word you’ve been saying, but we
must discuss it quietly. I expect they gave you a dreadful fright, come
to luncheon, whatever day you like,” (with Mme. de Guermantes
things always resolved themselves into luncheons), “you will let me
know your day and time,” and, lifting her red skirt, she set her foot on
the step. She was just getting into the carriage when, seeing this foot
exposed, the Duke cried in a terrifying voice: “Oriane, what have you
been thinking of, you wretch? You’ve kept on your black shoes! With
a red dress! Go upstairs quick and put on red shoes, or rather,” he
said to the footman, “tell the lady’s maid at once to bring down a pair
of red shoes.” “But, my dear,” replied the Duchess gently, annoyed to
see that Swann, who was leaving the house with me but had stood
back to allow the carriage to pass out in front of us, could hear,
“since we are late.” “No, no, we have plenty of time. It is only ten to;
it won’t take us ten minutes to get to the Parc Monceau. And, after
all, what would it matter? If we turned up at half past eight they’ld
have to wait for us, but you can’t possibly go there in a red dress and
black shoes. Besides, we shan’t be the last, I can tell you; the
Sassenages are coming, and you know they never arrive before
twenty to nine.” The Duchess went up to her room. “Well,” said M. de
Guermantes to Swann and myself, “we poor, down-trodden
husbands, people laugh at us, but we are of some use all the same.
But for me, Oriane would have been going out to dinner in black
shoes.” “It’s not unbecoming,” said Swann, “I noticed the black shoes
and they didn’t offend me in the least.” “I don’t say you’re wrong,”
replied the Duke, “but it looks better to have them to match the
dress. Besides, you needn’t worry, she would no sooner have got
there than she’ld have noticed them, and I should have been obliged
to come home and fetch the others. I should have had my dinner at
nine o’clock. Good-bye, my children,” he said, thrusting us gently
from the door, “get away, before Oriane comes down again. It’s not
that she doesn’t like seeing you both. On the contrary, she’s too fond
of your company. If she finds you still here she will start talking
again, she is tired out already, she’ll reach the dinner-table quite
dead. Besides, I tell you frankly, I’m dying of hunger. I had a
wretched luncheon this morning when I came from the train. There
was the devil of a béarnaise sauce, I admit, but in spite of that I
sha’nt be at all sorry, not at all sorry to sit down to dinner. Five
minutes to eight! Oh, women, women! She’ll give us both indigestion
before to-morrow. She is not nearly as strong as people think.” The
Duke felt no compunction at speaking thus of his wife’s ailments and
his own to a dying man, for the former interested him more,
appeared to him more important. And so it was simply from good
breeding and good fellowship that, after politely shewing us out, he
cried “from off stage”, in a stentorian voice from the porch to Swann,
who was already in the courtyard: “You, now, don’t let yourself be
taken in by the doctors’ nonsense, damn them. They’re donkeys.
You’re as strong as the Pont Neuf. You’ll live to bury us all!”
Modern Library of the World’s Best Books
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please use number at right of title
ADAMS, HENRY The Education of Henry Adams 76

AIKEN, CONRAD A Comprehensive Anthology of
American Verse 101
AIKEN, CONRAD Modern American Poetry 127
ANDERSON, SHERWOOD Winesburg, Ohio 104
BALZAC Droll Stories 193
BEERBOHM, MAX Zuleika Dobson 116
BEMELMANS, LUDWIG My War with the United States 175
BENNETT, ARNOLD The Old Wives’ Tale 184
BIERCE, AMBROSE In the Midst of Life 133
BOCCACCIO The Decameron 71
BRONTË, CHARLOTTE Jane Eyre 64
BRONTË, EMILY Wuthering Heights 106
BUCK, PEARL The Good Earth 2
BURTON, RICHARD The Arabian Nights 201
BUTLER, SAMUEL Erewhon and Erewhon Revisited 136
BUTLER, SAMUEL The Way of All Flesh 13
CABELL, JAMES BRANCH Jurgen 15
CALDWELL, ERSKINE God’s Little Acre 51
CANFIELD, DOROTHY The Deepening Stream 200
CARROLL, LEWIS Alice in Wonderland, etc. 79
CASANOVA, JACQUES Memoirs of Casanova 165
CELLINI, BENVENUTO Autobiography of Cellini 3
CERVANTES Don Quixote 174
CHAUCER The Canterbury Tales 161
CHAUCER Troilus and Cressida 126
CONFUCIUS The Wisdom of Confucius 7
CONRAD, JOSEPH Heart of Darkness (In Great Modern
Short Stories 168)
CONRAD, JOSEPH Lord Jim 186
CONRAD, JOSEPH Victory 34
CORNEILLE and RACINE Six Plays of Corneille and Racine 194
CORVO, FREDERICK BARON A History of the Borgias 192
CUMMINGS, E. E. The Enormous Room 214
DANTE The Divine Comedy 208
DAUDET, ALPHONSE Sapho 85
DEFOE, DANIEL Moll Flanders 122
DEWEY, JOHN Human Nature and Conduct 173
DICKENS, CHARLES A Tale of Two Cities 189
DICKENS, CHARLES David Copperfield 110
DICKENS, CHARLES Pickwick Papers 204
DINESEN, ISAK Seven Gothic Tales 54
DOS PASSOS, JOHN Three Soldiers 205
DOSTOYEVSKY, FYODOR Crime and Punishment 199
DOSTOYEVSKY, FYODOR The Brothers Karamazov 151
DOSTOYEVSKY, FYODOR The Possessed 55
DOUGLAS, NORMAN South Wind 5
DREISER, THEODORE Sister Carrie 8
DUMAS, ALEXANDRE Camille 69
DUMAS, ALEXANDRE The Three Musketeers 143
DU MAURIER, GEORGE Peter Ibbetson 207
EDMAN, IRWIN The Philosophy of Plato 181
EDMONDS, WALTER D. Rome Haul 191
ELLIS, HAVELOCK The Dance of Life 160
EMERSON, RALPH WALDO Essays and Other Writings 91
FAULKNER, WILLIAM Sanctuary 61
FEUCHTWANGER, LION Power 206
FIELDING, HENRY Joseph Andrews 117
FIELDING, HENRY Tom Jones 185
FINEMAN, IRVING Hear, Ye Sons 130
FLAUBERT, GUSTAVE Madame Bovary 28
FORESTER, C. S. The African Queen 102
FORSTER, E. M. A Passage to India 218
FRANCE, ANATOLE Crime of Sylvestre Bonnard 22
FRANCE, ANATOLE Penguin Island 210
FRANKLIN, BENJAMIN Autobiography, etc. 39
GALSWORTHY, JOHN The Apple Tree (In Great Modern Short
Stories 168)
GAUTIER, THEOPHILE Mlle. De Maupin, One of Cleopatra’s
Nights 53
GEORGE, HENRY Progress and Poverty 36
GIDE, ANDRÉ The Counterfeiters 187
GISSING, GEORGE New Grub Street 125
GISSING, GEORGE Private Papers of Henry Ryecroft 46
GLASGOW, ELLEN Barren Ground 25
GOETHE Faust 177
GOETHE The Sorrows of Werther (In Collected
German Stories 108)
GOGOL, NIKOLAI Dead Souls 40
GRAVES, ROBERT I, Claudius 20
HAMMETT, DASHIELL The Maltese Falcon 45
HAMSUN, KNUT Growth of the Soil 12
HARDY, THOMAS Jude the Obscure 135
HARDY, THOMAS The Mayor of Casterbridge 17
HARDY, THOMAS The Return of the Native 121
HARDY, THOMAS Tess of the D’Urbervilles 72
HART, LIDDELL The War in Outline 16
HAWTHORNE, NATHANIEL The Scarlet Letter 93
HEMINGWAY, ERNEST A Farewell to Arms 19
HEMINGWAY, ERNEST The Sun Also Rises 170
HEMON, LOUIS Maria Chapdelaine 10
HOMER The Iliad 166
HOMER The Odyssey 167
HORACE The Complete Works of 141
HUDSON, W. H. Green Mansions 89
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HUGHES, RICHARD A High Wind in Jamaica 112
HUGO, VICTOR The Hunchback of Notre Dame 35
HUNEKER, JAMES G. Painted Veils 43
HUXLEY, ALDOUS Antic Hay 209
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IBSEN, HENRIK A Doll’s House, Ghosts, etc. 6
JAMES, HENRY The Portrait of a Lady 107
JAMES, HENRY The Turn of the Screw 169
JAMES, WILLIAM The Philosophy of William James 114
JAMES, WILLIAM The Varieties of Religious Experience
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JEFFERS, ROBINSON Roan Stallion; Tamar and Other Poems
118
JOYCE, JAMES Dubliners 124
JOYCE, JAMES A Portrait of the Artist as a Young Man
145
KUPRIN, ALEXANDRE Yama 203
LARDNER, RING The Collected Short Stories of 211
LAWRENCE, D. H. The Rainbow 128
LAWRENCE, D. H. Sons and Lovers 109
LAWRENCE, D. H. Women in Love 68
LEWIS, SINCLAIR Arrowsmith 42
LEWISOHN, LUDWIG The Island Within 123
LONGFELLOW, HENRY W. Poems 56
LOUYS, PIERRE Aphrodite 77
LUDWIG, EMIL Napoleon 95
LUNDBERG, FERDINAND Imperial Hearst 81
MACHIAVELLI The Prince and The Discourses of
Machiavelli 65
MALRAUX, ANDRÉ Man’s Fate 33
MANN, THOMAS Death in Venice (In Collected German
Stories 108)
MANSFIELD, KATHERINE The Garden Party 129
MARQUAND, JOHN P. The Late George Apley 182
MARX, KARL Capital and Other Writings 202
MAUGHAM, W. SOMERSET Of Human Bondage 176
MAUGHAM, W. SOMERSET The Moon and Sixpence 27
MAUPASSANT, GUY DE Best Short Stories 98
McFEE, WILLIAM Casuals of the Sea 195
MELVILLE, HERMAN Moby Dick 119
MEREDITH, GEORGE Diana of the Crossways 14
MEREDITH, GEORGE The Ordeal of Richard Feverel 134
MEREJKOWSKI, DMITRI The Romance of Leonardo da Vinci
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MISCELLANEOUS An Anthology of American Negro
Literature 163
An Anthology of Light Verse 48
Best Ghost Stories 73
Best Amer. Humorous Short Stories 87
Best Russian Short Stories, including
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Eight Famous Elizabethan Plays 94
Five Great Modern Irish Plays 30
Four Famous Greek Plays 158
Fourteen Great Detective Stories 144
Great German Short Novels and
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Great Modern Short Stories 168
The Federalist 139
The Making of Man: An Outline of
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The Making of Society: An Outline of
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The Short Bible 57
Outline of Abnormal Psychology 152
Outline of Psychoanalysis 66
The Sex Problem in Modern Society
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MOLIERE Plays 78
MORLEY, CHRISTOPHER Human Being 74
MORLEY, CHRISTOPHER Parnassus on Wheels 190
NIETZSCHE, FRIEDRICH Thus Spake Zarathustra 9
ODETS, CLIFFORD Six Plays of 67
O’NEILL, EUGENE The Emperor Jones, Anna Christie and
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O’NEILL, EUGENE The Long Voyage Home and Seven

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Centrifugal Separations in

Biotechnology 2nd Edition Wallace

Nanofiber Filter Technologies for Filtration of

Essentials in Modern HPLC Separations 2nd Edition

Beginning Programming. All-in-One 2nd Edition Wallace

Pump Wisdom Essential Centrifugal Pump Knowledge for

MXene Membranes for Separations Haihui Wang

Shallow Equality and Symbolic Jurisprudence in

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Wallace Woon-Fong Leung

For Information on all Elsevier publications

Publisher: Susan Dennis

In God, I trust xvii

2 Principles of Centrifugal Sedimentation 27

3 Batch and Semibatch Centrifuges 49

4.2 Disk-Stack Centrifuge 75

5 Decanter Centrifuge 121

5.5 Differential Speed 124

6 Commercial Applications of Centrifugation in

7 Concentrating Solids by Centrifugation 161

8 Laboratory and Pilot Testing 173

9 Selection and Sizing of Centrifuges 203

10 Troubleshoot and Optimization 229

11 Visualization and Modeling of Flow and Separation in

11.5 Dimensionless Le Parameter 252

12 Disk-Stack Modeling 261

13 Performance Projection of Centrifuges in Bioseparation 273

13.4 Spintube 294

14 Rotating Membrane in Bioseparation 299

15 Flocculation With Decanter Centrifuges 331

15.3 Field Test 340

16 Case Studies of Monotonic and Unimodal Size

17 Classifying Bimodal Particle Size Distribution and Case

17.3 Processing Hybridoma Cell Broth 384

18 Integration of Unified Modeling With Practice in

18.3.5 Troubleshooting 429

Appendix A: Nomenclature 435

Since the Centrifugal Separation in Biotechnology (CSB) has been

flowable concentrate stream can be routed to a small diameter near the

parameters. These analytical forms include monotonic size distribution,

recombinant protein and other biotech processes, becomes available in

Wallace Woon-Fong Leung

In processing biological materials to produce high value-added

growing needs of centrifugation in combination with other separations and

was working on the manuscript among other responsibilities that also

Wallace Woon-Fong Leung

Centrifugal Separations in Biotechnology. DOI: https://doi.org/10.1016/B978-0-08-102634-2.00001-5

Figure 1.3 Escherichia coli cell schematic.

DNA technique. A schematic of E. coli bacteria cell is shown in

Extracellular proteins secreted from yeasts are produced for making

1.1.1 Common Host Cells for Secreting Recombinant Protein

Extracellular Intracellular Extracellular

Solid product Liquid product

Depth filter Centrifuge Centrifuge lysate Centrifuge

Drug substance and monoclonal antibodies

Figure 1.4A Drug substances produced from fermentation and down-

can be used for separation, clarification/polishing, thickening, classifica-

platforms with subsequent cellular expression of protein for labeling

1.1.2 Platform for Protein Expression

Extracellular Intracellular Intracellular

Figure 1.4B Three types of secreted recombinant protein by cells and

1.1.3 Extracellular Protein