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Centrifugal Separations in
Biotechnology
Centrifugal Separations in
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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 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 Introduction 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
16.1 Introduction 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
17.1 Introduction 382
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
18.1 Introduction 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.

Wallace Woon-Fong Leung

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.

Wallace Woon-Fong Leung

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
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 CHAPTER IX
THE WHITE ROOM

“The white room, excellency, was, until a year ago, the only chamber
in the house of Paul Zaloski which was set apart for the
entertainment of women. Elsewhere, the many rooms which opened
off from the cloisters and the silent corridors were so many tents for
soldiers—barrack-like dens, in which the only furniture was a bed,
and the only adornment a crucifix. There were periodical days when
Count Paul would break these crucifixes with the flat of his sword or
any weapon that came handy—for he was ever a man of a violent
temper, and he had a religion that he kept to himself. Old Father
Mark, the priest, was permitted rather than welcomed in the house.
He had been a servant of the Count’s father; and while he would not
swerve a hair’s-breadth from the formularies of his office, he did
many a good deed among the poor of Jajce, and had once
converted a Turk—a fact upon which he relied for a comfortable seat
in Paradise. He it was who replaced the crucifixes when the Count
broke them in his wrath; and he never failed after such an outburst to
spend hours before the altar in the chapel, praying that a curse might
not come upon the house.
“Side by side with these miserable apartments I have spoken of, the
white room was a thing to see. The great bed of it was heavy with
gold and painting; there was a canopy above, supported by carved
figures of angels. Many mirrors with gold frames almost hid the
panels of the wall, themselves decked with frescoes and medallions.
The carpet had come from the looms at Serajevo; the roomy chairs
were from Vienna; the fine cut glass from Venice. Twenty years
before the day of which I write, the house had many rooms such as
this to shew; but the people had plundered it when Austria came to
take their country, and the last of the Zaloskis was no lover of
gewgaws or of women’s finery. Camp beds, without crucifixes above
them, were good enough for him. While there was a room ready for
his sister, who came at Easter from Vienna and remained regularly
one month in the château, he cared nothing in what state the other
apartments might be.
“‘Let them rot,’ he would cry to the priest, when the good man spoke
of restoration; ‘let those buy carpets who have nails in their feet. I
have no money for fopperies. If you find my beds hard, there is an
hotel in Jajce, Father, with pretty chambermaids to tuck you in, and a
cellar full of sour Burgundy to split your head before matins. What!
you have no tongue for that? Then mind your business and leave me
to mine.’
“Father Mark used to twinge at these rough-and-ready rejoinders; or,
when the Count persisted, he would shut his ears and go out to pray
in the chapel. Not that he personally was any lover of fine stuffs or of
gilding; but he looked upon the house of the Zaloskis with the eye of
an artist and the soul of a great builder. He loved every stone in
those ramparts, which had stood invincible before Turk and heretic
through so many centuries. He had pride in the traditions of a family
which had witnessed for the faith with its blood, and had surrendered
nothing to the example of Eastern barbarism and teaching. Had he
been consulted he would have made the castle a centre of power
and of learning, whence his religion might have spread abroad an
influence to the uttermost ends of the province. The Count was the
only obstacle—the Count, whose heart was shut to women—the
man who was the last of his race, who cared nothing that an heir
should be born to him.
“But I am telling you of things which have small concern with this
story, excellency. Rather let me show you Christine, not asleep, but
awake and well clothed, and wondering, in the white room to which
she had been carried on that day the Count found her. Her aimless
flight from the mountains had brought her very near to death. What
food she had got by the way had been thrown to her by those who
had compassion upon her pretty face and her obvious need. And for
a week she lay in the Count’s house hovering between life and
death. I have heard it said that nothing was more remarkable than
the care bestowed by the master of the château upon this little
vagrant he had snatched from the roadside. Always an incurable
lover of the medical science, he found in this case an occupation
most congenial to him. Scarce an hour passed, they tell me, but he
was at her bedside; he watched her through the dangerous changes
of the night; he brought physicians from Jajce and Livno. And all this,
excellency, without one thought of the sweetness of the face before
him, or the pretty figure which lay racked with pain in the great gold
bed of the white room. Such things were nothing to him; but the
disease—that was worthy of his skill and of his knowledge. And
when at last he knew that he had won a victory—when the fever was
gone, and the pulse beat calmly, and the flush had left the cheeks—
then he returned to his own work with no more thought of little
Christine than of a wench in his scullery.
“‘She is no peasant’s daughter,’ he would say to the priest; ‘I must
have her history when she is well enough to tell it. She wears a silver
ring on her finger; but so does every slut in Dalmatia, for that matter.
You shall make her speak, Father, and we will send her to her home
—but that must be some days yet. Meanwhile—no little flirtations on
your own account; you understand me?’
“Father Mark held up his hands in horror at the suggestion; and the
Count, with a sly twinkle in his eye—such a twinkle as this,
excellency—went off to hunt the bear in the mountains of the Verbas.
It was just then that Christine was lying like one bewitched in the
great bed.
“From misery, beggary, and a couch of the brushwood she awoke to
see those splendours which were to her like the splendours of
Paradise itself. Remember her education—recall her training in
those fine superstitions which are the wealth of the islanders—and
then how shall we wonder that there was a time when she believed
that the spirits had carried her up to a mansion of the heavens?
Such comfort she had never known; of such marvels her imagination
had taught her nothing. For hours she lay dazed and fascinated and
spell-bound, asking herself if the angels in the paintings lived, if her
limbs really rested upon a bed, or were not rather floating in a gauze-
like cloud. Of memory of that which she had passed through there
was at the moment none. It was as though the years which had
come between her childhood and that pleasant hour had never been.
Her lover; her island home; the people who had cried upon her; the
shepherd; the hut in the hills—these she had forgotten. Her
prevailing sense was that of rest and gladness—of a great gladness
and of a perfect content.
“Once or twice during the first hours of waking she had seen a kindly
face bend over her. It was the face of Mother Theresa, the
housekeeper at the château. She, good soul, watched unceasingly
when the Count had resumed his old occupation. She had children
of her own; she fancied that she could read the story of this child.
And when at length she saw that her patient was coming back to life
again, her joy was that of a mother who has found a daughter.
“‘My little girl,’ she said, kissing the white forehead and holding the
shrunken hand—‘nay, sweet, you must not raise that pretty head; by-
and-bye we will get up together, dear, but rest now.’
“She pressed cooling drink to the parched lips and smoothed the
long brown hair; and, excellency, the words were sweet to her who
had never known what a mother’s word may be. They were sweet,
for love and kindness seemed to be breathed with them. Yet of their
meaning Christine knew nothing. She had never spoken any other
tongue than the dialect of Zlarin, which is wholly Italian. Mother
Theresa was an Austrian of Linz; and although she had a smattering
of the uncouth speech of Bosnia, German served all her purposes in
the château. As well might she have spoken Russian to her patient,
who did but lie and smile lovingly upon her, and open her eyes wider
at the wonders, and tell herself that here, indeed, was the palace of
her visions, here the destiny which she had crossed the mountains
to fulfil.
“In this happy state she lay for three days, but upon the third day she
rose from her bed. The Count was away at Serajevo then, and
Father Mark having business with his bishop, it befell that Mother
Theresa was left to do her will with the patient. So soon as Christine
was strong enough they went abroad in the park together, and the
first breath of autumn being upon the lake, the child shewed the old
mother how well she could sail a boat, and how familiar she was with
all things concerning the woods and waters. Nor, when they drove to
the town of Jajce together, did the number of the people or the
shapes of the buildings frighten her. You know Jajce, excellency?
Ah, she is a queen of the hills, a white city of the mountains; her
minarets rise up abundantly like silver spires above the
unsurpassable green of the heights; she listens ever to the foam of
the great cascade which thunders at her gates; the spray of the
waters bathes her as in a foam of jewels. In her streets are Turks
and Christians, Greeks and Jews. Friars raise their voices against
the allah il allah of the muezzin; a new hotel rubs shoulders with the
catacombs where lie the dead who fought against Mohammed. Yet
she is the one citadel which time has not touched nor civilisation
conquered. Her walls stand to-day as they stood when Kings of
Bosnia looked out from them upon the armies of the infidels. Her
people dress as they dressed when Corvin was their lord; her castle
still marks her glory and the glory of her chiefs. She is a city of the
East and of the West—a gem of the mountains, like to nothing that
was or is or shall be.
“Christine saw Jajce, and found new delights in its contemplation.
The invigorating winds of autumn now began to fill her blood with
youthful strength and vigour. The colour came again to her cheeks
when the crisp mountain air wooed them; her eyes sparkled with
health restored. And she was quick to make friends in the château.
Old Mother Theresa adored her; Hans, the steward, being convinced
that his master had no such thoughts as he feared, remembered that
he was once at Trieste, and had three words of Italian for his
dictionary. He called her carina, and treated her like a little schoolgirl
come home for the holidays. As for the priest, who made it his first
business to inquire, as well as he could in his broken Italian, what
was her faith and who were her parents, even he admitted that she
had brought a new spirit to lighten the gloom of the house of the
Zaloskis.
“‘My daughter,’ he would stammer as he endeavoured to impart to
her the mysteries of the catechism, ‘you know that there is hell-fire
for the wicked?’
“‘You say so, Father,’ she would answer him.
“‘But I wish you to believe that there is.’
“‘I will try, Father.’
“‘You must try always,’ he repeated, ‘try to think of the good God
above you and of the burning fire below—the fire which is, which is,
my child——’
“But the priest was ill-equipped when he came to the larger use of
Italian adjectives, and he would turn in despair to question her upon
that past concerning which she had as yet remembered so little.
“‘You have a home, my child?’
“‘Si, si,’ she answered him.
“‘Your father lives there?’
“‘I have no father but Father Andrea.’
“‘Is your mother dead?’
“‘I do not know; I have never seen her.’
“‘This ring, my child—is it a ring of betrothal or of marriage?’
“‘It is a ring of marriage.’
“‘Where, then, is your husband?’
“‘He left me in the mountains, in the hut of the shepherd Orio.’
“‘That was long ago?’
“She shook her head. Though health had restored her memory to
her, it had yet left but faint and blurred impressions of that week of
suffering in the hut. She realised little of the meaning of the tie she
had contracted; of sense of obligation there was none.
“‘Girl,’ said the priest, severely, ‘the Holy Gospels teach us that the
wife should cleave unto her husband. You are well punished for your
sins, as God always punishes those who break His commandments.
You must return to Zlarin when your strength is wholly restored. I
must speak to the Count when he is back again. I fear you have
acted very wrongly.’
“Her only answer was a merry laugh, excellency. He neither
frightened nor convinced her. She did not believe that the God he
spoke of would ever carry her back to that loneliness and misery
which she had fled. Nay, all exhortation was lost upon her; and when
the Count returned at the end of the week he found Father Mark full
of bitterness and despair.
“‘Oh, indeed,’ cried the priest, ‘a pretty guest you have brought to us!
She has a husband at Zlarin, and the holy truths are so many fairy
tales to her. This very morning she met me in the park and cried to
me: “Father Mark, tell me about hell-fire.” A child would have asked
in the same tone for the story of Blue Beard. She has demoralised
your house, and the devil is in the music she can make.’
“But the Count only laughed at him.
“‘Trust a priest to be busy when there is a petticoat in the place,’ said
he; ‘who is your rival, Father Mark?’
“At this, excellency, the holy father went again to the oratory to pray.
“Count Paul, although he had twitted the priest, had determined
secretly that he would make himself aware of his strange guest’s
history without further loss of time. He had been overmuch occupied
with the military affairs of the province during his absence in
Serajevo; but now that he was home again he began to see that he
had taken a somewhat serious responsibility upon his shoulders, and
that he must face it without further loss of time. He made up his mind
that he would speak to Christine on the morrow; but when the
morning came there was much to hear from his steward and
business to do with the Prefect of Jajce. It was not until the afternoon
that he thought again of his intention, and that thought was thrust
upon him curiously.
“He was riding up from the town, and had entered at a gate of the
park which is near the eastern end of the lake. There is a little wood
here, excellency, and great trees give shade even at the zenith of the
summer. The Count had turned his horse from the road, for the sun
is hot in Bosnia even when the autumn is come, and he was
cantering gently towards his house, when he heard the notes of a
violin, played very sweetly, yet with moments of passion and swift
cunning execution which betrayed the musician of large powers.
Surprised that such music was to be heard in his own park, he rode
straight to the thicket whence the sounds came, and speedily
unravelled the mystery. Old Mother Theresa had carried food and
drink to a little bower of the woods, and there was gathered a merry
party—Hans cutting odd capers to the music of the fiddle, the dame
applauding, the priest leaning against a tree with laughter and
disapproval marked together upon his frowning face, Christine sitting
cross-legged upon the grass and wringing, from the crazy old
instrument they had found her, music which would have set the
oldest foot beating to its rhythm. So absorbed, indeed, was the party
in the lilt of the dance that Count Paul remained for many minutes
unobserved; and in this interval he used his eyes quickly. Nor could
he at the first bring himself to think that the little girl he then saw, so
gay, so full of happiness, and of such incontestable beauty, was the
wan-faced starving creature he had found upon the Jajce road not a
month ago.
“Well, indeed, may this pretty apparition have caused him wonder.
You have seen Christine for yourself, excellency; have seen her now
that she has passed through the heat of her trial, and sorrow has left
its stamp upon her—you have seen her, and you know that she is a
woman whose face is not to be forgotten by him who has once
looked upon it; a woman whose lightest gesture is grace, whose
limbs are the limbs of painters’ dreams. Think of her, then, when the
whole sweetness of youth was written in her dancing eyes, and the
new joy of her life was bursting into blossom. Oh, surely this man
may well have stood dumb in his surprise, slow to believe that some
trick had not been played upon him. And to this conclusion her new
dress helped him. No longer did ragged finery draggle about her
limbs. No longer was the dust matted in her hair. She wore a pretty
gown that the dame had made for her with loving labour. Her shoes
were of yellow leather, and had buckles like the priest’s. Her vest
was scarlet, slashed with gold—for that had come all the way from
Serajevo, and was the secret gift of the well-meaning Hans. She had
a dainty white cap to hide the gold in her curls, and silver bracelets
rattled upon the arm which held the bow. And when to these were
added the lustre of her deep-black eyes, the ripe red flush upon her
cheeks, the pout of the little mouth and the comely shape of her
limbs, she was indeed a picture to please, a creature to invite the
love of all that set their eyes upon her.”
 CHAPTER X
COUNT PAUL AT HOME

“Here, then, excellency, is the little Christine whom the Count saw
as he stood at the edge of the bower and gazed upon the scene with
surprised eyes. Not until the capering Hans had sunk down
exhausted upon the turf, and Mother Theresa had clapped her hands
again and again, and the priest had raised his finger in warning, did
the group become aware of the master’s presence, and hasten to
put on some show of respect and greeting. As for Christine, Father
Mark had not failed to impress upon her the nature of her obligation
to the owner of the château, and no sooner had she seen him than
she ran forward, and holding out her hand very prettily, she took his
and pressed it to her lips.
“‘Herr Count,’ she said—for thus had they taught her to address him
—‘I thank you, oh, I thank you from my heart—the Blessed Mother
give me words—I will be your servant always—I am so happy here,
Herr Count, and it is to you that I must speak—you will not send me
back to Zlarin?’
“The Count listened to her earnest prattle, not knowing whether to be
amused or troubled. The touch of her lips upon his hand had been
very sweet, and, stern recluse that he was, he had no heart to resist
such attractive pleading.
“‘Child,’ said he—and he spoke Italian very readily—‘we will talk of
all these things presently. Come up now to the house, that I may
learn your story.’
“He offered her a stirrup leather, and holding to this she ran by his
side, awed by his presence, yet drawn to him by an instinctive
feeling that here she had found a friend. And he encouraged her to
talk, desiring to form some opinion which would help him to deal with
her future.
“‘Well, my little girl,’ he said cheerily, ‘and who taught you to play the
fiddle?’
“‘Pietro,’ said she, ‘Pietro who sings for the priest at Zlarin. Oh,
Pietro is a wise-head; he has great eyes and great ears and great
hands like yours, Herr Count. When he opens his mouth it is just like
looking into a pumpkin. He talks to his fiddle and his fiddle talks to
him. It is good to see Pietro at the feast, Herr Count—such notes out
of his mouth—oh, they are like cannon on the sea; such anger—oh,
it is something to watch Pietro beat his fiddle when he is angry. And
he laughs and nods his head all the time, and says: “What music I
make! What injustice that I do not play at Vienna!” He taught me the
scales, and when I could not do it he would beat his breast and
break my bow and smack my face; but when I pleased him he would
cry: “Brava! the whole city shall dance to music such as that.”’
“The Count had smiled when she spoke of his great hands,
excellency, for in truth he was a big man, uncouth of limb, large of
frame, and a very giant in strength. But forty years of life had robbed
him of all the vanities, and he was pleased to find one who spoke
truth and had no shame of her words. He could not help but
remember, as he looked down upon the picturesque little figure
running at his side, that life in the house of the Zaloskis had been a
poor round of official dulness these twenty years and more. Christine
brought him to a sudden recollection of a past day when a woman
had enchained him and drank of the heart’s blood of his affections,
leaving him at last a soured and broken man. He recalled the hour
when a glance from eyes, that were very like the eyes of his little
Italian girl, had warmed him to ecstasies of hope and love; his mind
went back to those fleeting years of passion when a woman’s hand
had led him, and a woman’s whim had betrayed him and had cast
him out with affections withered and soul embittered. They had given
him the name of woman-hater since those days. Strange, then, to
hear the babble of a pretty girl’s voice in his own park, to feel his
hand warm at her kiss. More strange that such things should have
been pleasing to him, that he should have decided in his mind not to
send Christine from the house if circumstances would permit him to
retain her there.
“He had come to this conclusion as he rode up to the gate of the
château, and giving his horse to a groom, led the way to his study.
This was a great vaulted apartment in the east wing—a room with
many pillars buttressing the arches of the roof, and windows opening
towards the green mountains of Jajce. The walls of it spoke
eloquently of the Count’s common employments—hunting
implements, swords, suits of mail, old guns which dying Turks had
dropped, spears, lances, pikes, adorned them. The tiled floor was
covered with the soft skins of wolf and bear; other skins were piled
upon the couches and the chairs. The heavy sideboard of oak shone
resplendent with silver cups and jugs—mighty jugs for mighty
drinkers. A small case for books, many implements of science, a
table littered with papers, seemed out of place in that museum of a
hundred wars—a soldier’s room, excellency, the room of a man
whose ancestors had fought unnumbered battles.
“To such a room Count Paul led Christine. Here he pulled off his big
riding-boots, and having lighted a cigar, he sat down at his table and
bade her squat upon the cushion of skins at his side. And then he fell
to questioning her.
“‘Child,’ said he, ‘they tell me that your father, Andrea, is now living at
Sebenico, and that you ran away from him when he wished you to
finish your education in a convent?’
“‘My father Andrea!’ she answered with surprise. ‘Oh, he is not my
father, Herr Count, he is only my friend.’
“‘How came it, then, that he wished to send you to a convent?’
“‘It was because of Ugo—of Ugo Klun, who was to take me to
Vienna. He is my husband, you know.’
“She said it, excellency, with no more concern than if Ugo had been
her dog.
“‘Your husband, girl!’ cried the Count, angry at her indifference; ‘you
have a husband, then?’
“‘I stood with Ugo before the altar upon the island Incoronata,’ she
answered, playing with the bracelets upon her wrist; ‘he called me
wife, and the priest blessed me. We rode that day to the mountains,
for I feared to go to Sebenico to the Sisters, and Ugo feared that
they would make a hussar of him. It was to the hut of Orio the
shepherd that he carried me, and there I was ill with the fever. When
I awoke Ugo had gone, and no one was with me in the hut. Then I
tried to find him in the hills, and I ate all the bread, and the road was
hot, and the sun burnt my face, and—oh! it would have been good to
die! You will not send me back to Zlarin, Herr Count?’
“She asked him pitifully, her black eyes raised to his, and yet he
could not promise her.
“‘That is for your husband to say. First we must find him, child. You
would wish that, of course?’
“Her answer astonished him.
“‘Herr Count,’ she cried, clinging to his knees, ‘how shall I tell you—
the Blessed Mother give me words! Ugo is my friend, he has been
good to me. I thought that we should be friends always, that he
would take me to the great city; but when I was with him in the hut
and he put his arm upon my neck and burnt my lips with kisses, I
knew that it could never be, that God had meant it otherwise. Oh, his
kisses hurt me; I shuddered at his touch—I, who wished to thank him
and to be his friend. Herr Count, blame me not; we cannot give these
things; we cannot love because we wish it. Do not send for him; let
me be your servant always. I will work for you, I will serve you. Oh, I
have known no love in all my life, God help me! I have been alone
always; there has been none to care; even my brother beat me.
When I was a little child they let me beg for bread. Herr Count, what
happiness if you should speak a word of love to me—if you should
hear me now! I cannot go back to Zlarin—I cannot! The holy angels
are my witnesses, Herr Count.’
“She had sunk slowly to the floor, and she lay now with her head
pillowed upon her arm and her right hand holding still the right hand
of the master. So deeply did she feel the words she spoke that her
whole body shook with her sobs and the floor was wet with her tears.
She had tasted so sweet a draught of happiness that the thought of
putting down the cup was bitter to her beyond words. And in her
exceeding grief, excellency, she brought the Count to a memory of a
day in his own life when he too had asked for the bread of love and
had hungered. Such tears as she then shed openly he had shed in
his heart, and they had frozen there, shutting out for twenty years all
the warmth of human affections.
“‘Come,’ he said, when he had waited awhile, and she had raised
her burning face to his, begging for an answer, ‘tell me more of your
husband, and then perhaps I shall answer you. You say that they
would have made a hussar of him—where, then, was he drawn?’
“‘He was drawn at Jajce—your own city. His father is the
woodlander, but Ugo left him a year ago, for he would not serve,
since that would have taken him from me. They must have heard
that he was at Sebenico, and that is why he left me in the mountains.
Herr Count, I cannot go back to him—I cannot go!’
“The Count heard her out, and then, rising from his seat, he
unlocked the cabinet which held the books, and took from it a
volume wherein the reports of the Prefect of Jajce were filed. Two of
these, the most recent, he scanned without comment, but at the third
he stopped, and an exclamation broke from his lips.
“‘Christine,’ said he—and that was the first time he had called her so
—‘you are telling me the truth?’
“‘I swear it,’ she cried.
“‘You do not wish to go back to this man?’
“‘God knows that I cannot go.’
“‘Then think no more of him, my child, for he fired upon his officer
and was shot by a trooper in the woods of the Verbas ten days ago.’
“The Count closed the book with a snap; but Christine, trembling and
very pale, and awed by the words she had heard, rose to her feet.
“‘He was my friend,’ she said, and that was all, for as she spoke she
ran from the room and listened no more. Grief and joy had
conquered; the desire of years seemed gratified in that hour, the veil
lifted off her life.
“Yet this was the cruelty of it—that even while the Count was telling
her of the man’s death Ugo Klun, hidden in a cave above the Verbas,
was saying to himself that the pursuit would soon be over, and that
he would return to hold little Christine in his arms again.
“Excellency, the report had lied that a corporal might profit.”
 CHAPTER XI
ANDREA BEARS WITNESS

“You have told me, signor, that you know neither Jajce nor her
mountains. May it be my privilege, if you are of a mind to see the
wonders of this incomparable land, to be your companion in the
venture! And when that day shall come, let it be a day neither of
dreadful summer nor of unrelenting winter; a day when the hills are
sweet with the air of spring; when the rills run down foamed with
golden light; when the grass of the woods is carpeted with the year’s
first flowers. Then we shall see the minarets of the city rising up like
silver towers above the fresh waters of the river; then will the thunder
of her cascade make music for our ears. She will win a victory over
our affections; we shall hold her in perpetual honour.
“I am led to speak in this way since I am still mindful of my last
journey to the town of the falling rivers, and of the many troubles that
beset my path when I sought Christine in her new home. You may
ask well enough how it was that I, having, in my misconception, sent
soldiers to save her from shame, did not at once search for her in the
mountains and carry her back to Sebenico. As God is my witness, I
did this, and all else that might bring back to me the daughter I loved.
Ten days I rode in the heights, asking of all that passed by for tidings
of my child; ten days I searched the woods and the villages between
Livno and my own city. But that very secrecy which the lad Ugo had
observed stood between me and my desires. None but the shepherd
knew where Christine had slept upon the night of her marriage.
Vague rumours of a sharp pursuit in the mountains, intelligence of
the fate of Orio, divergent accounts of the capture of the deserter, of
his attack upon his commanding officer and his death—these things
came slowly to our ears in a province where gossip is high treason
and the publication of news often a felony. Indeed, I had begun to
look upon little Christine as one snatched from my love and my life,
to say that I should never hear her pretty prattle or touch her hand
again, when—and that was five months after she had left the island
—the summons from Jajce came to me, and I learnt not only of her
new estate, but of her happiness and her fortune.
“She wrote the letter with her own hand—no longer the childish
scrawl which was her gift from the priest at Zlarin, but neater
characters, telling of some education and of culture. I read there that
she was not forgetful of the old time or the old friends; but it was
plain that the fervour of childhood had left her, and that she had put
on the sober dignity of womanhood. Such love as she expressed for
me was expressed in measured terms; she spoke of her great
gratitude to the Count and of his unceasing kindness to her; she
made mention of Father Mark and of her other friends; and she
concluded by exhorting me to cross the mountains to Jajce and there
to tell all I knew of her early life and circumstances—‘for,’ she said,
‘this I owe to my benefactor, and you alone can pay the debt.’
“I read her letter, and on the third day after, having suffered much by
the way, I was driving my sleigh over the frozen park of Count Paul’s
château. The scene was one to linger in the memory, excellency, a
scene to bear witness to the glory of God, and of nature, which is
God’s child. Peak upon peak, shining in the sun like domes of ice,
rose up above the path I followed; Jajce herself had put upon her a
mantle of snow; her frozen cascades were so many ropes of gold
and of jewels; the great lake had become a vast mirror, like a mirror
of silver; the hills themselves were full of the dismal howling of the
wolves. From such desolation it was good to pass to the warmth and
the welcome of the house of the Zaloskis; good to hear the barking
of the dogs, to see the bright flame of the fires, to feel the spreading
heat of the many stoves. But better than all was it to remember that
little Christine was then in the house, and that I was about to hear
her voice again.
“They received me in the great hall, excellency, the priest coming
forward to meet me, and whispering a word in my ear before he led
me to his master. He had a kindly face, open and sincere; and ill as
Christine had liked him at the first, I make sure that she has reason
now to love him. This, indeed, his words proved, as you shall learn
subsequently.
“‘My friend,’ said he, ‘you must first drink a glass of prune brandy,
and warm yourself with the soup which Hans shall bring us. I know
very well what the cold may be on the hills. Let us thank God for
these thick walls around us.’
“He poured me a glass of the liquor, which was like fire to the
stomach; and when I had drunk it I asked for Christine.
“‘She is in the library with the Count,’ said he, and I thought with just
a little bitterness; ‘it is astonishing what service she seems to be to
him. There is nothing like a woman’s hand, Signor Andrea, and hers
is very ready. I have said from the first that she is no peasant’s child.’
“‘You have spoken well,’ I replied; ‘she is no peasant’s child. Her
father could remember the day when he was first man in Zara; her
mother had a gift of music above the common. The vagaries of trade
and the coming of the Austrian—I speak freely, for I read your
nationality in your face—brought ruin to their house. God be praised
that she has found another home!’
“‘Amen to that,’ cried he. ‘You must forgive us if the singular nature of
her story has made us desirous to learn all that can be learnt. I took
this opportunity of speaking to you, since I would rather hear
anything that is to be told than have it come suddenly to the ears of
the Count. Christine’s influence over him is very remarkable. Heaven
forbid that I should speak against him—but he is another man since
this little guest crept into his life. You will understand that I should not
like him to hear ill of her.’
“‘Hear ill of her!’ cried I; ‘per Baccho, Father, I would like to know the
man that would speak it. Hear ill of her—Holy Mother, what talk!’
“‘Then you are able to say that she is a good girl?’
“‘A good girl—cospetto, she is as innocent as a child at her first
Communion. What! you would condemn her for running away to the
hills with a lout who had promised to save her from a convent? That
is pretty charity, Father!’
“‘I condemn her for nothing, signor; I am only anxious to know.’
“‘Then you know now,’ said I, and I spoke with some heat; ‘you have
the word of Andrea of Sebenico.’
“‘The glory be to God,’ said he; ‘and now I will take you to her, for
she has been asking for you all day.’
“He led the way from the hall, excellency, and passed down a
vaulted corridor of stone, from the further end of which I heard the
loud tones of a man’s voice—a rich and resonant voice, which would
have held cavalry at the charge. I thought it strange that Father
Mark, who hitherto had shewn some conceit of bearing and of
manner, should become timid and reserved so soon as he heard his
master speaking; but thus it was; and when we came to the door at
the end of the passage he was humble and as fearful as a
schoolboy.
“‘The Count is busy, I fear,’ said he, knocking timidly; ‘he is angry
with the Prefect, and is putting down notes of his reply. Ah! that is a
full stop; now we can go in.’
“Excellency, the full stop was a blow with the fist upon a cabinet or a
table—a blow like the fall of a hammer upon the iron. We took
advantage of its echoes to open the door, and a moment later little
Christine’s arms were about my neck. Ah! that was an hour to
remember, an hour of my life—to have the wanderer’s cheek against
my own, to hear her pretty words, to know that she could still call me
Father Andrea. Yet I forgot not in whose presence I stood, and so
soon as I had returned my child’s greetings I turned towards the
Count and saluted him with proper respect.
“‘Herr Count,’ said I, ‘this is a happy day for me,’ and with that I
continued to bow to him, until suddenly he stopped me peremptorily.
“‘Sit down, sit down, Signor Andrea,’ cried he; ‘you have been long in
the coming, but are none the less welcome for that. We will give you
a glass of hot wine, and then you shall talk to us. I’ll wager it was
cold out yonder. Christine, see that the old man is made
comfortable.’
“Excellency, I was hurt that he should have spoken of me in this way,
for, poor as I am, there is old blood in my veins; yet I saw that he
meant no hurt, and that his manner was the manner of a soldier, little
given to courtesies. Nay, it was impossible not to feel respect for one
who had so many of those gifts which men value. Rough of speech,
huge of person, not wanting kindly eyes, to be reckoned handsome
by the women, oddly dressed in a dark green military tunic—you
would have marked Count Paul even in a regiment of Guards. There
was a command in the lightest word he spoke, an invitation to
obedience which few dared to resist. Yet you felt that his approval
was something to be won and kept, that a kind word from him was
precious as the gifts of others.
“But while I say this of the man, it is difficult to speak of the new
Christine I found in the château of Jajce. Oh, she was changed
beyond words, signor. Five months had made a woman of her; had
robbed her of none of the graces of her childhood, yet had added
other graces to them. She was always a tall child; but she had grown
since I had seen her, and now stood up almost to the shoulder of the
Count. Her new dress, brought from Vienna; her new manner of
speech, for they had taught her much German; her pretty airs, like
the airs of a little princess, all became her exceedingly. I saw that
she was worthy of the château—was worthy of any fortune that could
come to her; and this I made bold to tell the Count when he began to
question me.
“‘Herr Count,’ said I, ‘she was the best of children, and I have loved
her as one of my own.’
“‘And yet you left her four years to starve upon the island of Zlarin,’
said he, drily.
“Christine, eager to see that my wants were attended to, had run
away while he spoke thus harshly. I did not fail to observe that the
Count followed her steps with his eyes—nay, that his eyes rested
upon her alone when she was in the room.
“‘Sir,’ said I, ‘it is true that necessity carried me from my duty to the
child during the space of four years; but now that I am able, I am
willing to repay. If it is your wish, I will take her back with me on the