Enhancing recombinant protein yield and quality using novel CHO GT cells in high density fed batch cultures 1

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Enhancing Recombinant Protein Yield & Quality
Using Novel CHO GT Cells in
High Density Fed-batch Cultures
WONG CHEE FURNG
A thesis submitted for the degree of
Doctor of Philosophy
Department of Paediatrics
Faculty of Medicine
National University of Singapore
2006
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Enhancing Recombinant Glycoprotein Yield & Quality
Using Novel CHO GT Cells in High Density Fed-batch Cultures
SUMMARY
Chinese Hamster Ovary (CHO) cells are regarded as one of the ‘work-horses’ for
complex biotherapeutics production. Currently, batch (BC) and fed-batch (FBC) cultures
are the main culture modes for a vast majority of industrial bioprocesses due to their ease
of operation and reliability. During both BC and FBC, loss in viability attributed to
apoptosis often results in lower recombinant protein yield and affects protein quality. It is
hypothesized that extension of culture life can potentially improve recombinant
glycoprotein yield and quality. Using an ‘in-house’ developed CHO cDNA array and a
mouse oligonucleotide array for time profile expression analysis of CHO BC and FBC, the
genetic circuitry that regulates and executes apoptosis induction were examined. Genes
such as Fadd, Faim, Alg-2, and Requiem were identified to be key apoptosis signaling
genes during CHO cell cultures. Four CHO GT (Gene Targeted) cell lines were developed,
in which each of these early apoptotic genes was either knocked down or overexpressed.
These novel cell lines were shown to be effective in prolonging culture life resulting in
higher cell densities and significantly enhancing glycoprotein yield and quality.
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ACKNOWLEDGEMENTS

Despite being listed as the sole author of this thesis, this project has had ample
contributions from a lot of people, all of whom I am extremely grateful to.
To my supervisors Dr. Heng Chew Kiat and Prof. Miranda Yap, thank you very
much for being your patient guidance these the past 3 years. To Dr. Heng, thanks for
caring more than just project schedules and timelines and to Prof. Yap, thanks for pushing
me to be more than just a ‘lab-rat’ and that there is more to science than just the lab.
Special thanks too to my friends in the Animal Cell Technology group at BTC,
who welcomed me into the group with open arms and had shared some exciting and
challenging times in the research arena with me. Kathy for her guidance and support, Chun
Loong for successfully ‘knocking’ bioreactor principles into my brain and his ‘weird’
insights, Yih Yean for his endless help and fish-tales, Vesna for keeping things in
perspective, Niki for her friendship and inputs and of course not forgetting, Victor, Yan
Ying, Janice, Sanny and Poh Choo.
My heartfelt appreciation to BTI’s Analytics group led by Dr. Goh Lin-Tang and
Dr. Lee May May for their support in glycosylation analysis. Many thanks to Sim Lyn
Chiin, Ong Boon Tee and Tracy.
The BTI Microarray group: Dr. Peter Morin Nissom, Jennifer Lo, Tan Kher Shing,
Ong Peh Fern, Breanna Cham and Chuah Song Hui who had helped so much in getting the
CHO and mouse chips up and going.
Of course, there’s the help given by the undergraduate students whom worked with
me for their industrial attachment. Many thanks to Andrew Wu, Wei Jan, Wong Ju Wei,
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Nick Lim, Zeon Na, Amanda Lanza, Ang Pei Ling, Emily Lau and Dennis Goh for their
help and enthusiasm!
Last but not least, my family for being so understanding and supportive throughout
the years. Thanks especially to my wife, Winnie for being beside me through the good and
the bad times. Many thanks to my parents for their love and nurture and allowing me to
pursue my own passion.
Thanks everyone! This project would not be what it is without all of you. All the
research work described in this thesis was carried out in the Bioprocessing Technology

Institute (BTI), funded by the Biomedical Research Council (BMRC) established under
the Agency for Science, Technology and Research (A*STAR).
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TABLE OF CONTENTS:
Summary
Acknowledgements
Table of Contents
List of Figures
List of Tables
CHAPTER 1 Introduction
1.1 Background
1.1 Thesis Objectives
1.3 Thesis Organization
CHAPTER 2 Literature Review
2.1 Biotherapeutics Production In Mammalian Cell Culture
2.1.1 Batch Cultures
2.1.2 Fed-Batch Cultures
(A) Feed Media Design
(B) Feeding Strategy
2.1.3 Accumulation of Toxic Waste Metabolites
2.1.4 Reduction of Metabolite Waste Production
2.2 The Importance of Protein Glycosylation
2.2.1 N-Glycosylation
2.2.2 Heterogeneity in N-Glycosylation
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2.2.3 Factors Affecting Glycosylation
(A) Host Expression System
(B) Culture Environment
(C) Extracellular Degradation
2.3 Cell Death In Bioreactors
2.3.1 Apoptosis vs. Necrosis
2.3.2 Triggers of Apoptosis in Bioprocesses
2.3.3 Caspases, the central executioners of apoptosis
2.3.4 Apoptosis Signaling
2.3.5 Suppressing Apoptosis in Culture
2.4 Transcriptome Analysis
2.4.1 Microarray Technology
2.4.2 Transcription Expression Profiling
CHAPTER 3 Materials & Methods
3.1 Cell Lines

3.1.1 CHO IFN-γ
3.1.1 CHO GT
O
FADD DN
3.1.1 CHO GT
O
FAIM
3.1.1 CHO GT
KD
ALG-2
3.1.1 CHO GT
KD
REQUIEM
3.2 Cell Culture Maintenance
3.2.1 Working Cell Bank
3.2.2 Culture Maintenance
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3.3 Bioreactor Culture Operations
3.3.1 Batch Culture Operations
3.3.2 Fed-batch Culture Operations
(A) Glutamine-controlled FBC
(B) Glucose-controlled FBC coupled with glutamine profile
feeding
3.4 Metabolite Analysis
3.4.1 Glucose, Glutamine, Glutamate and Lactate measurement
3.4.2 Ammonia measurement
3.4.3 Amino Acid Analysis
3.5 Recombinant Glycoprotein Yields Determination
3.5.1 Determining IFN-γ Yield by ELISA
3.5.2 Average Specific Rates Calculations
3.6 N-Glycosylation Quality Of IFN-γ
3.6.1 Immunoaffinity Purification of IFN-γ
3.6.2 IFN-γ Macro-heterogeneity: Site-occupancy
3.6.3_ IFN-γ Micro-heterogeneity: Structural Composition of
Oligosaccharides
(A) Determination of Oligosaccharide Species using Mass
Spectrometry

(i) Tryptic digestion and glycopeptides separation
(ii) Reverse phase HPLC separation of IFN-
γ
glycopeptides
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(iii) Glycopeptides analysis using MALDI/TOF mass
spectometry
(B) Quantification of Oligosaccharide Species Using High pH
anion-exchange chromatography (HPAEC)
(i) Enzymatic release of glycans from IFN-
γ

(ii) Preparation of Glycan Standards
(iii) Purification of Released Glycans
(iv) HPAEC Operation
3.6.4 Sialylation Assay
3.7 Cell Viability and Apoptosis Detection
3.7.1 Trypan Blue Exclusion Viability Assay
3.7.2 Morphological Detection of Apoptosis
3.7.3 Biochemical Detection of Apoptosis
3.8 Transcriptome Analysis
3.8.1 Total RNA Purification
3.8.2 Microarray Construction
(A) Slide coating
(B) Printing of DNA probes
(C) DNA Immobilization & Preparation of Microarray Slides
3.8.3 Microarray Hybridization
(A) cDNA Synthesis
(B) Dye-labeling of cDNA
(C) Microarray Hybridization and Wash
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3.8.4 Microarray Image and Data Analysis
3.8.5 Real-Time PCR Validation
3.9 Cloning of Apoptotic Genes
3.9.1 Bacterial Culture
(A) Bacterial Cells
(B) Culture Broth and Agar Plates
3.9.2 Vectors
(A) pCR
®
-TOPO
®
Vector
(B) pcDNA3.1(+) Vector
(C) pSUPER.neo Vector
3.9.3 Gene Cloning
(A) Gene specific Polymerase Chain Reaction (PCR)
(B) Rapid Amplification of cDNA ends (RACE)
(C) DNA Restriction & Ligation
(D) DNA Sequencing
(i) Cycle Sequencing PCR

(ii) Ethanol Purification of Cycle Sequencing PCR Product
(iii) Electrophoresis
3.9.4 CDNA Cloning of Fadd, Faim, Alg-2 and Requiem
(A) Fadd
(B) Faim
(C) Alg-2
(D) Requiem
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3.10 Vector Construction For the Creation of CHO GT cells
3.10.1 pcDNA3.1(+) cg FADD Dominant Negative
3.10.2 pcDNA3.1(+) cg FAIM
3.10.3 pSUPER.neo cg ALG-2
3.10.4 pSUPER.neo cg REQUIEM
3.11 Creation of Stable Cell Lines
3.11.1 Selecting for Stable Expression Transfected Pool
3.11.2 Selecting for Single Cell Cloning
3.12 Quantitative Real Time PCR
3.12 Determination of Statistical Significance
CHAPTER 4 High Density Fed-batch Cultures of CHO Cells
INTRODUCTION
RESULTS
4.1 CHO Cell Growth & Metabolism in FBC
4.1.1 Glutamine-controlled FBC (FBC
0.1
, FBC
0.3
and FBC
0.5
)
(A) Tight control of glutamine concentrations
(A) Higher viable cell density and specific growth rates
(A) Reduced ammonia production
(A) Reduced lactate production
4.1.2 Glucose-controlled FBC (FBC
0.3/0.35
and FBC
0.3/0.70

)
(A) Tight control of glucose concentrations
(A) Viable cell density and specific growth rates
(A) Reduction of lactate production
(A) Increased glutamine consumption and ammonia production
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4.2 Enhanced Recombinant IFN-γ yields during FBC
4.3 N-glycosylation Quality of Recombinant IFN-γ
4.3.1 Macro-heterogeneity: Site-occupancy
4.3.1 Micro-heterogeneity: Structure and composition of IFN-γ
glycans
(A) The major glycan species of IFN-γ from BC (Asn25: C08-F
and Asn97: C07, C13)
(A) Glutamine-controlled FBC maintained the distribution of
major glycan species but not the minor glycan species
(A) Glucose-controlled FBC maintained the distribution of the
major glycan species but not the minor glycan species
4.3.1 Sialylation of recombinant IFN-γ
4.3.1 Impact of culture viability on N-glycosylation Quality
(A) Low culture viability does not alter IFN-γ macro-
heterogeneity
(A) Low culture viability led to increased number of low
molecular weight N-glycans
(A) Low culture viability led to decreased IFN-γ sialic acid
content
DISCUSSIONS
4.4.1 Improving FBC through use of dynamic online feeding
4.4.1 N-Glycosylation in FBC
CONCLUSION
CHAPTER 5 Transcriptional Profiling of Apoptotic Pathways in
Batch and Fed-batch CHO Cell Cultures
INTRODUCTION
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RESULTS
5.1 Apoptosis Signaling During BC and FBC
(A) Up-regulation of FasL and Fadd in the Death Receptor-
mediated Apoptosis Signaling during BC and FBC
(A) Up-regulation of Bim and Bad in the Mitochondrial-mediated
Apoptosis Signaling during BC and FBC
(A) Down-regulation of Ire-1 and up-regulation of Alg-2 in the
Endoplasmic Reticulum (ER)-mediated Apoptosis Signaling
during BC and FBC
(D) Differential expression of inhibitors of apoptosis proteins
DISCUSSIONS
5.2.1 Apoptosis-related Cell Death in CHO cell BC and FBC
5.2.2 Strategies to delay onset of apoptosis in culture
CONCLUSIONS
CHAPTER 6 Targeting Early Apoptotic Genes in Batch and Fed-

Batch CHO Cell Cultures
INTRODUCTION
RESULTS
6.1 Cloning of Apoptotic Homologs from CHO cells
6.2 Creation of Gene Targeted CHO (CHO GT) cell lines
6.3 CHO GT Cells in BC
6.3.1 Growth of CHO GT Transfected Pools in BC
6.3.2 Proteolytic Activities of Caspases in BC
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6.4 CHO GT Cell Lines in FBC
6.5 N-glycosylation Quality of IFN-γ Produced by CHO GT
cells during FBC

6.5.1 Macro-heterogeneity of recombinant IFN-γ
6.5.1 Micro-heterogeneity of recombinant IFN-γ
6.5.1 Sialylation of recombinant IFN-γ
DISCUSSION
6.6.1 Targeting of Fadd, Faim, Alg-2 and Requiem to Prolong BC and
FBC
6.6.1 Strategies to Enhance Apoptosis Resistance
CONCLUSION
CHAPTER 7 Conclusions & Recommendations
7.1 Conclusions
7.2 Recommendations
7.2.1 Impact of improved N-glycosylation IFN-γ quality on its
bioactivity and pharmocokinetic properties
7.2.2 Characterization of recombinant IFN-γ cross-reactivity with
CHO cells
7.2.3 Analysis of differentially expressed genes
7.2.4 Characterizing the role of Fadd, Faim, Requiem and Alg-2
7.2.5 Combinatorial gene targeting to further enhance apoptosis
resistance
7.2.6 Augmenting recombinant production using epigenetic gene
silencing
Abbreviations
References
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APPENDICES
Appendix 1 Ammonia and Lactate effects on CHO cell growth and
viability
Appendix 2 Fas Associated Death Domain (Fadd)
Appendix 3 Fas Apoptisis Inhibitory Molecule (Faim)
Appendix 4 Apoptosis Linked Gene 2 (Alg-2)
Appendix 5 Requiem
Appendix 6 Publications
Wong CFD, Wong TKK, Goh LT, Heng CK and Yap MGS. 2005. Impact
of Dynamic Online Fed-Batch Strategies on Metabolism, Productivity
and N-Glycosylation Quality in CHO Cell Cultures. Biotechnol Bioeng
89: 164-177
Wong CFD, Wong TKK, Lee YY, Nissom PM, Heng CK and Yap MGS.
2006. Transcriptional Profiling of Apoptotic Pathways in Batch and Fed-
batch CHO Cell Cultures. Biotechnol Bioeng. Accepted 27
th

December
2005
Wong CFD, Wong TKK, Heng CK and Yap MGS. 2006. Targeting Early
Apoptotic Genes in Batch and Fed-Batch CHO cell Cultures. Biotechnol
Bioeng. Accepted 27
th
December 2005.
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LIST OF FIGURES:
Page
Figure 2.1 N-glycan structures.
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Figure 2.2 Morphological differences between apoptosis and necrosis.
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Figure 2.3 Example of scanned image of a section of a hybridized microarray.
Figure 3.1 Schematic representation and picture of a dynamic online fed-batch
culture bioreactor system set-up.
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Figure 3.2 Example chromatogram of oligosaccharides from IFN-γ resolved by
HPAEC
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Figure 3.3 Morphological classification of cells into apoptotic or non-

apoptotic populations
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Figure 3.4 Semi-automatic DNA dispensing cell used to print DNA probes
onto glass slides.
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Figure 3.5 Restriction map of pCR®2.1 (Adapted from Invitrogen product
brochure)
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Figure 3.6 Restriction map of pcDNA3.1 (+) and (-). (Adapted from
Invitrogen product brochure).
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Figure 3.7 Restriction map of pSUPER.neo (Adapted from Oligoengine
produce brochure).
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Figure 4.1 Residual glutamine concentrations in BC and FBC maintained at
0.1, 0.3 and 0.5mM glutamine.
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Figure 4.2 Viable cell densities of FBC controlled at 0.1, 0.3 and 0.5mM
glutamine and control BC.
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Figure 4.3 Ammonia accumulations during FBC controlled at 0.1mM, 0.3mM
and 0.5 mM glutamine compared to control BC.
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Figure 4.4 Glucose concentrations during FBC controlled at 0.1mM, 0.3mM
and 0.5 mM glutamine compared to control BC.
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Figure 4.5 Lactate accumulations during fed-batch cultures controlled at
0.1mM, 0.3mM and 0.5 mM glutamine compared to control BC.
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Figure 4.6 Residual glucose concentrations in FBC maintained at 0.70mM and
0.35mM glucose.
Figure 4.7 Viable Cell Densities of FBC controlled at 0.70mM and 0.35 mM
Glucose.
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Figure 4.8 Lactate Concentrations of FBC controlled at 0.70mM and 0.35
mM glucose.
Figure 4.9 Residual glutamine concentrations of FBC controlled at 0.70mM
and 0.35 mM glucose.
Figure 4.10 Ammonia accumulation in FBC controlled at 0.70mM and 0.35
mM glucose.
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Figure 4.11 Glycan site-occupancy in BC and FBC.
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Figure 4.12 Sialic acid content of IFN-γ harvested during high viability
(>95%) in BC and FBC.
Figure 4.13 Glycan site-occupancy during BC and FBC at low culture
viability (70-80%).
Figure 4.14 Comparison between sialic acid content of IFN-γ harvested during
low viability, 70-80% and high viability, >95% in BC and FBC.
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Figure 5.1 BC and FBC for expression profiling using microarrays.
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Figure 5.2 Apoptosis-related genes regulated during BC and FBC of CHO
cells

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Figure 5.3 Validation of microarray expression profiles of apoptosis signaling
genes across exponential, stationary and death phases of BC and FBC.
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Figure 5.4 Apoptosis signaling in BC and FBC of CHO cells.
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Figure 6.1 Apoptosis signaling via death receptor-, mitochondria- and ER-
mediated apoptosis signaling pathways during CHO cell culture.
Figure 6.2 Schematic representation of gene cloning approach using gene
specific PCR and RACE methods.
Figure 6.3 FADD Dominant Negative Strategy For Apoptosis Suppression.
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Figure 6.4 CHO GT
O
cells in BC. The viable cell density, viability and
percentage of apoptotic cells of CHO GT
O
FADD DN pool and CHO GT
O
FAIM pool in BC.
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Figure 6.5 CHO GT
KD
cells in BC. The viable cell density, viability and
percentage of apoptotic cells of CHO GT
KD
ALG-2 pool and CHO GT

KD
REQUIEM pool in BC.
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Figure 6.6 Recombinant human IFN-γ yields of CHO GT cells in BC.
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Figure 6.7 Specific productivities of recombinant human IFN-γ in CHO GT
cells in BC.
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Figure 6.8 Caspase-8, -9 and –3 activities during BC of CHO GT
O
cell lines
Figure 6.9 Caspase-8, -9 and –3 activities during BC of CHO GT
KD
cell lines
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Figure 6.10 Viable cell densities of CHO GT cell lines in FBC.
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Figure 6.11 Enhanced recombinant human IFN-γ yields in CHO GT cell lines
during FBC.
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Figure 6.12 Site-occupancy of IFN-γ purified from FBC of CHO GT cells
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Figure 6.13 Micro-heterogeneity of complex-type glycans of recombinant
human IFN-γ harvested from CHO GT cells.
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Figure 6.14 Sialylation of recombinant IFN-γ in CHO GT cell lines during
mid-exponential, stationary and death phase of FBC.
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LIST OF TABLES:
Page
Table 2.1 Examples of recombinant therapeutics proteins produced in CHO
cells.
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Table 2.2 Effects of the anti-apoptosis genetic engineering on CHO cell lines.
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Table 3.1 List of N-linked oligosaccharide standards and their volumes used
in the preparation of standard sets
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Table 3.2 Gene specific primers used in quantitative real time PCR
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Table 4.1 Setpoint concentrations of glucose and glutamine used for Batch
and Fed-batch cultures.
Table 4.2 Specific growth rates and consumption/production rates of
metabolites for FBC compared to BC
Table 4.3 Yields and Specific Productivity of Recombinant IFN-γ during BC
and FBC.
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Table 4.4 Sugar compositions and glycan structures of Asn25.
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Table 4.5 Sugar compositions and glycan structures of Asn97.
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Table 4.6 Micro-heterogeneity of IFN-γ glycans on Asn25 harvested during
HIGH VIABILITY (>95%).
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Table 4.7 Micro-heterogeneity of IFN-γ glycans on Asn97 harvested during
HIGH VIABILITY (>95%).

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Table 4.8 Micro-heterogeneity differences between IFN-γ glycans on Asn25
harvested during high viability and low viability (70-80%) of BC and FBC
0.3
.
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Table 4.9 Micro-heterogeneity differences between IFN-γ glycans on Asn97
harvested during high viability and low viability (70-80%) of BC and FBC
0.3
.
Table 6.1 Summary of similarity between C. griseus genes (Fadd, Faim, Alg-
2 and Requiem) with equivalent homologs from M. musculus and H. sapien.
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Table 6.2 Fold increase/decrease in gene expression in CHO GT cells and
control cell lines.
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1 INTRODUCTION
1.1 Background
One of the challenges faced by large-scale production of therapeutic proteins is the
need to achieve high cell density while maintaining high culture viability in order to obtain
high recombinant glycoprotein yield and quality. In most of these proceses, batch (BC)
and fed-batch (FBC) cultures are the main culture modes used for recombinant protein
production. The major cause of viability loss in BC is nutrient depletion. By addressing
nutrient depletion through nutrient feeding, FBC offer a solution towards higher cell
density and extended culture viability. However, overfeeding can lead to increased
accumulation of toxic metabolites such as ammonia and lactate that are detrimental to cell
growth and viability. This can be alleviated through the control of glucose and glutamine at
low levels in the culture medium allowing for a metabolic shift towards lower lactate

production and an energetically more efficient glutamine metabolism without any loss in
productivity (Cruz et al., 1999; Europa et al., 2000, Lee et al., 2003). Thus, achieving high
cell density FBC through detailed analysis of nutrient consumption and feeding strategy is
highly desirable in terms of product yield improvement.
Currently, the influence of metabolic shifts on product glycosylation remains
relatively unknown. The structural heterogeneity of glycans on glycoproteins is sensitive
to culture environment such as nutrient starvation, metabolic waste accumulation, culture
viability, pH and temperature (Goochee and Monica, 1990; Yang and Butler, 2000;
Andersen et al., 2000; Baker et al., 2001). As glycans on glycoproteins are often critical for
a myriad of functions, some of which are crucial for its pharmacokinetic properties (Varki,
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1993; Jenkins et al., 1996; Jefferis, 2005), it is important to examine the impact of the
production process on glycosylation patterns to ensure product efficacy and consistency.
Despite nutrient feeding, FBC are still susceptible to loss in culture viability albeit
at a later time point compared to BC. The implication of this is that either a critical
nutrient is still missing or an insult is triggering cell death. In addition, viability loss not
only lowers productivity but may affect recombinant protein quality as well. For example,
degradative enzymes released during cell death can detrimentally affect the sialylation of
the recombinant protein resulting in reduced circulatory half-life of biotherapeutics in vivo
(Varki, 1993; Gramer et al., 1995).
It has been shown the major mode of cell death in culture is apoptosis, a genetically
controlled form of cellular suicide (Singh et al., 1994; Goswami et al., 1999). The most
common anti-apoptotic manipulation is BCL-2 protein overexpression (Fusseneger and
Bailey, 1998’ Laken and Leonard, 2001; Vives et al., 2003a; Arden and Betenbaugh, 2004).
However, in most cases, this resulted in limited protection from apoptosis induction
(Laken and Leonard, 2001). Furthermore, in spite of being the predominant mode of cell
death, apoptosis signaling during bioreactor cultures has not been examined extensively.
Investigation of apoptosis signaling is therefore crucial for a better understanding of cell
death in bioprocesses.
First described by Schena and co-workers (1995), DNA microarray technology is

based on the simultaneous hybridization of two different DNA populations (each labeled
either with red or green fluorescence) onto microarrays containing thousands of distinct
gene sequences. The ratio of fluorescence intensity then represents the ratio of expression
between the two different populations. Transcriptional profiling using DNA microarray
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offers a tool to simultaneously characterize these multiple gene expression changes in a
global manner. Consequently, genes that are involved in apoptosis signaling in culture can
be identified and specifically targeted to suppress apoptosis in BC and FBC of CHO cells.
1.2 THESIS OBJECTIVES
The main goal of the thesis is to enhance the recombinant glycoprotein yield and
quality in FBC of CHO cells through gene targeting. It is hypothesized that transcriptome
analysis could be used to decipher cell death signaling in BC and FBC, enabling genes
associated with the early onset of apoptosis to be identified and targeted to prolong
culture.
The scope of the thesis involved:
(1) Developing an enhanced high-density FBC based on a dynamic online feeding
strategy for CHO cells producing recombinant human interferon gamma (IFN-γ) and
determining the impact on IFN-γ production and glycosylation quality,
(2) Examining the signaling pathways that are responsible for apoptosis induction
in BC and FBC processes using transcriptome analysis and
(3) Based on the knowledge gained, develop novel apoptosis gene-targeted cell lines
through the targeting of key early apoptosis genes to extend culture viability.
1.3 THESIS ORGANIZATION
There are eight chapters in this thesis. Chapter 1 provides a brief introduction and
defines the objective and scope of the thesis. Chapter 2 reviews the literature on cell
culture processes, protein glycosylation and apoptotic cell death, as well as transcriptome
analysis. A detailed description of the materials and methods used is covered in Chapter 3
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and in Chapter 4, the development of an enhanced high-density fed-batch process and its
impact on cellular metabolism and protein N-glycosylation are described. In Chapter 5, the

results of the transcriptome profiling of apoptosis signaling pathways for BC and FBC are
reported. Chapter 6 describes the cloning of four early apoptosis signaling genes identified
using transcriptional profiling, and the subsequent targeting of the four genes through gene
‘knock-down’ or overexpression to generate novel apoptosis resistant CHO GT cells. This
chapter also detailed CHO GT’s ability to significantly improve recombinant glycoprotein
yield and quality. Chapter 7 summarizes the important conclusions resulting from this
study and provides recommendations for future work.
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2 LITERATURE REVIEW
2.1 Biotherapeutics Production In Mammalian Cell Culture
The early 1980s saw the biopharmaceutical industry starting to use recombinant
DNA technology for the production of large quantities of therapeutic proteins. During this
period, human growth hormone (hGH) was extracted from human pituitaries for the
treatment of dwarfism. Discovery of possible Creutzfeldt-Jacob disease transmission using
human pituitaries purified hGH in 1985 quickly shifted treatment to the use of
recombinant hGH which offers a much safer treatment alternative. The biopharmaceutical
industry’s global market value now stands in excess of US$30 billion and has more than
500 biopharmaceuticals undergoing clinical trials (Walsh, 2003). These new
biopharmaceuticals offers treatment for diseases ranging from cancer, autoimmunity,
cardiovascular and infectious diseases.
Today, 60-70% of all the recombinant protein pharmaceuticals are produced in
mammalian cells (Chu and Robinson, 2001; Wurm, 2004). Mammalian cells have been used
extensively for complex biologics production due to their ability to produce properly
folded and glycosylated version of the proteins. Although yeast, insect and plant cells are
capable of glycosylating proteins, only mammalian cells are capable of producing
glycoforms similar to those required for human therapeutics. Among the mammalian cells,
Chinese hamster ovary (CHO) cells are one of the most commonly used cell lines for the
production of therapeutically important proteins (Table 2.1). CHO cells have been
successfully used for the production of important biologics such as erythropoietin (Sasaki
et al. 1987), Factor VIII (Kaufman et al. 1988) and follicle stimulating hormone (Gerbert &

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Gray 1994). They are well studied and characterized, have high transfection efficiency and
good amplification mechanisms like DHFR system (Simonsen and McGrogan, 1994).
Table 2.1 Examples of Recombinant Therapeutics Proteins Produced in CHO cells
(Adapted from Walsh, 2003)
Product
Recombinant
Protein
Therapeutic Usage
Commercial Drug
Name
Factor VIII
Hemophilia A
ReFacto, Bioclate
Factor IX
Hemophilia B
Benefix
Blood factors
Tissue plasminogen
activator
Myocardial infarction
Tenecteplase,
TNKase, Activase
Follicle-stimulating
hormone
Infertility
Follistim, Puregon,
Gonal F
Choriogonadotrophin
Assisted reproductive

techniques
Ovitrelle
Hormones
Thyrotrophin-α
Detection/ trement of
thyroid cancer
Thyrogen
Hematopoietic
growth factors
Erythropoietin
Treatment of anemia
Aranesp, Nespo,
Neorecormon
Interferons
Interferon-β
Relapsing/remitting
multiple sclerosis
Rebif, Avonex
rIgG1kMab that
binds to IgE
Asthma
Xolair
Monoclonal
antibody-based
products
Targets CD20
antigen
Non-Hodgkin
lymphoma
Zevalin

α-galactosidase
Fabry disease
Fabrazyme
TNF receptor-IgG
fragment fusion
protein
Rheumatoid arthritis
Enbrel
Others
Dornase-α
Cystic fibrosis
Pulmozyme
To be economically feasible, recombinant protein production processes often need
to achieve high cell yield, steady productivity and consistent glycosylation. Industrial
processes for large-scale production using mammalian cell culture are suspension serum-
free batch and fed-batch cultures in stirred-tank reactors (Chu and Robinson, 2001;
Andersen and Krummen, 2002; Wurm, 2004). The popularity of these culture modes stem
from their relative ease and simplicity of operation. Perfusion cultures have recently gained
25
popularity as they can achieve higher cell densities than BC and FBC and can be
maintained for many weeks, even months. However, perfusion cultures are very
sophisticated processes, which require a high degree of control and the need to maintain
contamination-free conditions for longer periods of time.
2.1.1 Batch Cultures (BC)
The usual practice in BC is to supply all the nutrients needed by the cells for the
duration of the culture at the beginning of a culture. Unlike bacterial cells, mammalian cells
have complex nutritional requirements for cell growth in vitro. Different cell lines also have
different nutritional requirement, necessitating the development of different basal media
that contains different amounts of essential nutrients. Detailed spent medium analysis
allowed for the development of highly fortified basal media enriched in multiple nutrient

components that were limiting in the basal media. This allows for further improvement in
cell density and protein yield. However, many medium components can inhibit cell growth
when added at concentrations higher than those commonly found in basal medium (Hassell
et al., 1991, Bibila and Robinson, 1995, Lao and Toth, 1997).
2.1.2 Fed-batch Cultures (FBC)
FBC allows for periodic nutrient feeding to prevent nutrient depletion. Periodic
feeding also prevents exposure to inhibitory concentrations of excessive nutrients. Due to
the complexity of mammalian cell metabolism, optimization of timing and mode of addition
of nutrient feeds is often performed empirically (Bibila and Robinson, 1994). Most FBC
strategies rely upon a combination of physiological reasoning, nutrient depletion analysis
and reiterative feed design to maximize cell growth, culture longevity and recombinant
protein production.