EFFECT OF AMINO ACID LIMITATION AND SUPPLEMENTATION IN CHINESE HAMSTER ...

151 Pages·2015·3.16 MB·English

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EFFECT OF AMINO ACID LIMITATION AND SUPPLEMENTATION IN
CHINESE HAMSTER OVARY FED-BATCH CULTURES
by
Navid Ghaffari
B. Eng. Ryerson University, Toronto, Canada, 2011

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF APPLIED SCIENCE

in

THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES
(Chemical and Biological Engineering)

THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)
February 2015

© Navid Ghaffari, 2015
Abstract
Fed-batch processes are the industrial norm for the production of recombinant proteins such as
monoclonal antibodies (MAb) from Chinese hamster ovary (CHO) cells. Optimization of such
processes is an important objective of industry process development groups. Amino acid
availability is a key factor that is controlled to achieve the desired product yield and quality. In
order to improve fed-batch productivity, the individual effects of limiting the three depleted
amino acids were investigated for three antibody expressing CHO cell lines. Specifically, the
effects of limiting glutamine, asparagine and cysteine on the cell growth, metabolism, antibody
productivity and quality were investigated. Cysteine limitation was found to be detrimental to
both the cell proliferation and the productivity for all three CHO cell lines. In contrast,
asparagine limitation had no significant effect on either their growth or productivity. Glutamine
limitation resulted in a reduction in growth but not in cell specific productivity, again for all
three cell lines. Neither glutamine nor asparagine limitation significantly affected the MAb
glycosylation. However, the fucosylation ratio was reduced in the absence of cysteine. It was
confirmed that cysteine is a rate limiting factor for the productivity and growth of the three CHO
cell lines. Replenishing cysteine after 1 day of the limitation allowed the cells to regain their
growth and productivity; however, this was not observed after 2 days of cysteine limitation.
Under cysteine limitation there was increased oxidative damage to the mitochondria, possibly
caused by reduced synthesis of co-enzyme A which is essential for functionality of the TCA
cycle. Finally, a fed-batch protocol was developed to improve the MAb productivity of CHO-
DXB11 cells and the results were compared to the results with a commercial feed. Although use
of the commercial feed resulted in higher maximum cell and final MAb concentrations,
maintaining the levels of cysteine yielded cell specific production rates that were comparable to
the commercial feed culture. Overall, the results of this study showed that amino acid limitations
have varied effects on the performance of CHO cell cultures, such that it is important to focus
process development efforts on the critical amino acids.

ii
Preface
The results presented in chapter 4 of this thesis will be submitted in form of a manuscript to a
peer reviewed journal. I have been the contributor to the experimental design and the execution
of the results presented in this work. For decisions regarding the general direction of this project,
I was the major contributor along with my supervisors, Dr. James Piret and Dr. Bhushan
Gopaluni and Dr. Malcolm Kennard. The analysis of amino acids was performed using HPLC by
Chris Sherwood (Dr. Piret Laboratory, University of British Columbia). The autophagic flux
analysis was performed by Dr. Mario Jardon (Dr. Sharon Gorski Laboratory, British Columbia
Cancer Agency). The glycan analysis was performed by Natalie Krahn (Dr. Mike Butler
Laboratory, University of Manitoba). The Raman spectroscopy was performed by me (Dr. Robin
Turner Laboratory, University of British Columbia). The RNA analysis (Appendices) was
performed by Izabella Gadawska and the mitochondrial oxidative damage by Judith Booth (Dr.
Hélène Côté Laboratory, University of British Columbia).

iii
Table of Contents

Abstract ........................................................................................................................................... ii
Preface............................................................................................................................................ iii
Table of Contents ........................................................................................................................... iv
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................. x
List of Abbreviations .................................................................................................................... xv
Acknowledgements ..................................................................................................................... xvii
Dedication .................................................................................................................................. xviii
1. Introduction ................................................................................................................................. 1
1.1 Production of Recombinant Proteins Using Mammalian Cells ............................................ 1
1.2 Different Derivatives of Chinese Hamster Ovary Cells ........................................................ 2
1.3 Culturing CHO Cells ............................................................................................................. 3
1.3.1 Fed-Batch Processes ....................................................................................................... 3
1.3.2 Review of CHO Cell Fed-batch Processes ..................................................................... 4
1.4 Role of Amino Acids on Performance of CHO Cell Cultures .............................................. 6
1.4.1 Glutamine ....................................................................................................................... 7
1.4.2 Asparagine ...................................................................................................................... 9
1.4.3 Cysteine ........................................................................................................................ 10
1.5 Physiological Responses of Mammalian Cells to the Environmental Factors .................... 14
1.5.1 Apoptosis ...................................................................................................................... 15
1.5.2 Autophagy .................................................................................................................... 17
1.5.3 Necrosis ........................................................................................................................ 22
1.6 Research Objectives ............................................................................................................ 23
iv
2. Materials and Methods .............................................................................................................. 26
2.1 Cell Lines ............................................................................................................................ 26
2.2 Cell Culture ......................................................................................................................... 26
2.2.1 Inoculum ....................................................................................................................... 27
2.2.2 Experiments .................................................................................................................. 27
2.2.3 Cell Banking ................................................................................................................. 29
2.3 The Enzyme-Linked Immunesorbent Assay (ELISA) ........................................................ 29
2.4 LC3 Autophagic Flux Assay ............................................................................................... 31
2.5 Free Amino Acids Measurement......................................................................................... 31
2.6 Apoptosis Detection Using Flow Cytometry ...................................................................... 32
2.7 Glycan Analysis .................................................................................................................. 32
2.8 Mitochondrial Damage Analysis ......................................................................................... 33
2.9 Messenger-RNA and Ribosomal-RNA Measurement ........................................................ 33
2.10 Raman Spectroscopy ......................................................................................................... 33
2.11 Cell Count/Viability .......................................................................................................... 34
2.12 Glucose, Lactate, Glutamine, and Glutamate Measurements ........................................... 35
2.13 Calculations ....................................................................................................................... 35
3. CHO Cell Fed-Batch Culture Adaptation ................................................................................. 37
3.1 Fed-Batch Protocol for the Production of EG2-hFc from CHO-DXB11 Cells .................. 37
3.2 Monitoring Amino Acid Consumption and Production in Batch and Fed-Batch Culture .. 37
3.3 Influence of Rapamycin or 3-MA Treatment on Performance of Fed-Batch Cultures of
CHO-DXB11 Cells ................................................................................................................... 41
3.4 Effect of 3-MA treatment or gradual increase of osmolality on productivity of CHO-
DXB11 Cells ............................................................................................................................. 46
3.5 Chapter 3 Conclusions ........................................................................................................ 47
v
4. Effects of Cysteine, Asparagine and Glutamine Limitations and Supplementation in Chinese
Hamster Ovary Fed-Batch Cultures .............................................................................................. 49
4.1 Response of Chinese Hamster Ovary (CHO) Cells to Amino Acid Limitations ................ 49
4.2.1 Protein Production ........................................................................................................ 57
4.2.2 Product Quality ............................................................................................................. 59
4.3 Transient Cysteine Limitation in CHO-DXB11 Cells ........................................................ 63
4.4 Cysteine Dose Response of CHO-DXB11 Cells ................................................................ 65
4.5 Cysteine Limitation Causes Oxidative Damage to Mitochondria ....................................... 65
4.6 Cell Death Mechanism under Cysteine Limitation ............................................................. 66
4.7 Treatments with Rapamycin or Glutathione can Increase Tolerance of the Cells for
Cysteine Limitation ................................................................................................................... 67
4.7.1 Effect of Rapamycin Treatment on Glycosylation of EG2-Hfc Produced from CHO-
DXB11 Cells.......................................................................................................................... 69
4.8 Cellular Autophagy Response to Amino Acid Limitation .................................................. 71
4.9 Transition to Fed-Batch CHO-DXB11 Cultures ................................................................. 73
4.9.1 Cell Growth and Viability ............................................................................................ 73
4.9.2 Glucose and Lactate Concentration .............................................................................. 75
4.9.3 Final Product Concentration and Specific Productivity ............................................... 78
4.10 Conclusions ....................................................................................................................... 80
5. Conclusions and Future Directions ........................................................................................... 82
References ..................................................................................................................................... 84
Appendices .................................................................................................................................. 100
Appendix A – Supplementary GBraphs .................................................................................. 100
A.1 Glutamine and Glutamate Concentration and Uptake Rates (Section 4.2) .................. 100
A.2 Analysis of Caspase 3/7 of CHO-DXB11 under Cysteine Limitation and in Response to
Rapamycin Treatment (Section 4.7) .................................................................................... 103
vi
A.3 HPLC Glycan Analysis (Section 4.2.2) ........................................................................ 104
A.4 Ribosomal and Messenger RNA Levels of CHO-DXB11 Cells (Section 3.3) ............ 106
A.5 Comparison of Glutamine Metabolism and Autophagy in Three CHO Cell Lines ..... 107
Appendix B – Design of Experiments..................................................................................... 110
Appendix C – Raw Data for Chapter 4 ................................................................................... 111

vii
List of Tables
Table 1. 1 Published results on feeding protocols. ......................................................................... 4
Table 2. 1 Cell lines used for performing experiments ................................................................. 26
Table 2. 2 ELISA primary antibodies for different MAbs with the respective coating buffers ... 29
Table 2. 3 Respective washing and coating buffers for different MAbs ...................................... 30
Table 2. 4 Respective dilution buffers and range of standard concentrations for each MAb ....... 30
Table 2. 5 Respective secondary Ab and dilution ratio for each MAb ......................................... 30
Table 2. 6 Respective substrate and substrate buffer for each MAb ............................................ 31
Table 2. 7 Description of Raman peaks of interest in the spectra................................................. 34
Table 4.1 HPLC analysis of glycans released from EG2-hFc produced from CHO-DXB11 cells
....................................................................................................................................................... 60
Table 4.2 HPLC analysis of glycans released from IgG1 produced by CHO-K1SV cells ........... 61
Table 4.3 HPLC analysis of glycans released from IgG1 produced by CHO-S cells .................. 62
Table 4.4 HPLC analysis of glycans released from EG2-hFc produced from CHO-DXB11 cells
under rapamycin treatment ........................................................................................................... 70
Table B. 1 Design of experiment of fed-batch cultures for Section 4.9 ..................................... 110
Table B. 2 Design of experiment of fed-batch cultures for Section 3.3 ..................................... 110
Table C. 1 Cell concentration, viability and product concentration data used for graphs presented
in Section 4.2 – CHO-DXB11 cells ............................................................................................ 111
Table C. 2 Glucose and lactate data used for graphs presented in Section 4.2 – CHO-DXB11
cells ............................................................................................................................................. 112
Table C. 3 Glutamine and glutamate data used for graphs presented in Section A.1 – CHO-
DXB11 cells ................................................................................................................................ 113
Table C. 4 Cell concentration, viability and production concentration data used for graphs
presented in Section 4.2 – CHO-K1SV cells .............................................................................. 114
Table C. 5 Glucose and lactate data used for graphs presented in Section 4.2 – CHO-K1SV cells
..................................................................................................................................................... 115
Table C. 6 Glutamine and glutamate data used for graphs presented in Section A.1 – CHO-K1SV
cells ............................................................................................................................................. 116
viii
Table C. 7 Cell concentration, viability and production concentration data used for graphs
presented in Section 4.2 – CHO-S cells ..................................................................................... 117
Table C. 8 Glucose and lactate data used for graphs presented in Section 4.2 – CHO-S cells .. 118
Table C. 9 Glutamine and glutamate data used for graphs presented in Section A.1 – CHO-S
cells ............................................................................................................................................. 119
Table C. 10 Cell concentration, viability and product concentration of CHO-DXB11 cells for
graphs presented in Section 4.3 .................................................................................................. 120
Table C. 11 Cell concentration, viability and product concentration of CHO-DXB11 cells for
graphs presented in Section 4.4 .................................................................................................. 121
Table C. 12 Cell concentration and viability data of CHO-DXB11 cells under glutathione
treatment for graphs presented in Section 4.7 ............................................................................. 121
Table C. 13 Cell concentration and viability data of CHO-DXB11 cell under rapamycin
treatment for graphs presented in Section 4.7 ............................................................................. 122
Table C. 14 Cell concentration, viability and product concentration of fed-batch cultures of
CHO-DXB11 cells presented in Section 4.9.1............................................................................ 123
Table C. 15 Lactate concentration data of fed-batch cultures of CHO-DXB11 cells presented in
Section 4.9.2................................................................................................................................ 125
Table C. 16 Glucose concentration data of fed-batch cultures of CHO-DXB11 cells presented in
Section 4.9.2................................................................................................................................ 126
Table C. 17 Cell concentration, viability and product concentration of fed-batch cultures of
CHO-DXB11 cells presented in Section 3.2............................................................................... 127
Table C. 18 Cell concentration, viability and product concentration of fed-batch cultures of
CHO-DXB11 cells presented in Section 3.3............................................................................... 128
Table C. 19 Cell concentration, viability and product concentration of fed-batch cultures of
CHO-DXB11 cells presented in Section 3.4............................................................................... 130
Table C. 20. Amino acid concentration of CHO-DXB11 cell during batch cultivation in
BIOGRO CHO medium .............................................................................................................. 133

ix
List of Figures
Figure 1. 1 Major glutamine metabolic pathways in mammalian cells .......................................... 8
Figure 1. 2 Major asparagine metabolic pathways ....................................................................... 10
Figure 1. 3 Cysteine metabolic pathway ....................................................................................... 12
Figure 1. 4 Glutathione (GHS) anti-oxidative reactions ............................................................... 13
Figure 1. 5 Autophagic pathway schematic .................................................................................. 18
Figure 1. 6 Micro autophagy pathway .......................................................................................... 19
Figure 1. 7 Micro autophagy pathway .......................................................................................... 20
Figure 3. 1 The percentage decrease of amino acids in CHO-DXB11 batch culture; n=1. .......... 38
Figure 3. 2 The percentage decrease of amino acids in fed-batch culture of CHO-DXB11 cells;
n=1. ............................................................................................................................................... 38
Figure 3. 3 Schematic of serine metabolism in mammalian cells ................................................. 39
Figure 3. 4 Cell growth profiles of CHO-DXB11 cells in fed-batch cultivation fed with Feed A
and Feed A plus different amino acid solutions............................................................................ 40
Figure 3. 5 Normalized total antibody produced at the end of fed-batch cultures of CHO-DXB11
cells fed with Feed A and supplemented with different amino acid solutions. ............................ 41
Figure 3. 6 Growth and viability profiles of rapamycin treated CHO-DXB1 cells grown in
BIOGRO CHO in batch mode. ..................................................................................................... 42
Figure 3. 7 Normalized final antibody and specific production of CHO-DXB11 cells in batch
mode treated with different doses of rapamycin and different times............................................ 43
Figure 3.8 Cell growth profiles of fed-batch cultures of CHO-DXB11 cell treated with rapamycin
and different concentrations of 3-MA, having 0 mM 3-MA as control........................................ 44
Figure 3.9 The monoclonal antibody concentration profiles of CHO-DXB11 cells treated with
rapamycin and 3-MA, having 0mM 3-MA as control. ................................................................. 45
Figure 3.10 Specific productivity of CHO-DXB11 fed-batch cultures treated with rapamycin and
3-MA, having 0mM 3-MA as control. .......................................................................................... 45
Figure 3. 11 Cell growth profiles of CHO-DXB11 cell in fed-batch cultivation in response to 3-
MA treatment and increasing osmolality (HiOsmo). .................................................................... 46
Figure 3. 12 Total EG2-hFC produced by CHO-DXB11 cells and specific production from the
time of treatment with 3-MA. Error bars represent SEM; n=2. .................................................... 47
x