Dielectric and Precursor Analysis to Study Metabolic Effects on CHO Cell Viability and Antibody ...

247 Pages·2016·9.12 MB·English

Dielectric and Precursor Analysis
to Study Metabolic Effects on CHO Cell Viability
and Antibody Glycosylation

by

Katrin Braasch

A Thesis submitted to the Faculty of Graduate Studies of
The University of Manitoba
in partial fulfillment of the requirements of the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology
University of Manitoba
Winnipeg

Copyright © 2015 by Katrin Braasch
ACKNOWLEDGMENTS

I would like to thank my supervisor, Dr. Michael Butler, for his constant support and
guidance throughout my Grad Studies, as well as all the opportunites. I would also like to thank
Dr. Deborah Court and Dr. David Levin, members of my advisory committee, for their helpful
advice and support. Furthermore a thank you to my external examiner, Dr. Michael Kallos, for
his criticism and advise to improve this thesis. In addition, I would like to thank the following
people:
 Dr. Douglas Thomson, Dr. Gregory Bridges, Dr. Marija Nikolic-Jaric, Elham Salimi,
Dr. Tim Cabel, Ashlesha Bhidea, Bahareh Saboktakin Rizi, Kaveh Mohammad, and
Samaneh Afshar for their insight, collaboration and support with the dielectric portion
of this project.
 Vincent Jung and Dr. Maureen Spearman for their technical support and helpful advice.
 To all members of the Butler lab, past and present, that I had the opportunity to work
with: Natalie Krahn, Sarah Chan, Dr. Ben Dionne, Dr. Venkata Tayi, Neha Mishra,
Carina Villacres, Viridiana Urena Ramirez, Bo Liu, Alan Froese, Teslin Sandstrom and
Rachel Rudney. Thank you for your support and constant discussion.

I am also grateful to my husband, Paul, and my family for their encouragement and
support throughout this time.

The work for this thesis was supported by a Natural Science and Engineering Research
Council network research grant. A Manitoba Graduate Scholarship and a Faculty of Science
Graduate Scholarship are also gratefully acknowledged.
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ABSTRACT
The main goal in biopharmaceutical production is achieving high volumetric productivity
while maintaining product quality (i.e. glycosylation). The objectives of this project were to
explore the use of dielectric analysis in the early detection of cell demise and to analyze the
impact of nucleotide / nucleotide sugar precursor feedings in biopharmaceutical production and
glycosylation.
Measurements of changes in the polarizability of individual cells can be performed in a
dielectrophoretic (DEP) cytometer designed at the University of Manitoba. In this instrument the
trajectory of individual cells was tracked according to their polarizability and recorded as a force
index (FI). The identified sub-populations from a batch bioreactor and apoptosis-induced
cultures were correlated with the fluorescent markers of apoptosis analyzed in a flow cytometer.
Discrete cell sub-populations were identified as cells passed through the various stages of
apoptosis. In the batch and the starvation culture the early changes in the measured FI of cells
correlated with the Annexin V fluorescent assay associated with early phase apoptosis. For the
oligomycin and staurosporine cultures changes in the FI could be correlated to modifications in
the mitochondrial metabolism linked with early apoptosis for both inducers.
In fed-batch experiments 10 mM galactose alone or 20 mM galactose in combination
with 1 mM uridine or 1 mM uridine + 8 μM MnCl was added to the basal and feed medium for
2
two CHO cell lines to determine their impact on the biopharmaceutical production and the
glycosylation process. The results showed that the addition of all three precursors combined
increased UDP-Gal, which increased and maintained the galactosylation index during the
bioprocess for CHO-EG2 and CHO-DP12 cultures by 25.4% and 37.9%, respectively, compared
to the non-supplemented fed-batch culture. In both cell lines saturation was reached when a
iii
further increase in the UDP-Gal concentration did not increase the galactosylation. A negative
impact on cell growth was observed with the uridine addition in the CHO-EG2 culture, which
was linked to the CHO-EG2 cell line being DHFR-/-.
This work presents a dielectric detection method to monitor early changes in the cell
metabolism and information for shifting and maintaining galactosylation during
biopharmaceutical production.
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LIST OF ABBREVIATION
7-AAD 7-aminoactinomycin
ADCC Antibody-dependent cellular cytotoxicity
ADP Adenosine diphosphate
AEC Adenylate Energy Charge (ATP + 0.5 ADP)/(ATP + ADP + AMP)
AMP Adenosine monophosphate
ATP Adenosine triphosphate
CDC Complement-dependent cytotoxicity
CDP Cytidine diphosphate
CHO Chinese hamster ovary
CTP Cytidine triphosphate
DEP Dielectrophoresis
ELISA Enzyme linked Immuno Sorbent Assay
EDTA (Ethylene dinitrilo)-tetraacetic acid
FI Fucosylation index
GDP Guanosine diphosphate
GI Galactosylation index
GTP Guanosine triphosphate
HILIC Hydrophilic interaction liquid chromatography
HPLC High performance liquid chromatography
IVCD Integral viable cell density
NTP Nucleotide triphosphate
NTP ratio (ATP + GTP)/((UDP-GlcNAc + CTP) + UTP)
v
NTP/U ratio NTP ratio/ U-ratio
PBS Phosphate-buffered saline
PS Phosphatidylserine
q Specific glucose consumption rate
G
q Specific lactate production rate
L
SI Sialylation index
UDP-Glc Uridine diphosphate-glucose
UDP-GalNac Uridine diphosphate-N-acetylgalactosamine
UDP-GlcNac Uridine diphosphate-N-acetylglucosamine
UDP-GNac Sum of UDP-GalNac and UDP-GlcNac
U-ratio UTP/UDP-GNac
UTP Uridine triphosphate
Y Yield coefficient; moles of lactate produced per mole of glucose
Lac/Glc
utilized
μ Maxium growth rate
max
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TABLE OF CONTENTS
Acknowledgements ii
Abstract iii
Abbreviations v
Table of Contents vii
List of figures xv
List of tables xx
List of copyright material xxii

Chapter 1 – Introduction 1
1.1 Biopharmaceuticals 1
1.2 Antibodies 3
1.2.1 Antibody DP12 4
1.2.2 Antibody EG2 5
1.3 Mammalian cell culture 5
1.3.1 Batch and fed-batch culture 6
1.3.2 Growth media 8
1.3.3 Monitoring of cell culture 10
1.4 Apoptosis 10
1.4.1 Intrinsic (mitochondrial) pathway 12
1.4.2 Extrinsic (receptor mediated) pathway 13
1.5 Cell Culture Density and / or Viability Determination 14
1.5.1 Coulter Counter 14
vii
1.5.2 Image based analysis 15
1.5.3 Flow cytometer 15
1.5.4 Dielectric based analysis 17
1.5.4.1 Dielectric properties of mammalian cells 17
1.5.4.2 Bulk dielectric measurement 17
1.5.4.3 Single cell dielectric measurement 19
1.5.5 Adenylate energy charge 22
1.6 Intracellular nucleotide and nucleotide sugars 23
1.6.1 Monosaccharide Metabolism 23
1.6.2 Building blocks for glycosylation 24
1.6.3 Nucleotide / Nucleotide Sugar Analysis 25
1.7 Glycosylation 27
1.7.1 N-linked glycans 27
1.7.2 Importance of glycans 29
1.7.3 Biomanufacturing 30
1.8 Aims of PhD 30

Chapter 2 – Materials and Methods 34
2.1 Chemicals and reagents 34
2.2 Cell culture 34
2.2.1 Cell line 34
2.2.2 Culture medium 35
2.2.3 Culture Maintenance 35
viii
2.2.3.1 Viable cell determination 35
2.2.4 Experimental Cultures and Sampling 36
2.2.4.1 Viability assay comparison in a batch culture 36
2.2.4.2 Apoptosis induction experiments 36
2.2.4.3 Precursor feeding experiments 38
2.2.5 Specific growth rate and integral viable cell density 40
2.3 Analysis of media components 40
2.3.1 Glucose consumption and lactate production 41
2.3.2 Glutamine consumption and glutamate production 42
2.4 Cell Density and Viability Determination 42
2.4.1 Coulter Counter 42
2.4.2 Image Analysis 43
2.4.3 Flow Cytometry Analysis 43
2.4.4 Dielectric Measurements 44
2.4.4.1 Capacitance Probe 44
2.4.4.2 Dielectrophoretic Cytometer 44
2.5 Protein quantification 45
2.5.1 ELISA for EG2 45
2.5.2 ELISA for DP12 46
2.6 Glycan Analysis 47
2.6.1 Glycan release 47
2.6.2 Glycan labeling and clean up 48
2.6.3 HPLC using HILIC 49
ix
2.6.4 Glycan analysis 50
2.6.5 Index calculations 50
2.6.5.1 Galactosylation Index (GI) 50
2.6.5.2 Fucosylation Index (FI) 51
2.6.5.3 Sialylation Index 51
2.7 Nucleotide and Nucleotide sugar analysis 51
2.7.1 Quenching 52
2.7.2 Extraction 53
2.7.3 HPLC 55
2.7.3.1 HPLC Program and Set-Up 55
2.7.3.2 Nucleotide / Nucleotide Sugar Standards 56
2.7.3.3 HPLC Sample Preparation 56
2.7.3.4 Data Analysis 56
2.8 Statistical Analysis 58

Section A
Monitoring cell viability using image, flow cytometer and dielectric analysis

Introduction 59

Chapter 3 – The comparison of different viability assays for cell culture 60
3.1 Introduction 60
3.2 Results 62
x