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. ii 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. iv 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 vi 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