Supplementation Strategies for Tuning Glycosylation of Monoclonal Antibodies and Enhancing ...

204 Pages·2017·7.79 MB·English

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Supplementation Strategies for Tuning Glycosylation of Monoclonal Antibodies and Enhancing
Growth in Mammalian Cell Culture by Omics Analysis

by
Eric John McQuillan Blondeel

A thesis
presented to the University of Waterloo
in fulfillment of the
thesis requirements for the degree of
Doctor of Philosophy
in
Chemical Engineering

Waterloo, Ontario, Canada, 2018
©Eric John McQuillan Blondeel 2018
Examining Committee Membership
The following served on the Examining Committee for this thesis. The decision of the
Examining Committee is by majority vote.

External Examiner Dr. Mario Jolicoeur
Professor
École Polytechnique de Montréal

Supervisor(s) Dr. Marc Gordon Aucoin
Associate Professor

Internal Member Dr. Hector Budman
Professor

Internal-external Member Dr. Brian Dixon
Professor

Other Member(s) Dr. Maud Gorbet
Associate Professor

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Author’s Declaration
This thesis consists of material all of which I authored or co-authored: see Statement of
Contributions included in the thesis. This is a true copy of the thesis, including any required final
revisions, as accepted by my examiners.

I understand that my thesis may be made electronically available to the public.

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Statement of Contributions
The majority of the work presented in this thesis has been performed either directly by me, or
directed by me as the primary researcher.
The work presented in Chapter 3 is an accepted manuscript published in the Journal of
Biotechnology entitled, “An omics approach to rational feed: Enhancing growth in CHO cultures
with NMR metabolomics and 2D-DIGE proteomics.” and is co-authored with Raymond Ho,
Steffen Schulze, Dr. Stanislav Sokolenko, Simon R. Guillemette, Dr. Igor Slivac, Dr. Yves
Durocher, Dr. Guy J. Guillemette, Dr. Brendan J. McConkey, Dr. David Chang, and Dr. Marc G.
Aucoin. While I am the primary author of this manuscript, the research represents two
complementary experiments conducted in collaboration with the McConkey and Durocher
laboratories as part of the NSERC MAbNet antibody research network. The metabolomics
experiments and necessary culturing was performed by myself, Steffen Schulze, Dr. Stanislav
Sokolenko, and Simon Guillemette, while the proteomics experiments and necessary culturing
was performed by Raymond Ho and Dr. Igor Slivac.
The work presented in Chapter 4 is an accepted manuscript published in the Journal of
Biotechnology entitled, “Tuning a MAb glycan profile in cell culture: Supplementing N-
acetylglucosamine to favour G0 glycans without compromising productivity and cell growth.”
and is co-authored with Dr. Katrin Braasch, Thomas McGill, Dr. David Chang, Christina Engel,
Dr. Maureen Spearman, Dr. Michael Butler, and Dr. Marc G. Aucoin. While I am the primary
author of this manuscript, this research represents an experiment conducted in collaboration with
the Butler laboratory as part of the NSERC MAbNet antibody research network. Myself and
Thomas McGill performed the experiment and necessary culturing, while Dr. Katrin Braasch and
Dr. Maureen Spearman performed intracellular metabolomic analysis of nucleotide sugar

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precursors and glycan analysis of the EG2 antibody product from samples prepared by Thomas
and myself. Christina Engel replicated parts the experiment on an alternative CHO cell platform.
The work presented in Chapter 5 is a manuscript submitted to the journal Biotechnology
Advances entitled, “Supplementing Glycosylation: A Review of Applying Nucleotide-Sugar
Precursors to Growth Medium to Affect Therapeutic Recombinant Protein Glycan Profiles.” and
is co-authored with Dr. Marc G. Aucoin. I am the primary author of this manuscript reviewing
work by several authors of this field.

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Abstract
Two fundamental objectives of bioprocess engineering are to increase productivity and
improve product quality. Glycosylation is a critical and variable factor in the quality of several
therapeutic proteins, particularly those with immune-system interactions such as monoclonal
antibodies (mAbs). In this thesis, supplementation of nutrients to growth medium of CHO1A7
mammalian cell cultures are examined towards enhancing cell growth, by increasing peak cell
density, and improving product quality, by tuning glycosylation of EG2-hFc heavy-chain
camelid antibodies. Targeted profiling via 1D-1H-NMR metabolomics and differential
expression analysis via 2D-DIGE proteomics, elucidate factors to create a nutrient cocktail to
enhance culture growth. Eight target nutrients corresponding with five identified metabolic
systems for CHO cells including anaplerotic TCA-replenishment; NADH/NADPH
replenishment; tetrahydrofolate cycle C1 cofactor conversions; limitations to lipid synthesis; and
redox modulation; resulted in a ~75% improvement to peak cell densities. Towards improving
product quality, nucleotide-sugar precursors, capable of shifting glycan distributions were
supplemented to growth medium to tune glycosylation of EG2-hFc towards a single G0
glycoform. Growth inhibition from glucosamine-based precursors was mitigated given a priori
knowledge from metabolomic analysis of the system – identifying cytosolic acetyl-CoA as a
sensitive metabolic pool for CHO1A7 cell growth. Additional nucleotide-sugar precursor nutrients
were examined to better resolve conflicting reports of effects to glycan distributions. These
conflicts are subsequently attributed to five key factors: differences across cell platforms;
differences between glycan sites of expressed proteins; the fermentation and sampling timeline;
glutamine levels; finally, no standardized metrics for reporting shifts in glycan distributions with
respect to controls.

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Acknowledgements
I would like to express thanks to the University of Waterloo and the Department of Chemical
Engineering for the opportunity to pursue graduate studies and complete my doctoral program. I
would also like to thank the Government of Canada by way of NSERC and the MAbNet
antibody research network for supporting my research. A major thank-you to my co-authors, as
well as the Butler Lab, the McConkey Lab, the Durocher Lab, and members of MAbNet for their
collaboration, help, and above all friendship. I feel privileged to be counted among these
colleagues. I would further like to express my thanks to Andrew and Margaret Stevens for their
scholarship to the chemical engineering department that supported me financially during my
research.
An enormous thank you to my supervisor Professor Marc Aucoin for taking a chance on me
to join his research group, remaining patient, and not giving up on me. Thank you for your
guidance and support and always doing your best to help me. Thanks also to all my colleagues in
the Aucoin Lab, Steve, Stan, Andreas, Steffen, Sandi, Megan, Jian, Zen-Zen, Altamash, and Jann
– thanks for putting up with me.
Thank you to my PhD committee members Dr. Maud Gorbet, Dr Hector Budman, Dr. Brian
Dixon, and Dr. Mario Jolicoeur for evaluating my thesis, and my suitability to receive a doctoral
degree.
Thank you to Dr. Brendan MacDonald, Dr. Thorsten Dieckmann, Dr. Bo Cui, Dr. Bernie
Dunker, for all your help, support, and encouragement; I wouldn’t be here without it. I would
also like to thank Dr. Marc Gibson, Dr. John Dick, and Dr. Morteza Ahmadi for commiserating
with me and encouraging me to power through the hardest stretches.

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Thank you to Velocity, Dean Pearl Sullivan, Jay Shah, Larry Smith, Mike Kirkup and the
Faculty of Engineering for encouraging me to reach beyond my grasp. Thanks as well to Y
Combinator, and my group partners Geoff Ralston and Tim Brady for encouraging me to grasp
further still.
Thank you to my partner Jen for sticking with me and supporting my dreams; Mom, Dad,
and my brothers for all their support and love. Thank you to all my friends who have suffered
through years of pretending to listen to what my research is about.
Thanks to Moufeed, my best friend and partner on the wild adventure of the past 4 years.
Thanks also to Christian, and the rest of the ExVivo gang.
Also, thank you to Jan Venne and the UWaterloo NMR Facility, as well as the administrative
and support staff at the Department of Chemical Engineering: Judy, Rose, Liz, Ingrid, Bert, Tom,
Ralph and Rick for helping me whenever I needed it.

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Dedication
To whomever should read this, I may no longer even be living, but I invite you to join me in
a great conversation that was started before I got here, and will continue long after both of us
have left.
Hopefully I’ve said something useful…

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Table of Contents
Examining Committee Membership ............................................................................................... ii
Author’s Declaration ...................................................................................................................... iii
Statement of Contributions ............................................................................................................ iv
Abstract ................................................................................................................................. vi
Acknowledgements ....................................................................................................................... vii
Dedication ................................................................................................................................. ix
Table of Contents ............................................................................................................................ x
List of Figures ............................................................................................................................... xv
List of Tables ............................................................................................................................... xix
List of Abbreviations ................................................................................................................. xxiii
Chapter 1 Introduction ............................................................................................................... 1
1.1 Objective of Research........................................................................................................... 4
1.2 Hypothesis of Research ........................................................................................................ 5
Chapter 2 Literature Review...................................................................................................... 6
2.1 Antibodies in vivo ................................................................................................................. 6
2.2 Antigen Binding ................................................................................................................... 8
2.2.1 Monoclonal Antibodies (mAbs) ........................................................................................... 9
2.2.2 Antibody Fragments and Fusion Proteins .......................................................................... 10
2.3 Glycosylation ...................................................................................................................... 11
2.3.1 Eukaryotic Glycosylation ................................................................................................... 12
2.3.2 N-linked Glycosylation ...................................................................................................... 13
2.3.3 Antibody Fc & Immune Effector Function ........................................................................ 17
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