Heterologous Protein Production in CHO Cells: Methods and Protocols

256 Pages·2017·5.896 MB·English

by  Paula Meleady (eds.)

Methods in
Molecular Biology 1603
Paula Meleady Editor
Heterologous
Protein
Production
in CHO Cells
Methods and Protocols
M M B
ethods in olecular iology
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Heterologous Protein
Production in CHO Cells
Methods and Protocols
Edited by
Paula Meleady
National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland
Editor
Paula Meleady
National Institute for Cellular Biotechnology
Dublin City University
Dublin, Ireland
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6971-5 ISBN 978-1-4939-6972-2 (eBook)
DOI 10.1007/978-1-4939-6972-2
Library of Congress Control Number: 2017935545
© Springer Science+Business Media LLC 2017
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Cover Illustration: The front cover image, kindly provided by Alan Costello (National Institute for Cellular Biotechnology,
Dublin City University), shows Chinese hamster ovary (CHO) cells with inducible green fluorescent protein (GFP)
expression (from Chapter 6).
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Preface
Since their introduction into the market over 20 years ago, biotherapeutics have consti-
tuted a large and growing percentage of the total pharmaceutical market, as well as approxi-
mately 25% of the R&D pipeline in industry. These biotherapeutics are having a huge
global impact on the treatment of challenging and previously untreatable chronic disease.
Currently biopharmaceuticals generate global revenues of $163 billion, making up about
20% of the pharma market, and predicted to grow to over $320 billion by 2020. The num-
ber of approved products in Europe and the USA has steadily increased to 2016 in 2014,
of which 37 have “blockbuster” status, i.e., sales over $1 billion per year, with monoclonal
antibodies (Mabs) representing the most lucrative single product class [1]. Most signifi-
cantly, nearly 50% of these biopharmaceutical products are produced in a single production
host, i.e., Chinese hamster ovary (CHO) cells. Improving the efficiency of production of
these biologics will be critical in controlling costs to healthcare systems as more of these
drugs come to market.
There has been considerable success in developing high-producing CHO cell culture
processes using approaches such as optimization of media formulation, improvements in
expression vector design, and also improvements in the design of bioreactors. The next
generation of improvements is expected to be made via genetic engineering of the host
(CHO) cell itself to increase or decrease the expression of endogenous genes depending on
the desired outcome, in order to improve the efficiency of the production of therapeutic
protein product. In order to enhance the production capabilities and efficiency of the host
cell line, an increased understanding of cellular physiology of CHO cells is of critical impor-
tance. There are substantial research efforts in progress focusing on the ‘omic analysis and
systems biology of CHO cells to understand CHO cell physiology. The publication of the
draft CHO-K1 genome in 2011 represented a major milestone in CHO systems biology.
This information has been supplemented further with the publication of draft genomes for
Chinese hamster and the CHO-S, CHO DG44 and CHO DXB11 cell lines. Availability of
the genome sequence will facilitate the interpretation and analysis of transcriptomic and
proteomic data to assess the physiological state of the cells under different growth and pro-
duction systems. Combining all levels of regulation through systems biology models will
unveil the underlying complexity inherent in CHO cell biology and will ultimately enhance
and accelerate CHO productive capabilities in the coming decades.
This book includes reviews and protocols for genetic manipulation of CHO cells for
recombinant protein production, including “difficult-to-express” therapeutics. A method is
also included on the use of the recently described genome editing tool, CRISPR/Cas9, and
how this can be applied to CHO cells. The book also includes a review and protocols for
characterization of CHO cells using ‘omic approaches and how these methods can be used
to improve efficiency of recombinant protein production during cell line development.
Analytical methods for characterization of recombinant protein product, such as glycosyl-
ation and host cell protein analysis, are also described in this book.
v
vi Preface
I am deeply grateful to all authors for giving up their valuable time and for contributing
to the book. I would also like to thank the series editor, Prof. John Walker, for help and
guidance during the process of getting the book to publication.
Dublin, Ireland Paula Meleady
Reference
1. Walsh G (2014) Biopharmaceutical benchmarks 2014. Nat Biotechnol 32(10):992–1000
Contents
Preface.......................................................... v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Strategies and Considerations for Improving Expression of “Difficult
to Express” Proteins in CHO Cells................................. 1
Christina S. Alves and Terrence M. Dobrowsky
2 Glycoengineering of CHO Cells to Improve Product Quality ............. 25
Qiong Wang, Bojiao Yin, Cheng-Yu Chung, and Michael J. Betenbaugh
3 Large-Scale Transient Transfection of Chinese Hamster Ovary Cells
in Suspension ................................................. 45
Yashas Rajendra, Sowmya Balasubramanian, and David L. Hacker
4 Cloning of Single-Chain Antibody Variants by Overlap-E xtension PCR
for Evaluation of Antibody Expression in Transient Gene Expression........ 57
Patrick Mayrhofer and Renate Kunert
5 Anti-Apoptosis Engineering for Improved Protein Production
from CHO Cells............................................... 71
Eric Baek, Soo Min Noh, and Gyun Min Lee
6 Conditional Knockdown of Endogenous MicroRNAs in CHO Cells
Using TET-ON-SanDI Sponge Vectors ............................. 87
Alan Costello, Nga Lao, Martin Clynes, and Niall Barron
7 Application of CRISPR/Cas9 Genome Editing to Improve
Recombinant Protein Production in CHO Cells ....................... 101
Lise Marie Grav, Karen Julie la Cour Karottki, Jae Seong Lee,
and Helene Faustrup Kildegaard
8 Improved CHO Cell Line Stability and Recombinant Protein Expression
During Long-Term Culture ...................................... 119
Zeynep Betts and Alan J. Dickson
9 Selection of High-Producing Clones Using FACS for CHO
Cell Line Development.......................................... 143
Clair Gallagher and Paul S. Kelly
10 The ‘Omics Revolution in CHO Biology: Roadmap to Improved
CHO Productivity ............................................. 153
Hussain Dahodwala and Susan T. Sharfstein
11 A Bioinformatics Pipeline for the Identification of CHO Cell Differential
Gene Expression from RNA-Seq Data............................... 169
Craig Monger, Krishna Motheramgari, John McSharry, Niall Barron,
and Colin Clarke
vii
viii Contents
12 Filter-Aided Sample Preparation (FASP) for Improved Proteome
Analysis of Recombinant Chinese Hamster Ovary Cells.................. 187
Orla Coleman, Michael Henry, Martin Clynes, and Paula Meleady
13 Phosphopeptide Enrichment and LC-MS/MS Analysis to Study the
Phosphoproteome of Recombinant Chinese Hamster Ovary Cells.......... 195
Michael Henry, Orla Coleman, Prashant, Martin Clynes,
and Paula Meleady
14 Engineer Medium and Feed for Modulating N-Glycosylation
of Recombinant Protein Production in CHO Cell Culture ............... 209
Yuzhou Fan, Helene Faustrup Kildegaard, and Mikael Rørdam Andersen
15 Glycosylation Analysis of Therapeutic Glycoproteins Produced
in CHO Cells ................................................. 227
Sara Carillo, Stefan Mittermayr, Amy Farrell, Simone Albrecht,
and Jonathan Bones
16 Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses ... 243
Catherine E.M. Hogwood, Lesley M. Chiverton, and C. Mark Smales
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Contributors
Simone Albrecht • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
chriStinA S. AlveS • Biogen Inc ., Cambridge, MA, USA
mikAel rørdAm AnderSen • Department of Systems Biology, Technical University
of Denmark, Kgs . Lyngby, Denmark
eric bAek • Department of Biological Sciences, KAIST, Daejeon, Republic of Korea
SowmyA bAlASubrAmAniAn • Laboratory of Cellular Biotechnology (LBTC), École
Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
niAll bArron • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
michAel J. betenbAugh • Department of Chemical and Biomolecular Engineering,
Johns Hopkins University, Baltimore, MD, USA
Zeynep bettS • Faculty of Science and Literature, Department of Biology, Kocaeli
University, Izmit, Kocaeli, Turkey
JonAthAn boneS • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
SArA cArillo • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
leSley m. chiverton • Industrial Biotechnology Centre and School of Biosciences,
University of Kent, Canterbury, Kent, UK
cheng-yu chung • Department of Chemical and Biomolecular Engineering,
Johns Hopkins University, Baltimore, MD, USA
colin clArke • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
mArtin clyneS • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
orlA colemAn • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
AlAn coStello • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
huSSAin dAhodwAlA • Vaccine production program (VPP), VRC/NIAID/NIH,
Gaithersburg, MD, USA; SUNY Polytechnic Institute, Albany, NY, USA
AlAn J. dickSon • Faculty of Life Sciences, The University of Manchester, Manchester, UK
terrence m. dobrowSky • Biogen Inc ., Cambridge, MA, USA
yuZhou FAn • Department of Systems Biology, Technical University of Denmark, Kgs .
Lyngby, Denmark; The Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, Hørsholm, Denmark
Amy FArrell • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
clAir gAllAgher • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
ix
x Contributors
liSe mArie grAv • The Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, Hørsholm, Denmark
dAvid l. hAcker • Laboratory of Cellular Biotechnology (LBTC), École Polytechnique
Fédérale de Lausanne (EPFL), Lausanne, Switzerland; Protein Expression Core Facility
(PECF), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
michAel henry • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
cAtherine e.m. hogwood • Industrial Biotechnology Centre and School of Biosciences,
University of Kent, Canterbury, Kent, UK
pAul S. kelly • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
kAren Julie lA cour kArottki • The Novo Nordisk Foundation Center for
Biosustainability, Technical University of Denmark, Lyngby, Denmark
helene FAuStrup kildegAArd • The Novo Nordisk Foundation Center for
Biosustainability, Technical University of Denmark, Lyngby, Denmark
renAte kunert • Department of Biotechnology, Vienna Institute of BioTechnology,
University of Natural Resources and Life Sciences-Vienna, Vienna, Austria
ngA lAo • National Institute for Cellular Biotechnology, Dublin City University, Dublin,
Ireland
gyun min lee • Department of Biological Sciences, KAIST, Daejeon, Republic of Korea
JAe Seong lee • The Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, Lyngby, Denmark
pAtrick mAyrhoFer • Department of Biotechnology, Vienna Institute of BioTechnology,
University of Natural Resources and Life Sciences-Vienna, Vienna, Austria
John mcShArry • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland
pAulA meleAdy • National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
SteFAn mittermAyr • National Institute for Bioprocessing Research and Training
(NIBRT), Dublin, Ireland
crAig monger • National Institute for Bioprocessing Research and Training (NIBRT),
Dublin, Ireland; National Institute for Cellular Biotechnology, Dublin City University,
Dublin, Ireland
kriShnA motherAmgAri • National Institute for Bioprocessing Research and Training
(NIBRT), Dublin, Ireland; National Institute for Cellular Biotechnology, Dublin City
University, Dublin, Ireland
Soo min noh • Department of Biological Sciences, KAIST, Daejeon, Republic of Korea
prAShAnt • National Institute for Cellular Biotechnology, Dublin City University, Dublin,
Ireland
yAShAS rAJendrA • Laboratory of Cellular Biotechnology (LBTC), École Polytechnique Fédérale
de Lausanne (EPFL), Lausanne, Switzerland; Biotechnology Discovery Research, Lilly
Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, USA
SuSAn t. ShArFStein • SUNY Polytechnic Institute, Albany, NY, USA
c. mArk SmAleS • Industrial Biotechnology Centre and School of Biosciences, University of
Kent, Canterbury, Kent, UK
Qiong wAng • Department of Chemical and Biomolecular Engineering, Johns Hopkins
University, Baltimore, MD, USA
boJiAo yin • Department of Chemical and Biomolecular Engineering, Johns Hopkins
University, Baltimore, MD, USA