M e t h o d s in Molecular Biology

Transcription

1 M e t h o d s in Molecular Biology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes:

2 Heterologous Protein Production in CHO Cells Methods and Protocols Edited by Paula Meleady National Institute for Cellular Biotechnology, Dublin City University,

3 Editor Paula Meleady National Institute for Cellular Biotechnology Dublin City University ISSN ISSN (electronic) Methods in Molecular Biology ISBN ISBN (ebook) DOI / Library of Congress Control Number: Springer Science+Business Media LLC 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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). Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

4 Preface Since their introduction into the market over 20 years ago, biotherapeutics have constituted a large and growing percentage of the total pharmaceutical market, as well as approximately 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 The number 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 significantly, 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 importance. 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 production 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 glycosylation and host cell protein analysis, are also described in this book. v

5 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. Paula Meleady Reference 1. Walsh G (2014) Biopharmaceutical benchmarks Nat Biotechnol 32(10):

6 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 Qiong Wang, Bojiao Yin, Cheng-Yu Chung, and Michael J. Betenbaugh 3 Large-Scale Transient Transfection of Chinese Hamster Ovary Cells in Suspension Yashas Rajendra, Sowmya Balasubramanian, and David L. Hacker 4 Cloning of Single-Chain Antibody Variants by Overlap- Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression Patrick Mayrhofer and Renate Kunert 5 Anti-Apoptosis Engineering for Improved Protein Production from CHO Cells Eric Baek, Soo Min Noh, and Gyun Min Lee 6 Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors Alan Costello, Nga Lao, Martin Clynes, and Niall Barron 7 Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells 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 Zeynep Betts and Alan J. Dickson 9 Selection of High-Producing Clones Using FACS for CHO Cell Line Development Clair Gallagher and Paul S. Kelly 10 The Omics Revolution in CHO Biology: Roadmap to Improved CHO Productivity Hussain Dahodwala and Susan T. Sharfstein 11 A Bioinformatics Pipeline for the Identification of CHO Cell Differential Gene Expression from RNA-Seq Data Craig Monger, Krishna Motheramgari, John McSharry, Niall Barron, and Colin Clarke vii

7 viii Contents 12 Filter-Aided Sample Preparation (FASP) for Improved Proteome Analysis of Recombinant Chinese Hamster Ovary Cells 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 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 Yuzhou Fan, Helene Faustrup Kildegaard, and Mikael Rørdam Andersen 15 Glycosylation Analysis of Therapeutic Glycoproteins Produced in CHO Cells Sara Carillo, Stefan Mittermayr, Amy Farrell, Simone Albrecht, and Jonathan Bones 16 Characterization of Host Cell Proteins (HCPs) in CHO Cell Bioprocesses Catherine E.M. Hogwood, Lesley M. Chiverton, and C. Mark Smales Index

8 Contributors Simone Albrecht National Institute for Bioprocessing Research and Training (NIBRT), 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, 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), Sara Carillo National Institute for Bioprocessing Research and Training (NIBRT), 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), Martin Clynes National Institute for Cellular Biotechnology, Dublin City University, Orla Coleman National Institute for Cellular Biotechnology, Dublin City University, Alan Costello National Institute for Cellular Biotechnology, Dublin City University, 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), Clair Gallagher National Institute for Cellular Biotechnology, Dublin City University, ix

9 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, 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, 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), Paula Meleady National Institute for Cellular Biotechnology, Dublin City University, Stefan Mittermayr National Institute for Bioprocessing Research and Training (NIBRT), Craig Monger National Institute for Bioprocessing Research and Training (NIBRT), ; National Institute for Cellular Biotechnology, Dublin City University, Krishna Motheramgari National Institute for Bioprocessing Research and Training (NIBRT), ; National Institute for Cellular Biotechnology, Dublin City University, 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