Methods in Molecular Biology 1104 Ralf Pörtner Editor Animal Cell Biotechnology Methods and Protocols Third Edition M M B ™ ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Animal Cell Biotechnology Methods and Protocols Third Edition Edited by Ralf Pörtner Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Hamburg, Germany Editor Ralf Pörtner Institute of Bioprocess and Biosystems Engineering Hamburg University of Technology Hamburg , G ermany ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-62703-732-7 ISBN 978-1-62703-733-4 (eBook) DOI 10.1007/978-1-62703-733-4 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2013955043 © Springer Science+Business Media, LLC 2 014 This work is subject to copyright. 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Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Preface/F oreword Mammalian cells are used in industry as well as in research for a variety of applications. Examples are the production of monoclonal antibodies or proteins for diagnostic or thera- peutic use, production of viral vaccines as well as cultivation of tissue cells for artifi cial organs or for gene therapy. Besides the techniques required for establishing specifi c cell lines, techniques are required for optimized cultivation in small and large scale, cell charac- terization and analysis, purifi cation of biopharmaceuticals and vaccines. Animal Cell Biotechnology: Methods and Protocols constitutes a comprehensive manual of state-of-the-art techniques for setting up mammalian cell lines for production of biophar- maceuticals and optimizing critical parameters for cell culture considering the whole cas- cade from the lab to the fi nal production. Scientists with long-refi ned expertise describe cutting-edge techniques for the production of therapeutic proteins and vaccines. Capturing the major advances that have occurred in both science and the technology of these biophar- maceuticals, this important book covers the powerful new techniques used in cell line development and selection, protein expression, optimizing their growth in defi ned media, operating the fermentation at high time-space-yield, and in analysis. Topics include basic techniques for establishment of production cell lines, transient protein expression, tech- niques for optimization of process parameters, cell characterization and analysis, production- scale cultivation techniques, advanced applications of biopharmaceuticals and vaccines, and fi nally legislatory issues. The volume is divided into fi ve parts that refl ect the processes required for different stages of production. In Part I, basic techniques for establishment of production cell lines are addressed, especially high-throughput synchronization, insect cell lines, transient gene and protein expression, DNA Profi ling and Characterization. Part II addresses tools for process and medium optimization as well as microcarrier technology. Part III covers monitoring of cell growth, viability and apoptosis, metabolic fl ux esti- mation, quenching methods as well as NMR-based techniques. Part IV details cultivation techniques, such as microfl uidic perfusion culture, disposable bioreactors, confi guration of bioreactors, spectroscopic and microscopic techniques as well as seed train optimization. Part V describes special applications, including vaccine production, baculovirus protein expression, chromatographic techniques for downstream as well as membrane techniques for virus separation. In summary, this volume constitutes a comprehensive manual of state-of-the-art and new techniques for setting up mammalian cell lines for production of biopharmaceuticals, and optimizing critical parameters for cell culture considering the whole cascade from the lab to the fi nal production. Inevitably, some omissions will occur in the test, but the authors v vi Preface/Foreword have sought to avoid duplications by extensive cross-referencing to chapters in other vol- umes of this series and elsewhere. We hope the volume provides a useful compendium of techniques for scientists in industrial and research laboratories that use mammalian cells for biotechnology purposes. The editor is grateful for the support of all the contributors, the series editor Prof. John Walker, Hertfordshire , UK and the publishers who have made this volume possible. Hamburg , Germany Ralf Pörtner Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi PART I CELL LINE DEVELOPMENT AND SELECTION 1 High-Throughput Synchronization of Mammalian Cell Cultures by Spiral Microfluidics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Wong Cheng Lee, Ali Asgar S. Bhagat, and Chwee Teck Lim 2 Insect Cell Line Development Using Flp-Mediated Cassette Exchange Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 João Vidigal, Fabiana Fernandes, Ana S. Coroadinha, Ana P. Teixeira, and Paula M. Alves 3 Scalable Transient Gene Expression in Adherent Mammalian Cells Using Polyethylenimine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Lukas Fliedl and Christian Kaisermayer 4 Scalable Transient Protein Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Henry C. Chiou, Sanjay Vasu, Chao Yan Liu, Isabel Cisneros, Meredith B. Jones, and Jonathan F. Zmuda 5 DNA Profiling and Characterization of Animal Cell Lines. . . . . . . . . . . . . . . . 57 Glyn N. Stacey, Ed Byrne, and J. Ross Hawkins PART II TECHNIQUES FOR PROCESS DEVELOPMENT 6 Tools for High-Throughput Process and Medium Optimization . . . . . . . . . . . 77 Martin Jordan and Matthieu Stettler 7 Designing Media for Animal Cell Culture: CHO Cells, the Industrial Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Karlheinz Landauer 8 Feed Optimization in Fed-Batch Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Ana Rita Costa, Maria Elisa Rodrigues, Mariana Henriques, Rosário Oliveira, and Joana Azeredo 9 Screening and Optimization of Chemically Defined Media and Feeds with Integrated and Statistical Approaches . . . . . . . . . . . . . . . . . . . 117 Zhihua Xiao, Michelle Sabourin, Graziella Piras, and Stephen F. Gorfien 10 Evaluation of Solid and Porous Microcarriers for Cell Growth and Production of Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Maria Elisa Rodrigues, Ana Rita Costa, Mariana Henriques, Joana Azeredo, and Rosário Oliveira vii viii Contents 11 Microbioreactors and Scale-Down Models: Growth of CHO Cells Using the Pall Micro24 MicroReactor System. . . . . . . . . . . . . . . . . . . . . . . . . 149 Steve R.C. Warr PART III CELL CHARACTERISATION AND ANALYSIS 12 Monitoring Cell Growth, Viability, and Apoptosis. . . . . . . . . . . . . . . . . . . . . . 169 Michael Butler, Maureen Spearman, and Katrin Braasch 13 Metabolic Flux Estimation in Mammalian Cell Cultures . . . . . . . . . . . . . . . . . 193 Chetan T. Goudar, Richard K. Biener, James M. Piret, and Konstantin B. Konstantinov 14 Quenching Methods for the Analysis of Intracellular Metabolites. . . . . . . . . . . 211 Judith Wahrheit and Elmar Heinzle 15 NMR Methods for Metabolomics of Mammalian Cell Culture Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Nelly Aranibar and Michael D. Reily 16 1H-NMR Protocol for Exometabolome Analysis of Cultured Mammalian Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 Tiago M. Duarte, Nuno Carinhas, Ana Carina Silva, Paula M. Alves, and Ana P. Teixeira PART IV CULTIVATION TECHNIQUES 17 Microfluidic Perfusion Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Koji Hattori, Shinji Sugiura, and Toshiyuki Kanamori 18 Disposable Bioreactors for Inoculum Production and Protein Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Regine Eibl, Christian Löffelholz, and Dieter Eibl 19 Configuration of Bioreactors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Dirk E. Martens, Evert J.van den End, and Mathieu Streefland 20 Process Analytical Technology (PAT) in Insect and Mammalian Cell Culture Processes: Dielectric Spectroscopy and Focused Beam Reflectance Measurement (FBRM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 Damir Druzinec, Katja Weiss, Christiane Elseberg, Denise Salzig, Matthias Kraume, Ralf Pörtner, and Peter Czermak 21 Cell Assessment by At-line Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Alexander Babitzky, Patrick Lindner, and Thomas Scheper 22 Seed Train Optimization for Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Björn Frahm PART V SPECIAL APPLICATIONS 23 Vaccine Production: Upstream Processing with Adherent or Suspension Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Yvonne Genzel, Jana Rödig, Erdmann Rapp, and Udo Reichl Contents ix 24 Efficient Processes for Protein Expression Using Recombinant Baculovirus Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 Heike Cremer, Ingrid Bechtold, Marion Mahnke, and René Assenberg 25 Chromatographic Techniques in the Downstream Processing of Proteins in Biotechnology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419 Ruth Freitag 26 Virus Separation Using Membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Tanja A. Grein, Ronald Michalsky, and Peter Czermak Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493