(BQ) Part 1 book “Update on production of recombinant therapeutic protein - Transient gene expression” has content: Transient gene expression in different expression systems, recent advances in transient gene expression protocol.
Smithers Rapra Technology Ltd, 2013 Over the past decade, the transient gene expression (TGE) technology platform has been actively pursued to produce a wide range of therapeutic proteins, monoclonal antibodies, and vaccines for mainly preclinical assessment, due to its short development times and low overall cost This book updates the latest advances in the field, with focusing on systematic description of the technology from cell lines, cell culture conditions, vector construction, expression strategy, current protocols, optimisation of the procedure, and potential for clinical application As a conclusion, the author foresees that therapeutic biopharmaceutics will be manufactured for clinical development using TGE technology in the near future because of its fast development time, good protein expression, acceptable quality of product and due to the progress which has been made in analytical methodology and process quality control Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression Published by The objectives of this book are to summarise current TGE protocols, to describe optimisation of the technology through the latest advances, and to explore clinical applications of the technology It gives the reader a good insight into the latest development and future application of the technology platform, including: • • • • Jianwei Zhu The current protocols from small to large scale for different cells Optimisation methods in construction designing, transfection procedures, and cell culture conditions Overall quality of the product from the transient gene expression Future clinical application of the technology platform Jianwei Zhu Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 Web: www.polymer-books.com Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression Update on Production of Recombinant Therapeutic Protein: Transient Gene Expression Jianwei Zhu A Smithers Group Company Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 http://www.polymer-books.com First Published in 2013 by Smithers Rapra Technology Ltd Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK © 2013, Smithers Rapra Technology Ltd All rights reserved Except as permitted under current legislation no partof this publication may be photocopied, reproduced or distributed in anyform or by any means or stored in a database or retrieval system, without the prior permission from the copyright holder A catalogue record for this book is available from the British Library Every effort has been made to contact copyright holders of any material reproduced within the text and the authors and publishers apologise if any have been overlooked ISBN: 978-1-84735-976-6 (hardback) 978-1-84735-977-3 (ebook) Typeset by Integra Software Services Pvt Ltd C ontents Acknowledgements vi Preface vii Contributors ix Transient Gene Expression in Different Expression Systems 1.1 Introduction 1.2 Transient Gene Expression versus Stable Gene Expression 1.3 Transient Gene Expression in Different Systems 1.3.1 Mammalian Cell Systems 1.3.2 Plant Systems 1.3.3 Insect Cell Systems 1.3.4 Stem Cell Systems 11 References 12 Recent Advances in Transient Gene Expression Protocol 17 2.1 Vectors 18 2.1.1 Viral Vector 19 2.1.2 Nonviral Vectors 23 2.2 Construction for Expression 25 2.2.1 Promoter 25 2.2.2 Other Construction Components 26 2.2.3 Plasmid Preparation and Quality 26 iii Update on Production of Recombinant Therapeutic Protein 2.3 2.4 2.5 Nonviral Gene Delivery 28 2.3.1 Electroporation Methods 29 2.3.2 Chemical Methods 30 Cell Lines used in Transient Gene Expression 35 2.4.1 Human Embryonic Kidney 293 Cells 40 2.4.2 Chinese Hamster Ovary Cells 42 2.4.3 Other Cell Lines 43 Current Transient Gene Expression Protocols 47 2.5.1 Shake Flask Protocol for Volumes of Normal and High Density Cell Cultures Greater than One Litre [108] 49 2.5.2 Protocol for Large-scale Transient Transfection in the Wave Bioreactor [71, 110] 51 2.5.3 High Density Large-Scale Transfection of Mammalian Cells [109] 55 2.5.4 100 L Transient Gene Expression Protocol [4] 58 2.5.5 Purification of Products from Transient Gene Expression 61 References 70 Optimisation of Transient Gene Expression for Therapeutic Protein Production 81 3.1 Optimisation of the Transient Gene Expression Conditions 86 3.2 3.1.1 Medium Optimisation 87 3.1.2 Optimisation of Transient Gene Expression Conditions and Procedures 93 3.1.3 Construction Optimisation 96 3.1.4 Coexpression of Growth Factors 103 Extension of Protein Production after Transfection 104 3.2.1 iv Stable Transfection Pool 105 Contents 3.3 3.2.2 Transfection Pools with Genetic Modification 109 3.2.3 Plasmid Replication 112 3.2.4 Antiapoptosis 114 Optimisation of the Technology in Other Aspects 119 3.3.1 Product Improvement 119 3.3.2 BacMam 121 References 122 Clinical Applications of the Transient Gene Expression 135 4.1 4.2 Quality Assessment of the Product Manufactured by Transient Gene Expression 136 4.1.1 Glycosylation Analysis 136 4.1.2 Product Quality Consistency and Process Reproducibility 142 4.1.3 Further Analysis of Transient Expression Systems 147 Clinical Development of Therapeutic Recombinant Proteins using Transient Gene Expression 149 4.2.1 Acceleration of Screening Drug Candidates at the ‘Proof-of-Principal’ Stage 149 4.2.2 Therapeutic Proteins in Clinical Development using Other Systems 150 4.3 Quality Requirements for Clinical Products 151 4.4 Clinical Manufacturing of Recombinant Therapeutic Proteins using Transient Gene Expession 153 4.4.1 In-process Quality Control 157 4.4.2 Product Quality Characterisation 157 References 158 Abbreviations 161 Index 167 v A cknowledgements The authors would like to extend acknowledgement to the Biopharmaceutical Development Program of Frederick National Laboratory for Cancer Research where the editor experienced and accumulated knowledge of the transient gene expression technology platform The authors also like to thank many individuals who directly or indirectly contributed to this book including, particularly, Dr Baohong Zhang for having assisted in reference listing and Tammy Schroyer for having assisted in figures Some of the original data were from the presentations made by Dr Man-shiow Jiang and Dr Matt Zustiak at a number of scientific symposiums vi P reface Mammalian cells have become the dominant system for producing 70% of approved recombinant therapeutic proteins modified by human-like post-translational modification with respect to molecular structures and biochemical properties There is a growing number of therapeutic biological molecule candidates in the pipeline awaiting preclinical and clinical evaluation Due to its short development times and low overall cost, transient gene expression (TGE) has been actively pursued over the past decade to produce a wide range of therapeutic proteins, monoclonal antibodies and vaccines, mainly for preclinical assessment Over the last ten years, the remarkable progress in TGE makes this approach attractive for supplying materials for preclinical development and which have potential clinical applications As the TGE technology platform reached the g/L expression level milestone, as cell culture and transfection can be scaled up to over 100 L for production, and as products made by TGE were consistent and reproducible, this technology platform has been widely employed as an initial stage of biopharmaceutical development such as screening for expression strategy in terms of construction design, molecular candidate selection, and manufacturing products for characterisation, which will potentially be developed for clinical applications This book will update the latest advances in the field Particular attention will be paid to systematic description of the technology from cell lines, cell culture conditions, vector construction, expression strategy, current protocols, transfection procedure, optimisation of the procedure, and potential for clinical application This book is vii Update on Production of Recombinant Therapeutic Protein composed of four chapters While TGE is used in several expression systems that are briefly introduced in Chapter 1, this book describes the production of biotherapeutics using the mammalian cell TGE technology platform In Chapter 2, current protocols are summarised with detailed analysis of the critical steps including vector, plasmid preparation, gene delivery methods and cell lines used Further optimisation of TGE procedures is described in Chapter through cell culture conditions and procedure, genetic construction and cell line engineering Finally, application of the TGE technology in clinical development of biopharmaceuticals is updated, analysed, and rationalised in Chapter As a conclusion, the author foresees that therapeutic biopharmaceutics will be manufactured for clinical development using TGE technology in the near future because of its fast development time, good protein expression, acceptable quality of product and due to the progress which has been made in analytical methodology and process quality control The objectives of this book are to summarise current TGE protocols, to describe optimisation of the technology through the latest advances, and to analyse and explore clinical applications of the technology A further aim is to provide up-to-date information and reference sources for those who are working in the field to utilise in their projects It is hoped that the book will be of interest to those in the field of conducting research and development in the field of biotherapeutics, from basic science laboratories to process and product development in the biopharmaceutical industry It will be a particularly useful reference for those who are at undergraduate and graduate levels studying biopharmaceutical development and preparing themselves for creating the next generation of innovative biopharmaceutics It is also an invaluable information package for those in the biopharmaceutical industry who are actively developing potential new biotherapeutics through efficient methodologies viii C ontributors Hua Jiang Novavax, Inc., 9920 Belward Campus Drive, Rockville, MD 20850, USA Charles Y Zhu Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA Jianwei Zhu Shanghai Jiao Tong University and Biopharmaceutical Development Program, National Cancer Institute at Frederick, SAIC Frederick, Inc., 3704 Spicebush Way, Frederick, MD 21704, USA ix Update on Production of Recombinant Therapeutic Protein rate; high binding capacity; less expensive than Super Q-5PW; easy to sanitise; excellent scalability: • For these reasons, Q-Sepharose FF is used downstream from the Super Q to serve as an intermediate purification and concentrating step It is important to note that the Super Q flow-through step prior to Q-Sepharose FF removes a significant amount of impurities and hence reduces the column size of Q-Sepharose by at least three-fold as compared to the size of Q-Sepharose column that would be required without the Super Q flow through step • The Q Sepharose FF resin (GE Healthcare) column is packed into a XK 50 × 200 mm column to a final bed height of 13.7 cm and a final pbv of 265 mL After the column is cleaned and sanitised, the column is flushed with >3 pbv of WFI and charged with >3 pbv of M NaCl to ensure complete flush off of hydroxide ions from the column The column is further charged with >5 pbv of charging buffer (50 mM Tris HCl, M NaCl, 0.1 mM EDTA, 0.01% Tween 80, pH 7.4) and equilibrated with >3 pbv of EB (buffer A: 50 mM Tris HCl, mM EDTA, 50 mM NaCl, 0.1% Tween 80, pH 7.4) Prior to loading on to the Q-Sepharose FF chromatography column, the Super Q 650M FT and wash pool are diluted with one volume (1:1 dilution) of dilution buffer (50 mM Tris HCl, mM EDTA, 0.01% Tween 80, pH 7.4) to lower the conductivity to less than 11 mS/cm The Q-Sepharose load is applied to the Q-Sepharose FF column at 61 cm/h (20 mL/min) and all remaining steps are performed at this same flow rate Once this material is loaded onto the AKTA machine, the sample line is flushed with pbv of EB to ensure complete sample loading on to the column The column is then washed with 0-5% buffer B (50 mM Tris HCl, mM EDTA, M NaCl, 0.01% Tween 80, pH 7.4) over pbv and washing is continued for pbv at 5% buffer B During this washing stage, a pre-peak (most likely impurities) comes 66 Recent Advances in Transient Gene Expression Protocol out The product (main peak) is eluted with 5-20% buffer B over pbv The eluted peak is collected into a separate PETG bottle once the A280 reading has climbed above mAU and the collection is ended once the A280 reading has dropped below 10 mAU The column is then stripped with 100% buffer B and the resulting peak is also collected (strip peak) The majority of the r-protein is in the main peak of Q-Sepharose chromatography The pre-peak (5% buffer B wash) may contain a small amount of the r-protein but the ratio of the optical density at 280 nm versus that at 254 nm can be very low indicating that this fraction may contain significant amounts of DNA/RNA Therefore, this fraction (pre-peak; 0-5% buffer B wash) should not be pooled for further purification The Q-Sepharose FF step does not only further purify the product protein but it also serves as a concentration step by reducing the volume There may be very little of the target protein in the strip peak indicating that most of the target protein is eluted off at 10-50% buffer B • All of the fractions are weighed The total protein concentration is determined by A280 and the theoretical extinction coefficient of the r-protein is calculated from the r-protein sequence The individual pools are analysed by SEC-HPLC and SDS-PAGE • Heparin-Sepharose High Performance (HP) column chromatography: The Heparin-Sepharose column could be used for further purification and a final polishing step The capacity of the Heparin-Sepharose column is approximately 1-10 mg/mL depending upon the specific r-protein Recombinant proteins may bind to the Heparin-Sepharose column at an ionic strength of approximately 13 mS/cm and can be eluted at an ionic strength of approximately 40 mS/cm: Prepacked Heparin Sepharose HP columns (16 ì 25 mm) are used (GE Healthcare) After the column is cleaned and 67 Update on Production of Recombinant Therapeutic Protein sanitised, the column is flushed with >3 pbv of WFI and charged with >3 pbv M NaCl to ensure complete flush of hydroxide ions from the column The column is further charged with >5 pbv of charging buffer (50 mM Tris HCl, M NaCl, 0.1 mM EDTA, 0.01% Tween 80, pH 7.4) and equilibrated with >3 pbv of EB (buffer A, 50 mM Tris HCl, mM EDTA, 100 mM NaCl, pH 7.4) • Before loading, the Q-Sepharose main peak is diluted with one and a half time (1:1.5 dilution) of dilution buffer (50 mM Tris HCl, mM EDTA, 0.01% Tween 80, pH 7.4) to lower the conductivity to less than 11 mS/cm The resulting Heparin-Sepharose load is applied to the Heparin-Sepharose columns at 300 cm/h (10 mL/min) and all the remaining steps are performed at this same flow rate except for the elution step, which is performed at 150 cm/h (5 mL/min) Once the material is loaded on to the AKTA machine, the sample line is flushed with pbv of EB to ensure complete sample loading on to the column The column is then washed with buffer A (50 mM Tris HCl, mM EDTA, 50 mM NaCl, pH 7.4) over pbv The target product is eluted with 0-50% buffer B over 30 pbv The eluted peak is collected into a separate PETG bottle once the A280 reading climbs above 10 mAU and the collection is ended once the A280 reading below 10 mAU The ‘elution pre-peak’ and ‘post peak’ are also collected The column is stripped with 100% buffer B and the resulting peak is also collected All of the fractions are weighed The total protein concentration is determined by A280 and the theoretical extinction coefficient of the product is calculated The individual pools are analysed by SEC-HPLC and SDS-PAGE The majority of an r-protein is in the main peak pool of Heparin-Sepharose chromatography This step removes the remaining nucleic acid and high molecular weight impurities and also significantly reduces the volume 68 Recent Advances in Transient Gene Expression Protocol Pooled fractions Assay SDS-PAGE, SEC-HPLC Conditioned Medium Super Q Chromatography Flow through SDS-PAGE, SEC-HPLC Q-Sepharose Chromatography Main peak SDS-PAGE, SEC-HPLC Polishing Step Main peak SDS-PAGE, SEC-HPLC 0.2 µm Filtration Vialling SEC-HPLC SDS-PAGE A280 LAL N-terminal Sequence, SEC-HPLC, SDS-PAGE, Immunoblot, A280, LAL Size exclusion-High performance liquid chromatography Sodium dodecyl sulfate polyacrylamide gel electrophoresis Absorbance at the wavelengths of 280 nm Limulus amoebocyte lysate Figure 2.5 Purification process flow chart for recombinant proteins A purification process flow chart which includes three column steps is described in Figure 2.5 Assay items for process quality control are listed on the right side of the flow chart to facilitate understanding of the corresponding process steps At each step of the purification, SEC-HPLC and SDS-PAGE are used to monitor the product For the FVP, N-terminal sequencing and immunoblotting are used for identity verification; SEC-HPLC, SDS-PAGE, A280, and RP-HPLC are used to determine both the quantity and purity of the product Finally, LAL assay is used for safety reasons to determine the residual endotoxin level The products purified using this protocol should be of an appropriate material for in vivo, and in vitro biological activity studies, as well as animal studies 69 Update on Production of Recombinant Therapeutic Protein References L Baldi, D.L Hacker, M Adam and F.M Wurm, Biotechnology Letters, 2007, 29, 677 N Muller, M Derouazi, F van Tilborgh, S Wulhfard, D.L Hacker, M 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Optimisation of Transient Gene Expression for Therapeutic Protein Production 81 3 .1 Optimisation of the Transient Gene Expression Conditions 86 3.2 3 .1. 1 Medium Optimisation 87 3 .1. 2... Optimisation of Transient Gene Expression Conditions and Procedures 93 3 .1. 3 Construction Optimisation 96 3 .1. 4 Coexpression of Growth Factors 10 3 Extension of Protein Production after... 14 2 4 .1. 3 Further Analysis of Transient Expression Systems 14 7 Clinical Development of Therapeutic Recombinant Proteins using Transient Gene Expression 14 9 4.2 .1 Acceleration of