Fed-batch Fermentation A Practical Guide to Scalable Recombinant Protein Production in Escherichia Coli PDF
Preview Fed-batch Fermentation A Practical Guide to Scalable Recombinant Protein Production in Escherichia Coli
Fed-batch fermentation Related titles Therapeutic risk management of medicines (ISBN 978-1-907568-48-0) An introduction to pharmaceutical sciences: Production, chemistry, techniques and technology (ISBN 978-1-907568-52-7) Formulation tools for pharmaceutical development (ISBN 978-1-907568-99-2) Woodhead Publishing Series in Biomedicine: Number 42 Fed-batch fermentation A practical guide to scalable recombinant protein production in Escherichia coli G arner G. M oulton amsterdam (cid:129) boston (cid:129) cambridge (cid:129) heidelberg (cid:129) london new york (cid:129) oxford (cid:129) paris (cid:129) san diego san francisco (cid:129) singapore (cid:129) sydney (cid:129) tokyo Woodhead Publishing is an imprint of Elsevier Woodhead Publishing is an imprint of Elsevier 80 High Street, Sawston, Cambridge CB22 3HJ, UK 25 Wyman Street, Waltham, MA 02451, USA Langford Lane, Kidlington, OX5 1GB, UK Copyright © 2014 Woodhead Publishing. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; e-mail: [email protected] . Alternatively, you can submit your request online by visiting the Elsevier website at http://elsevier.com/locate/ permissions , and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verifi cation of diagnoses and drug dosages should be made. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Control Number: 2014938060 ISBN 978-1-907568-92-3 (print) ISBN 978-1-908818-33-1 (online) For information on all Woodhead Publishing publications visit our website: http://store.elsevier.com/ Typeset by Refi neCatch Limited, Bungay, Suffolk Printed and bound in the United Kingdom Cover illustration: From the U.S. Department of Energy Genomic program website, http://genomicscience.energy.gov List of fi gures and tables Figures 1.1 N ucleotide bases made up of pyrimidines and purines as well as the addition of the sugar ribose (RNA) or deoxyribose (DNA) and a phosphate group 5 1.2 C hargaff’s rule 6 1.3 B ase pairing in DNA is complementary 7 1.4 C onversion of simple sugars to ethanol and carbon dioxide 9 1.5 T he E. coli cell 15 1.6 G lycolytic pathway and acetyl-CoA formation 16 1.7 T CA cycle and the formation of acetyl CoA from acetate 18 1.8 I sopropyl β -D-1-thiogalactopyranoside (IPTG) 21 2.1 G eneric plasmid 34 2.2 T ypical cloning of foreign gene into recombinant plasmid 37 2.3 I soproply-β -D-thio-galactoside (IPTG) shown with the arrow pointing to the sulfur–carbon bond that is not hydrolysable 39 vii Fed-batch fermentation 2.4 T ranscription of DNA 40 2.5 M icrograph of many transcription events taking place on a DNA molecule 41 2.6 E . coli micrograph 42 2.7 E . coli cell wall structure and components 45 2.8 T ransformation of a bacterial cell culture with a plasmid 47 2.9 D raw a “T” on the bottom of your Petri dish, as shown 58 2.10 Touch the inoculating loop to the upper left-hand corner and then move it across the agar from left to right, as shown 59 2.11 Touch the loop to the area previously streaked and then move the loop across the agar, as shown 60 2.12 Touch the loop on the previously streaked area and then move the loop across the agar onto the third area, as shown 60 2.13 Incubate the streak plate until you can see individual colonies 61 3.1 Exponential growth curve for bacterial growth 64 3.2 O xygen transport within the cell 76 3.3 % DO versus time 79 3.4 l n (C* − C ) versus Δ time (s) 79 L 3.5 O xygen transfer rate and K a determination 80 L 3.6 1 0-liter bioreactor for E. coli fermentation 84 3.7 D issolved oxygen electrode: polarographic sensor 88 3.8 T he pH electrode: Calomel electrode 91 3.9 T ypical fed-batch fermentation growth curve 105 viii List of fi gures and tables 3.10 Analysis of residual acetate, glucose and phosphate during the growth of the recombinant culture 106 3.11 Typical induction gel at prior to induction and at 3 hours post-induction 108 4.1 P rokaryotic ribosomal composition 112 4.2 T ranslation of protein in prokaryotes 114 4.3 A tetrapeptide (V-G-S-A) with the amino terminus of the peptide on the left and the carboxyl terminus on the right 118 4.4 A mino acid names, structures and one letter symbol associated with each 120 4.5 P rimary, secondary, tertiary and quaternary structures of proteins 122 4.6 B acterial GroES/GroEL complex 123 4.7 A ggregation pathways i n vivo 133 Tables 4.1 C odons for amino acids and start and stop sequences 113 4.2 P rotein complexes within prokaryotic and eukaryotic cells 126 ix About the author Gus G. Moulton is Chief Scientifi c Offi cer of BioBench LLC, a contracting facility for purifi cation and fermentation development in Seattle, USA. Gus started the company in 2011 and is now pursuing this full time. BioBench’s primary focus is initial development for product screening and vaccine Phase I clinical trials. Moulton has more than 20 years of process development experience in the biotechnology community. During the last 13 years he has been responsible for setting up and running fermentation labs to generate medium to high cell density fermentations. He performed these services for both Corixa Corporation, a former cancer vaccine company bought by GlaxoSmithKline plc, and the Infectious Disease Research Institute (IDRI), a nonprofi t organization which develops diagnostic tests and vaccines to diagnose and treat diseases in third-world countries, such as India, Brazil and in Africa. During Moulton’s career at Corixa he was initially responsible for purifi cation development of the most critical antigens, and subsequently for setting up and developing recombinant E. coli fermentation processes at the 30 liter scale for Phase I clinical vaccine trials for HER2/neu. He also developed an upstream and downstream process for the purifi cation of the recombinant antigen TcF to be used in the diagnostic test for Chagas disease. The upstream process was designed per GLP standards for in-house use, while the downstream process was designed for and successfully transferred to Viral Antigens, Inc. xi Fed-batch fermentation During Moulton’s tenure at IDRI he again set up a fermentation lab for development of recombinant E. coli production of foreign antigens. Most fermentation development work Moulton performed at IDRI was for vaccine development against leishmaniasis – a disease caused by protozoan parasites of the genus Leishmania and transmitted by the bite of certain species of the sand fl y (subfamily Phlebotominae) – and tuberculosis caused by Mycobacterium tuberculosis . While at IDRI, Moulton developed a unique feed recipe in which he supplemented phosphate for a recombinant E. coli fermentation using rich media that tripled the fi nal cell density without any signifi cant increase in process cost or time. Moulton also developed an M. smegmatis recombinant system that should easily be scalable using a wave reactor. This project can be used to produce Mtb antigens for both diagnostics and vaccine development. Over the last 13 years Moulton has successfully developed over 30 fermentation processes. xii 1 Introduction to fermentation DOI: 10.1533/9781908818331.1 Abstract: The use of yeast or microbial cells for the production of a foreign protein has changed the approach of medical research to fi nding healthcare solutions. The application of recombinant systems has become mainstream in treatment of disease. One of the most important aspects of this new scientifi c discipline is the ability to design a cell line or strain, in the case of bacterial or yeast recombinant systems that can be grown under controlled conditions, to produce signifi cant quantities of a recombinant protein. Recently, E . coli has been the predominant bacteria in research and production laboratories and plays a key role in the development of modern biological engineering and industrial microbiology, enabling foreign proteins to be produced in a prodigious and cost-e ffective way. This type of cell growth and production is called fermentation and its history and use will be discussed along with current developments and applications of recombinant technology. Key words: E. coli , fermentation, recombinant DNA, yeast, nucleic acids, bacteria, RNA, phosphate plasmid DNA, recombinant protein, media, fed-b atch, inclusion body, acetate, glucose, IPTG, cell factory. 1 © Elsevier Limited, 2014
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