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 Garner G Moulton amsterdam • boston • cambridge • heidelberg • london new york • oxford • paris • san diego san francisco • singapore • sydney • 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: permissions@elsevier.com 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 verification 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 RefineCatch 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 figures and tables Figures 1.1 Nucleotide bases made up of pyrimidines and purines as well as the addition of the sugar ribose (RNA) or deoxyribose (DNA) and a phosphate group 1.2 Chargaff’s rule 1.3 Base pairing in DNA is complementary 1.4 Conversion of simple sugars to ethanol and carbon dioxide 1.5 The E coli cell 15 1.6 Glycolytic pathway and acetyl-CoA formation 16 TCA cycle and the formation of acetyl CoA from acetate 18 Isopropyl β-D-1-thiogalactopyranoside (IPTG) 21 2.1 Generic plasmid 34 2.2 Typical cloning of foreign gene into recombinant plasmid 37 Isoproply-β-D-thio-galactoside (IPTG) shown with the arrow pointing to the sulfur–carbon bond that is not hydrolysable 39 1.7 1.8 2.3 vii Fed-batch fermentation 2.4 Transcription of DNA 40 2.5 Micrograph 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 Transformation of a bacterial cell culture with a plasmid 47 Draw a “T” on the bottom of your Petri dish, as shown 58 2.9 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 Oxygen transport within the cell 76 3.3 %DO versus time 79 3.4 ln (C* − CL) versus Δtime (s) 79 3.5 Oxygen transfer rate and KLa determination 80 3.6 10-liter bioreactor for E coli fermentation 84 3.7 Dissolved oxygen electrode: polarographic sensor 88 3.8 The pH electrode: Calomel electrode 91 3.9 Typical fed-batch fermentation growth curve viii 105 List of figures 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 hours post-induction 108 4.1 Prokaryotic ribosomal composition 112 4.2 Translation 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 Amino acid names, structures and one letter symbol associated with each 120 Primary, secondary, tertiary and quaternary structures of proteins 122 4.6 Bacterial GroES/GroEL complex 123 4.7 Aggregation pathways in vivo 133 4.4 4.5 Tables 4.1 4.2 Codons for amino acids and start and stop sequences 113 Protein complexes within prokaryotic and eukaryotic cells 126 ix About the author Gus G Moulton is Chief Scientific Officer of BioBench LLC, a contracting facility for purification 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 nonprofit 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 purification 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 purification 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 fly (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 final cell density without any significant 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 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 finding healthcare solutions The application of recombinant systems has become mainstream in treatment of disease One of the most important aspects of this new scientific 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 significant 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-effective 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-batch, inclusion body, acetate, glucose, IPTG, cell factory © Elsevier Limited, 2014 ... Biomedicine: Number 42 Fed-batch fermentation A practical guide to scalable recombinant protein production in Escherichia coli Garner G Moulton amsterdam • boston • cambridge • heidelberg • london... coli recombinant fermentation responses to some recombinant proteins limit their productivity during expression It is obvious that there is a need to learn how to manipulate growth conditions to. .. were not comparable to the same protein made from natural source and thus these recombinant proteins were not safe for human use The recombinant proteins had two major obstacles to overcome: proteolysis