AN ENGINEERING STUDY OF THE DESIGN, INTEGRATION AND CONTROL OF ANTIBODY FRAGMENT PRODUCTION PROCESSES A thesis submitted to the University of London for the degree of Doctor of Philosophy by Leigh Caroline Bowering June 2000 Department o f Biochemical Engineering University College London Torrington Place London WC1E 7JE l To Mum and Dad 2 ACKNOWLEDGEMENTS I would like to thank Dr Nigel Titchener-Hooker and Professor Peter Dunnill for their supervision and support during this project. Special thanks must also go to Dr Neil Weir at Celltech Chiroscience Limited for his invaluable guidance and enthusiasm throughout the course of this research. Much of this work would not have been possible without the technical support of Billy Doyle, Ian Buchanan and Clive Osborne; their help has been greatly appreciated. I would like to extend my gratitude to the numerous researchers at University College London who have provided intellectual input and experimental support. In particular I would like to acknowledge Nick Murrell for his help with the large-scale fermentation and centrifugation experiments, Nik Willoughby for keeping me company during the numerous overnight fermentations and advising on expanded bed operation, and John Maybury for his input into the scale-down and modelling of the extraction and centrifugation processes. Special thanks must go to the many friends I made during my time in London, especially Natalie and Helen for keeping me smiling, my colleagues on the first floor for the constant supply of tea, coffee and cakes, and to Layth for providing the support and encouragement I needed for the successful completion of this thesis. Finally, I must thank my parents for their continued support throughout my career, without which none of this would have been possible. The financial support of the BBSRC and Celltech Chiroscience Limited is gratefully acknowledged. 3 ABSTRACT This project investigates process options for the production of antibody fragments from Escherichia coli and quantifies the effects of processing decisions on the integration of a complete bioprocess sequence. Fab’ antibody fragments were produced by E. coli fermentation at scales of up to 45OL. Antibody expression was directed to the periplasmic space, however during initial fermentations over 50% of the Fab’ leaked into the extracellular broth over the course of the induction period. Alterations to the fermentation were made to allow greater control of product location, and with the modified protocol 80-90% of the product was consistently retained within the periplasm. This fermentation strategy formed the basis for future downstream purification studies. A novel method for the recovery of periplasmic proteins has been characterised and modelled at scales from 65mL to 100L. 85% recovery of periplasmic Fab’ was achieved following resuspension of cells in a Tris-EDTA extraction buffer at 60°C. Operation at high temperature also resulted in purification of the process stream by degradation of both contaminating E. coli proteins and incomplete or partially degraded Fab’ fragments. Clarification of the process stream following periplasmic extraction was compared using a novel tubular bowl and an intermittent discharge disc-stack centrifuge. Operating at 95% biomass removal, 94% Fab’ was recovered using the tubular bowl, compared to 73% with the disk-stack centrifuge. The improved recovery obtained with the tubular bowl was shown to be due directly to the greater level of liquid recovery. However lower throughputs were required for equivalent clarification when using this machine. The optical biosensor has been assessed as a technique for the monitoring of Fab’ in real time. The biosensor gave comparable Fab’ accumulation profiles to ELISA during fermentation. During chromatographic purification, the sensor provided an 4 accurate indication of Fab’ breakthrough during column loading and correctly identified product containing fractions during column elution. The thesis concludes with a series of mass balance studies which compare the relative efficiencies of traditional purification processes and more novel process alternatives conducted at pilot scale. The results show that novel techniques such as whole broth extraction (performing the periplasmic extraction process on whole fermentation broth) and expanded bed adsorption offer potentially viable process alternatives, however the operational problems and reduced reliability compared to more conventional routes means further adaptation or optimisation is required before such techniques will be selected over conventional processing strategies. 5 TABLE OF CONTENTS ACKNOWLEDGEMENTS...........................................................................................3 ABSTRACT ....................................................................................................................4 TABLE OF CONTENTS...............................................................................................6 LIST OF FIGURES.......................................................................................................12 LIST OF TABLES.........................................................................................................16 1. INTRODUCTION.................................................................................................. 19 1.1 Antibody engineering ............................................................................................ 19 1.1.1 Project significance.........................................................................................19 1.1.2 Structure of antibodies and antibody fragments ............................................20 1.1.2.1 Structure of whole antibodies ..................................................................20 1.1.2.2 Structure of antibody fragments .............................................................. 20 1.1.3 Commercial applications of engineered antibodies..................................... 23 1.2 Methods for the production of antibody fragments .............................................25 1.2.1 Production of whole antibodies and antibody fragments .............................25 1.2.1.1 Antibody fragments by proteolytic digestion..........................................25 1.2.1.2 Production of monoclonal antibodies by mammalian cell culture 25 1.2.1.3 E. coli production of antibody fragments................................................26 1.2.2 Expression strategies for E. coli.....................................................................27 1.2.2.1 Direct cytoplasmic expression.................................................................27 1.2.2.2 Secreted fusion proteins...........................................................................28 1.2.2.3 Functional periplasmic expression.......................................................... 28 1.2.2.4 Secretion to the extracellular media........................................................ 30 1.2.2.5 External cell surface expression............................................................. 30 1.2.3 Fermentation strategies.................................................................................. 31 1.2.3.1 Batch and fed-batch cultures ....................................................................31 1.2.3.2 Development of growth media.................................................................32 1.2.3.3 Induction strategies using the lac promoter............................................33 1.2.4 Effect of protein engineering on antibody fragment expression................. 35 1.2.5 Alternative host organisms for antibody fragment expression .................... 36 1.3 Recovery and purification of antibody fragments from Escherichia coli fermentation broths .................................................................................................. 37 1.3.1 Objectives of downstream processing ........................................................... 37 1.3.2 Biomass Separation.........................................................................................39 1.3.2.1 Centrifugation...........................................................................................39 1.3.2.2 Microfiltration...........................................................................................40 1.3.3 Release of cell associated antibody fragments ..............................................41 1.3.3.1 Mechanical cell disruption.......................................................................41 1.3.3.2 Specific periplasmic release.....................................................................42 1.3.4 Purification...................................................................................................... 44 1.3.4.1 Affinity purification................................................................................. 44 1.3.4.2 Facilitated affinity purification............................................................... 45 1.3.4.3 Ion exchange chromatography.................................................................46 6 1.4 Bioprocess monitoring...........................................................................................46 1.4.1 Techniques available for bioprocess monitoring ..........................................47 1.4.1.1 Chromatographic assays..........................................................................48 1.4.1.2 Biosensors.................................................................................................48 1.4.1.3 Flow-injection