Bowering, LC; (2001) An engineering study of the design, integration and control of antibody fragment production processes. Doctoral thesis (PhD), UCL (University College London).

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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 450L. 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.

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