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Title: Production of recombinant antibody fragments in microorganisms
Author: Harrison, Joanna Shan
ISNI:       0000 0001 3539 0019
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1996
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Recombinant DNA technology has enabled expression in microorganisms of antibody fragments containing the selective binding site only. These proteins and other engineered molecules based upon the antigen binding site are especially attractive for medical applications. This thesis presents results of experimental research into the expression and recovery of antibody fragments in an active form, by fermentation of recombinant Escherichia. coli at pilot plant scale. Fed-batch fermentation for production of a single-chain Fv fragment (scFv) routinely achieved a high cell density of 50 g dry cell weight/L by controlled exponential feeding of the carbon source (glucose) at a controlled specific growth rate. This was followed by induction of the tac promoter by addition of IPTG accompanied by a linear feed of yeast extract. Manipulation of yeast extract feed concentration resulted in titres of 200 mg of recombinant antibody fragment per litre of fermenter culture with 78% of this yield retained in the periplasm. The ability to control location will ease integration of fermentation with product recovery operations. An important aspect relating to production is accurate measurement of product concentration within a short time of sample collection from the process. This has been investigated by comparison of a novel optical biosensor technique with a standard and more time consuming enzyme-linked immunosorbent assay (ELISA). This was performed by monitoring the production of Fv fragments during a batch fermentation process. Production profiles generated by the two techniques were found to be similar although quantification of exact concentration of Fv by the biosensor technique was not possible. Preliminary studies into the recovery of antibody fragments from E. coli fermenter cultures have utilised pilot scale equipment to investigate process options for the recovery of both periplasmic and extracellular antibody fragments. Although specific methods for the release of periplasmic antibody fragments were addressed, a complete recovery scheme was not elucidated. Purification from a liquid stream capitalised on the selective binding characteristics of recombinant antibody fragments for their target antigen. Finally, expression of inactive antibody fragments in the yeast Saccharomyces cerevisiae was investigated, where it was necessary to both isolate and reactivate the protein. With these limitations, this organism was less suited to production at increased scale.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: E. coli fermenter; Yeast; Engineered molecules