Berry, Mark John; (1996) Analysis and recovery of Fv antibody fragments produced in Esherichia coli. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cloned antibody binding sites (known as Fv antibody fragments) are attractive to the fine chemicals industry for several applications: in particular, as ligands in affinity chromatography. However, optimised systems for producing Fv fragments are not currently available. This thesis aims to establish new methods for the analysis and recovery of Fv antibody fragments that have been expressed in Escherichia coli cultures by recombinant DNA technology, thereby enabling improved control of the processes required for Fv production. An important objective was to prepare and test generic analytical reagents that bind all Fv fragments regardless of their specificity. Tracer antibodies were raised against a common peptide motif and used to design a generally applicable immunoassay for analysing Fv protein in microbial culture. The immunoassay was used as a component part in a new analytical system that can determine total Fv and active Fv independently, thus providing the fermentation scientist with a complete picture of the immunoreactivity or "authenticity" of the Fv throughout the process. Using an Fv specific for human gonadotropin as an example, the immunoreactivity of the Fv in the fermenter was found to approach 100%. A new process for recovering active Fv fragments from microbial cultures was designed and tested, using an Fv specific for estrone-3-glucuronide as an example. The new process was based on antigen affinity chromatography and used two antigen analogues: one as a column ligand, the other as a specific eluant. The recovery efficiency of active Fv was several-fold superior to that obtained with conventional methods. Overall, the results show that the analysis and recovery of Fv fragments are related in analytical and production systems: both require the preparation and synthesis of reagents that can discriminate between Fv and non-specific biomass and between active Fv and inactive Fv.

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