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A high performance bench scale process for isolation from inclusion bodies, refolding and dimerisation of a thiol-engineered recombinant therapeutic protein

Schofield, DM; Nesbeth, DN; (2017) A high performance bench scale process for isolation from inclusion bodies, refolding and dimerisation of a thiol-engineered recombinant therapeutic protein. Biotechnology and Bioprocess Engineering , 22 (4) pp. 423-430. 10.1007/s12257-016-0385-0. Green open access

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Abstract

The use of laboratory procedures is often inefficient for materialisation of recombinant therapeutic proteins in Escherichia coli (E. coli) for pre-clinical evaluation. Approaches such as scaling out shake flask cultivation can be laborious, inefficient and expensive. These inefficiencies can be compounded if the protein requires post-translational modification such as multimerisation. We previously used laboratory methods to produce the < 60 kDa, recombinant biotherapeutic, RB1. We were aware, a priori, that dimerisation of RB1 could double the molecular weight of the protein and increase its systemic retention in the human body by avoiding renal filtration. Here we modified RB1 by substituting a native residue for an unpaired cysteine, generating eRB1, in order to favour its dimerisation. Laboratory methods failed to achieve > 20% disulphide-bridged homodimerisation or monomer of sufficient purity to enable chemi-dimerisation. As such we established a set of high performance, bench-scale, unit operations for cultivation of E. coli cells expressing eRB1, the isolation of eRB1 inclusion bodies, refolding and disulphide-based dimerisation of ≥ 40% of total eRB1 and finally successful chemi-dimerisation of remaining monomeric eRB1. The establishment of scalable procedures can now enable future investigations of eRB1 and other < 60 kDa biologics for which significant bench-scale production is required for pre-clinical evaluation.

Type: Article
Title: A high performance bench scale process for isolation from inclusion bodies, refolding and dimerisation of a thiol-engineered recombinant therapeutic protein
Open access status: An open access version is available from UCL Discovery
DOI: 10.1007/s12257-016-0385-0
Publisher version: http://doi.org/10.1007/s12257-016-0385-0
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Escherichia coli, inclusion body refolding, thiol engineering, recombinant biotherapeutic
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/1573477
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