Factors affecting protein refolding yields in a fed-batch and batch-refolding system

✍ Scribed by Gareth J. Mannall; Nigel J. Titchener-Hooker; Paul A. Dalby

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 478 KB
• Volume: 97
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.21377

✦ Synopsis

Abstract

The refolding of recombinant protein from inclusion bodies expressed in Escherichia coli can present a process bottleneck. Yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. This article studies the effect of five factors upon the dilution refolding of protein in a twin impeller fed‐batch system using refold buffer containing only the oxidized form of the redox reagent. Such a buffer is easier to prepare and more stable than a buffer containing both reduced and oxidized forms. The five factors chosen were: bulk impeller Reynolds number, mini‐impeller Reynolds number, injection rate of denatured protein, redox ratio, and guanidine hydrochloride (GdHCl) concentration. A 2^5^ factorial experiment was conducted at an industrially relevant protein concentration using lysozyme as the test system. The study identified that in the system used, the guanidine hydrochloride concentration, redox ratio, and injection rate were the most important factors in determining refolding yields. Two interactions were found to be important: redox ratio/guanidine hydrochloride concentration and guanidine hydrochloride concentration/injection rate. Conditions were also found at which high refolding yields could be achieved even with rapid injection and poor mixing efficiency. Therefore, a comparative assessment was carried out with minimal mixing in a simple batch‐refolding mode of operation, which revealed different behavior to that of fed‐batch. A graphical (windows of operation) analysis of the batch data suggested that optimal yields and productivity are obtained at high guanidine hydrochloride concentrations (1.2 M) and redox ratios of unity or greater. Biotechnol. Bioeng. 2007; 97: 1523–1534. © 2007 Wiley Periodicals, Inc.