Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation solubilisation and protein refolding yields.

Project title: Rapid microscale evaluation of the impact of fermentation conditions on inclusion body formation, solubilisation and protein refolding yields. Hypothesis: That protein refolding steps can be performed and optimized at a microlitre scale and that the technique, once established, can rapidly evaluate the impact of earlier bioreactor stages on whole bioprocess performance. Significance and Background: Microscale processing techniques offer the potential to speed up the delivery of new drugs to market to reduce development costs and thereby increase patient benefits. We and others have shown that in selected cases the study of bioprocess unit operations in microwell plate formats and the use of automation can significantly enhance experimental throughput and facilitate the parallel evaluation of a large number of process conditions (eg Nealon et al. 2005, Jackson et al., 2006, Lacki, 2007). While the majority of microscale studies have focused on microbial fermentation, by comparison little work has been done on downstream processing operations. This potential bottleneck requires considerable attention if significant step-wise process enhancements are to be gained. In particular the impact of fermentation conditions on whole process performance must be understood if downstream processing is not to become rate limiting (Micheletti and Lye, 2006). A particular example that could benefit from this approach is the refolding of recombinant protein from inclusion bodies (IB). Protein refolding yields at industrially relevant concentrations are restricted by aggregation of protein upon dilution of the denatured form. A number of studies have investigated chemical (Buswell and Middleberg, 2002, Mannall et al., 2007a) as well as physical (Mannall et al., 2005) factors affecting the dilution refolding in small (20-200ml) bioreactors, however for the majority of proteins a large number of refold conditions usually need to be tested in order to optimize this processing step. The use of microwell plates as a format in which to perform protein refold experiments has recently been preliminary investigated (Mannall et al., 2007b). Not only has it been shown that refold reactions scaled well between microwell and bench scale operations but it was also demonstrated that a significant amount of information can be gained in a short period of time using a small amount of the valuable IB-derived protein. In this project we propose to radically enhance the microwell approach for the rapid optimization of the protein refolding step by: 1) adopting a whole process approach to optimization which investigates the effect of fermentation conditions on subsequent refolding yields and product quality 2) establish the automation of the refolding step, both in terms of liquid handling operations and associated analytical methods, to speed up the investigation of multiple variables under different mixing conditions. Research Program: 1) Establish and demonstrate an automated microwell-based dilution-refolding system using an industrially relevant strain provided by Avecia Biologics 2) validate the dilution-refolding system by comparing the yields obtained to standard bench scale operations 3) generate E. coli fermentation broths using different carbon sources, induction time and window and harvest time strategies and then use the microwell format in investigating the effect of the upstream parameters on IB solubilisation and refolding yields. Jackson et al. (2005) J. of Membrane Sci., 276, 31-34. Nealon et al. (2006) Chem. Eng. Sci., 61, 4860-4870. Lacki et al. (2007) ACS National Mtg., Boston BIOT-472. Micheletti and Lye (2006) Curr. Opinion in Biotechnol., 17, 611-618. Buswell and Middleberg (2002) Biotech. Progress, 18, 470-475. Mannall et al. (2007a) Biotech. Bioeng., 97, 1523-1534. Mannall et al. (2006) Biotech. Bioeng., 93, 955-963. Mannall et al. (2007b) Biotech. Bioeng., submitted.