Mannall, G.J.; (2006) Characterisation of the effect of process factors upon protein refolding yield. Doctoral thesis , University of London.

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Abstract

Expression of recombinant protein in bacteria such as Escherichia coli can result in the formation of inclusion bodies. Active protein is derived from inclusion bodies by protein refolding. Refolding yields are often poor, and are a bottleneck in such recombinant protein processes. Work has addressed refolding process issues, but several areas remain poorly understood. This thesis aims to understand the importance of process parameters upon refolding yields. Previous studies highlighted that mixing can affect refolding, but have failed to establish why. Use of a two impeller system with operation of small paddle impeller (Re= 2000) in the proximity of the injection point, revealed the importance of energy dissipation experienced by injected denatured protein upon lysozyme refolding yields. A factorial experiment studying the effect of factors on lysozyme refolding yield in fed- batch revealed effects and interactions between physical and chemical process parameters. GdHCl concentration (1.2M) and redox ratio (2:1 red:ox) had the greatest effect. A graphical (windows of operation) approach revealed high GdHCl concentration (1.2 M) and redox ratios at or above unity gave greatest yields in the minimum time. Industry typically refolds from impure inclusion bodies. A series of studies using trypsinogen IB detailed the effect of process contaminants and the efficacy of steps to remove them, upon refolding. Analysis of the effect of centrifugation conditions on IB purity demonstrated the compromise between the levels of IB purity and recovery achieved, together with the removal of key contaminants and the refolding yield obtained, with an optimum of Q/I= 23.08 x 10'g m/s. A second study looked at IB washing and revealed that maximising the area for washing is key to IB purity but not to refolding yield. Use of Triton X was key to maximising refolding yields.

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