Multi-variable operational characteristic studies of on-column oxidative protein refolding at high loading concentrations

Chromatographic-based protein refolding techniques have proven to be superior to conventional dilution refolding methods, due to the higher loading concentration and simultaneous purification. Among these techniques, Size Exclusion Chromatography (SEC) has in particular been demonstrated as an effective method for refolding of variety of proteins. To date existing studies of protein refolding at high concentrations (>1 mg/mL) in SEC have primarily been conducted as single factor studies, in which a single parameter is varied to assess impact on operating performance, which does not allow for determination of the interactions of different operating parameters and optimized operating conditions. In this work a multi-variable investigation of size exclusion protein refolding at high protein concentration using lysozyme as a model protein was performed, in order to quantify the interaction of factors and optimize performance. It was observed when l-arginine is used as an additive the refolding yield becomes independent of the protein concentration and refolding buffer pH, providing that a redox couple is used to assist the reformation of disulfide bridges. Furthermore, the pore accessibility for small molecules was reduced at the presence of this additive particularly at higher protein concentrations indicating slower removal of these molecules and a possible additional mechanism of aggregation prevention. Using the subsequent optimized refolding buffer, a refolding yield of more than 90% was obtained for up to 40 mg/mL loading concentration of lysozyme which has only been reported for a urea gradient SEC (8–2 M) with lower equilibration and elution flow rates due to high viscosity of buffer containing high concentrations of urea.