Abstract

Bikunin is a glycosylated protein that aggregates extensively during mammalian cell culture, resulting in loss of activity, loss of native secondary structure, and the formation of nonnative disulfide bonds. We investigated the use of high hydrostatic pressure (1000-3000 bar) for the refolding of bikunin aggregates. The refolding yield obtained with pressure-modulated refolding at 2000 bar was 70 (+/-5%) by reverse-phase chromatography (RP-HPLC), significantly higher than the value of 55 (+/-6%) (RP-HPLC) obtained with traditional guanidine HCl "dilution-refolding." In addition, we determined the thermodynamics of pressure-modulated refolding. The change in volume for the transition of aggregate to monomer DeltaV(refolding) was calculated to be -28 (+/-5) mL/mole. Refolding was accompanied by a loss of hydrophobic exposure, resulting in a positive contribution to the DeltaV(refolding). These findings suggest that the disruption of electro-static interactions or the differences in size of solvent-free cavities between the aggregate and the monomer are the prevailing contributions to the negative DeltaV(refolding).