Glycerol-assisted hydrophobic interaction chromatography improving refolding of recombinant human granulocyte...

Efficient refolding of recombinant proteins in the forms of inclusion bodies at higher concentration remains challenging. Here, we report a strategy of a dual-gradient hydrophobic interaction chromatography (HIC) mode to refold recombinant human granulocyte colony-stimulating factor from its inclusion bodies at high protein concentration. The strategy was taken to meet the demand of dynamic refolding proceeding by gradually decrease the denaturant (guanidine-HCl) concentration and gradually increase the hydrophilicity of media (column of Poros PE 20) with glycerol as additive to provide a mild refolding surroundings. Compared with dilution method, this dual-gradient HIC process gave about 8.5-fold of increase in specific activity and 30% increase in soluble protein recovery. Furthermore, much higher protein concentration could be obtained at the same time.