Abstract: Protein refolding is a key process for the production of recombinant proteins from E. coli. One of the effective techniques for protein refolding is through liquid chromatography named as protein folding liquid chromatography (PFLC). Mobile phase composition is one of the most important factors affecting the efficiency of protein renaturation in PFLC. Optimization of mobile phase composition in PFLC for protein renaturation with simultaneous purification is thus much more important than that in usual liquid chromatography. This study presents an approach with regard to the increase of protein production through optimizing the mobile phase composition of PFLC. Recombinant human interferon-γ (rhIFN-γ) was purified with simultaneous renaturation by high performance hydrophobic interaction chromatography (HPHIC) using a stationary phase with an end group of PEG-200. Effects of mobile phase composition, gradient elution mode, and flow rate on the recoveries of mass and bioactivity of rhIFN-γ were investigated. It was found that these factors were very important for refolding with simultaneous purification of rhIFN-γ by PFLC. This study highlights the importance of optimizing the mobile phase composition in PFLC for increasing the recovery of protein. Taking 3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4 (pH 7.0) and 0.05 mol/L KH2PO4 (pH 7.0) as mobile phases A and B, respectively, the highest mass recovery was obtained under a nonlinear gradient elution for 35 min.

Key words: gradient elution mode, mass recovery, mobile phase composition, purification, recombinant , renaturation, high performance hydrophobic interaction chromatography （HPHIC）