Development of Simple and Fast Method for Preparation and Purification of Monopegylated Recombinant Human Granulocyte Colony-stimulating Factor (rhG-CSF, Filgrastim) with High Efficiency

Background: PEGylation is a valuable strategy for enhancing the pharmacokinetic properties of recombinant methionyl human granulocyte colony-stimulating factor (rh-Met-G-CSF, filgrastim) which is used to treat chemotherapy induced neutropenia. So development of a cost-effective method is required to attain monoPEGylated G-CSF form. Here it is focused on the PEGylation reaction engineering and purification processes that should be optimized to reduce the costs. Methods: In this study methoxy polyethylene glycol propionaldehydes (mPEG-ALD) 20 kDa MW was utilized to produce monoPEGylated rhG-CSF. PEGylation reaction was carried out at 4 °C and pH 4.5 in the presence of sodium cyanoborohydride and three mPEG-ALD: protein molar ratios (3:1, 5:1 and 10:1). The PEGylation reaction was monitored with SDS-PAGE. Subsequently, isolation of the monoPEGylated form was achieved by cation exchange chromatography (CEC) method. The PEG attachment site was assessed by FTIR and structural characteristics of purified products (unPEGylated and PEGylated rhG-CSF) were investigated by CD and intrinsic fluorescence techniques. Results: The results showed optimal yield of monoPEGylated protein (60%). Also a purity around of 99% achieved for pegylated rhG-CSF. Assay by FTIR disclosed that PEGylation precisely was performed in N-terminus of rhG-CSF. Structural analysis by CD and intrinsic fluorescence indicated that rhG-CSF maintained secondary structure after modification by PEGylation. Conclusion: Overall, this study has expanded a pilot method to purify monoPEGylated rhG-CSF which is simple, fast, and economical method with high efficiency.