title: Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column.
abstract: Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.
authors.affiliation: Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou Medical College, Wenzhou 325035, China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Huang
authors.forename: Chaohui
authors.initials: C
authors.lastname: Ye
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Liu
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Wang
authors.forename: Huaibin
authors.initials: H
authors.lastname: Chen
authors.forename: Yanlong
authors.initials: Y
authors.lastname: Liu
authors.forename: Lu
authors.initials: L
authors.lastname: Tang
authors.forename: Hongxin
authors.initials: H
authors.lastname: Zhao
authors.forename: Junfeng
authors.initials: J
authors.lastname: Wang
authors.forename: Wenke
authors.initials: W
authors.lastname: Feng
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
mesh_terms: D000818:Animals; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006493:Heparin; D006801:Humans; D008297:Male; D051379:Mice; D008934:Mitogens / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D041681:NIH 3T3 Cells; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D051381:Rats; D017207:Rats, Sprague-Dawley; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012685:Sepharose; D012996:Solutions; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008934:Mitogens; D011994:Recombinant Proteins; D012996:Solutions; D016222:Fibroblast Growth Factor 2; D011092:Polyethylene Glycols; D006493:Heparin; D012685:Sepharose; D051496:Receptor, Fibroblast Growth Factor, Type 1