Solid-Phase N-Terminus PEGylation of Recombinant Human Fibroblast Growth Factor 2 on Heparin-Sepharose Column
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- 作者：Zhifeng Huang ; Chaohui Ye ; Zhijun Liu ; Xiaojie Wang ; Huaibin Chen ; Yanlong Liu ; Lu Tang ; Hongxin Zhao ; Junfeng Wang ; Wenke Feng ; Xiaokun Li
- 刊名：Bioconjugate Chemistry
- 出版年：2012
- 出版时间：April 18, 2012
- 年：2012
- 卷：23
- 期：4
- 页码：740-750
- 全文大小：518K
- 年卷期：v.23,no.4(April 18, 2012)
- ISSN：1520-4812
文摘
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.