Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column

Bioconjug Chem. 2012 Apr 18;23(4):740-50. doi: 10.1021/bc200550f. Epub 2012 Apr 2.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Proliferation / drug effects
  • Chromatography, Ion Exchange / methods*
  • Female
  • Fibroblast Growth Factor 2 / chemistry*
  • Fibroblast Growth Factor 2 / isolation & purification
  • Fibroblast Growth Factor 2 / metabolism
  • Fibroblast Growth Factor 2 / pharmacology
  • Heparin*
  • Humans
  • Male
  • Mice
  • Mitogens / chemistry*
  • Mitogens / isolation & purification
  • Mitogens / metabolism
  • Mitogens / pharmacology
  • NIH 3T3 Cells
  • Polyethylene Glycols / chemistry*
  • Protein Stability
  • Rats
  • Rats, Sprague-Dawley
  • Receptor, Fibroblast Growth Factor, Type 1 / metabolism
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Sepharose*
  • Solutions
  • Substrate Specificity
  • Temperature

Substances

  • Mitogens
  • Recombinant Proteins
  • Solutions
  • Fibroblast Growth Factor 2
  • Polyethylene Glycols
  • Heparin
  • Sepharose
  • Receptor, Fibroblast Growth Factor, Type 1