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.
Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl4-induced injury in rats compared to rhKGF-1.
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