A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1

• Published in: PloS one 2012-05, Vol.7 (5), p.e36423
• Main Authors: Huang, Zhifeng, Zhu, Guanghui, Sun, Chuanchuan, Zhang, Jingui, Zhang, Yi, Zhang, Youting, Ye, Chaohui, Wang, Xiaojie, Ilghari, Dariush, Li, Xiaokun
• Format: Article
• Language: English
• Subjects: Aldehydes - chemistry; Alkylation; Amino acids; Animals; Anticoagulants; Binding Sites; Biocompatibility; Biological activity; Biology; Biotechnology; Carbon tetrachloride; Carbon Tetrachloride - adverse effects; Cell proliferation; Chemical and Drug Induced Liver Injury - prevention & control; Chemical bonds; Chemistry; Chromatography; Columns (structural); Cytoprotection - drug effects; Engineering; Extracellular signal-regulated kinase; Fibroblast Growth Factor 7 - chemistry; Fibroblast Growth Factor 7 - pharmacokinetics; Fibroblast Growth Factor 7 - pharmacology; Fibroblasts; Gastrointestinal diseases; Gastrointestinal tract; Gastrointestinal tract diseases; Growth factors; Half-life; Health aspects; Hematology; Heparin; Homeostasis; Homogeneity; Humans; Injuries; Ion-exchange chromatography; Ischemia; Keratinocyte growth factor; Laboratories; Liver; Liver Failure, Acute - chemically induced; Liver Failure, Acute - prevention & control; Lungs; Male; MAP kinase; Medicine; Metabolic pathways; Peptide Fragments - chemistry; Pharmaceuticals; Pharmacokinetics; Pharmacology; Polyethylene glycol; Polyethylene Glycols - chemistry; Protein Stability; Protein structure; Proteins; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacokinetics; Recombinant Proteins - pharmacology; Reductive alkylation; Reproducibility of Results; Rodents; Skin; Stability; Structure-Activity Relationship; Substance P; Substrate Specificity; Therapeutic applications
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0036423

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 CCl(4)-induced injury in rats compared to rhKGF-1.