Abstract
Traditional PEGylation strategy of peptide and protein usually results in heterogeneous products with wide molecular weight distribution. Mono‐PEGylated consensus interferon (C‐IFN) with homogeneous molecular weight was prepared by a novel methoxypolyethylene glycol (mPEG) derivative. 2‐Fluoro‐1‐methylpyridinium toluene‐4‐sulfonate (FMP) was applied to activate mPEG and a novel functionalized mPEG derivative (FMP‐mPEG) was obtained. The reactivity of FMP‐mPEG toward amino groups of C‐IFN was evaluated, and the heterogeneity of PEGylated protein was identified by high‐performance size‐exclusion chromatography (HPSEC) and matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS). Finally, the PEGylated C‐IFN was separated from intact protein by cation‐exchange chromatography. The results indicated that PEGylation process was dependent on pH dramatically and the modification degree was increased with the increasing molar excess of FMP‐mPEG. Under optimized condition, mono‐PEGylated C‐IFN with molecule weight of 24.64 kDa was achieved. Further investigation of in vitro biological activities demonstrated that about 88% of antiviral activity was maintained by the mono‐PEGylated C‐IFN. Copyright © 2006 Society of Chemical Industry