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. (c) 2006 Society of Chemical Industry.