Characterization of a monoPEG20000-Endostar

In this study, we investigated the PEG attachment site of mono-PEGylated Endostar, a modified recombinant human endostatin approved in China for lung cancer N-terminal site-dilected mono-PEGylation of Endostar was accomplished using mPEG-propionaldehyde derivatives (Mw = 20 kDa) under slightly acidic pH conditions (pH 5 5) One-step cation exchange chromatography was used to purify the mono-PEGylated Endostar Following tryptic digestion, the peptide fragment containing PEG was separated by SDS-PAGE Barium iodide staining and Western blotting were used to detect the PEG moiety and the N-terminus of Endostar. respectively The peptide fragment stained by barium iodide showed a positive response to anti-(His) 6 mAb, demonstrating that PEG was located at the N-terminus of Endostar LC/MS was applied to verify the occurrence of mono-PEGylation at the N-terminus of Endostar. (C) 2010 Elsevier B V All rights reserved