Separation of exenatide analogue mono-PEGylated with 40 kDA polyethylene glycol by cation exchange chromatography

Random PEGylation usually resulted in product mixtures composed of mono-PEGylated isomers and multi-PEGylated attachments. Generally in PEGylation research, separation of the mono-PEGylated isomers was the prerequisite for finding the optimal PEGylation site. However, when peptides or proteins were PEGylated with polyethylene glycol as large as 40 kDA, the physicochemical properties like hydrophobicity and molecular size of the isomers would become too similar to make the routine separation methods, like RP-HPLC, size-exclusion chromatography, SDS-PAGE and capillary isoelectric focusing invalid. This article presented a useful method of successfully separating exenatide analogue (an incretin for diabetic therapy) isomers mono-PEGylated with 40 kDA polyethylene glycol by cation exchange chromatography, which would be a powerful tool for the PEGylation research.