Discarded free PEG-based assay for obtaining the modification extent of pegylated proteins

Free polyethylene glycol (PEG) is a byproduct produced during the process of pegylation and should be removed for the purification of pegylated proteins. In this paper, it was used to develop a new method for obtaining the modification extent of pegylated proteins. This method included two steps of operation. Firstly, the free PEG was separated from crude reaction mixture of pegylated proteins by CM-Sepharose FF. Then PEG was determined based on the formation of a complex with barium chloride and iodine solution. The effective detective range of PEG was 0–7.5 μg/ml. The modification extent was calculated according to a formula. This method is simple, sensitive, and applicable to all of the PEG derivatives. The most distinctive aspect is that it does not consume proteins.