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Functionalized polyethylene glycols (PEGs) are used to derivatize therapeutic proteins to increase their half-life, reduce immunogenicity, and increase resistance to proteolysis.Characterization of the functionalized PEG raw material is important for controlling the quality of the final PEGylated protein product.Of particular interest are bis-functionalized PEGs which may cause protein cross-linking.Here, analyses of impurity bis-maleimide-PEG (bis-mal-PEG) in mono-mal-PEG will be presented.Chromatographic separation and detection of low levels of bis-mal-PEG in mono-mal-PEG is challenging because of the polydispersity of the analytes and because of the relatively minor differences between the desired mono-mal-PEG and the bis-mal-PEG impurity.Adequate selectivity and detectability were achieved by derivatization of the mal-PEGs with cys-peptides containing a UV chromophore and multiple ionizable sites.The polar, ionizable nature of the peptides results in significant differences between the mono-and bis-derivatized PEGs allowing separation by both reversed-phase HPLC (RPLC) and ion-exchange chromatography.Choice of the derivatizing peptide and optimization of the derivatization will be discussed.