Separation and detection of bis-maleimide-polyethylene glycol and mono-maleimide-polyethylene glycol by reversed-phase high pressure liquid chromatography.

Monofunctional maleimide polyethylene glycol (mono-mal-PEG) with average molecular weight up to 40 kDa can be used as a raw material for the PEGylation of therapeutic proteins. A possible impurity in this raw material which needs to be controlled is the bisfunctional maleimide-PEG, which has a similar average molecular weight to mono-mal-PEG. Chromatographic separation and detection of low level bis-mal-PEG in mono-mal-PEG presents a major challenge because of the polydispersity of the analytes and the minor difference between the desired mono-mal-PEG and the bis-mal-PEG impurity. In this study, linear mal-PEGs were first derivatized with a specially designed cys-peptide containing a UV chromophore and multiple ionizable sites. Separations were then carried out by reversed-phase HPLC with UV detection at 360 nm. Mono-mal-PEG and bis-mal-PEG were well resolved using a Gemini C18 column with an aqueous-acetonitrile mobile phase. Retention times increased as PEG molecular weight increased from 10 kDa to 40 kDa, while selectivities decreased as PEG molecular weight increased. Results from systematically designed studies for optimization of critical parameters including gradient slope, column temperature, and acidic modifier in the mobile phase led to the selection of the final separation conditions. The developed method conditions were specific, accurate, and sensitive for detecting bis-mal-PEG as an impurity in mono-mal-PEG with limit of quantitation of 0.2% and may be used to assess the quality of mono-mal-PEG as a raw material for protein PEGylation.