Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS–CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG40K-, PEG43.5K-, and PEG47K-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS–CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.