Differences in electrophoretic behavior between linear and branched PEG-conjugated proteins.

The objective of this study was to characterize the differences in electrophoretic behavior between linear and branched PEG-conjugated proteins. Human growth hormone and alpha-lactalbumin modified by linear or branched PEGs with molecular weight of 10 kDa were analyzed by SEC, MALDI-TOF MS, SDS-PAGE, and microchip CGE (MCGE). Chromatographic and mass spectrometric differences between the linear and branched PEG-proteins on SEC and MALDI-TOF MS were small, but their electrophoretic behaviors on SDS-PAGE and MCGE were significantly different. In particular, MCGE showed significant differences in the peak width and the migration times of linear and branched PEG-proteins, in which the branched PEG-proteins exhibited a narrower peak and longer migration time than the linear PEG-proteins. This phenomenon may explain the longer circulation half-life for the branched PEG-proteins observed in previously reported in vivo studies. Consequently, this study indicates that MCGE may be a valuable tool for differentiating linear and branched PEG-proteins.