Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy.

Capillary electrophoresis (CE) was investigated for characterizing poly(ethylene glycol) (PEG) attachment ("PEGylation") and PEG removal ("dePEGylation") of proteins. Lysozyme was used as a model protein because it is one of the best understood enzymes, has a high ionic strength (high pI value; thus making it suitable for CE), and has a tertiary structure that is known with high resolution. Several PEG derivatives, both hydrolytically degradable and nondegradable and with varying reactivities toward amino groups, were used to couple to amino groups (six epsilon-amino and one alpha-amino) on the surface of the protein. Capillary electrophoresis was found to be useful in following both the PEGylation and dePEGylation of lysozyme. Capillary electrophoresis separation is based on the size of the conjugate, which is determined by the number and molecular weight of the PEG that is attached. Baseline resolution was obtained between the peaks for each PEG chain attached per protein molecule ("PEGmers") for PEG molecular weights >5000, although individual PEGmers could be recognized at lower molecular weights without baseline separation. Highly modified lysozyme showed complete inactivation, but when released from the degradable PEG, regained >60% of the original in vitro activity. The sites of PEGylation were determined using a tryptic map of the modified and unmodified protein. Typically, peptide fragments are separated by reversed-phase HPLC, but we show that CE can provide a complementary separation technique for determining sites of PEGylation. Capillary electrophoresis has advantages of high efficiency separations, rapid analysis, and ease of use.