PEG is usually attached to proteins through the amine group side chains of amino acids (usually Lysine) in order to suppress potential immunogenicity or prolong the half-life over the native protein. The mechanism for these desired outcomes relies on the fact that the addition of PEG makes it more difficult for the immune system to detect the drug and therefore clear it. PEG can also be also used to increase the solubility of hydrophobic protein drugs.
PEGylated proteins present unique challenges to analytical assessment and structural characterization. For example, heterogeneity in the PEG moiety can result in complex profiles from particular characterization methods such as intact mass analysis or Edman sequencing, where PEG may block to certain reactions when it is located at the N-terminus of the protein.For some of these investigations it may be necessary to chemically de-PEGylate prior to analysis.
Furthermore, the chemical process used to couple the PEG unit to the protein may result in adverse chemical reactions occurring to other parts of the protein such as oxidation or deamidation events, disulfide bridge scrambling or modifications to glycosylation, therefore it is particularly important for PEGylated molecules that a thorough characterization of the protein be undertaken.