Characterization of PEGylated biopharmaceutical products by LC/MS and LC/MS/MS

PEGylation of peptide and proteins is an important method of improving their pharmacokinetic, pharmacodynamic, and immunological profiles, and thus enhancing their therapeutic effect. However, PEGylation of peptides and proteins creates significant challenges for detailed structural characterization, such as PEG heterogeneity, site of PEG addition, and number of attached PEG moieties. Here, we present two methodologies for the structural characterization of PEGylated peptides and proteins. LC/MS methodology utilizing post-column addition of amines was developed to obtain accurate masses of PEGylated peptides and proteins, which can be used to assign the structures and number of attached PEGs. The PEGylated sites in PEGylated products could be elucidated with the tandem LC/MS methodology combining in-source fragmentation with CID-MS/MS. Both methodologies are applied to model PEGylated peptides to obtain the accurate masses and identify PEGylated sites.