Prediction and Characterization of Glycated Peptides and Proteins using Hydrophilic Interaction Liquid Chromatography coupled with Mass Spectrometry (HILIC-MS).

Glycation is an important post-translational modification (PTM) that has been linked to diabetes, cataract, Alzheimer's, and Rheumatoid arthritis. This reaction occurs between a reducing sugar and a primary amine at the N-terminus of a protein or at a lysine side chain. Ultimately, this interaction can lead to advanced glycation end products (AGEs) that are associated with several disease complications. Glycation can occur during the manufacturing and storage of therapeutic proteins, including monoclonal antibodies (mAbs), necessitating the characterization of this modification to ensure the safety and efficacy of therapeutic drug products. Hydrophilic Interaction Liquid Chromatography (HILIC) has been previously employed to characterize hydrophilic modifications. It can also be used to characterize glycated species, as the hydrophilic nature of the glycation product can lead to a characteristic shift in HILIC retention. This work focuses on deriving a retention coefficient that describes the extent of hydrophilicity imparted by glycation modification in HILIC using in vitro glycated peptide and protein samples. The HILIC retention coefficient can be used to predict the retention times of tryptic peptides with glycation modifications in complex, unknown protein samples, including immunoglobulins (IgGs).