Characterization of Glycation and Other PTMs in Biotherapeutics Using HILIC-MS

Glycation is an important post-translational modification that is linked to diabetes, cataract, Alzheimer’s, Rheumatoid arthritis and Parkinson’s disease.¹ This reaction occurs between a reducing sugar and a primary amine at the N-terminus of protein or the sidechain of Lysine residues.  Ultimately this interaction leads to advanced glycation end products (AGEs) that are associated with several disease complications.² Glycation could also occur during the manufacturing and storage of therapeutic proteins, including mAbs, necessitating the characterization and accurate quantitation of this modification to ensure safety and efficacy of therapeutic drug products.³ This work focuses on the analysis of in vitro glycated peptides and proteins. Hydrophilic Interaction Chromatography (HILIC) that has been used to characterize hydrophilic modifications⁴ is utilized in this analysis as the hydrophilic nature of the glycation product could lead to a characteristic shift in HILIC retention. Quantitation for glycation modification has been attempted in the literature.⁵ Relative quantitation is currently performed by comparing the area of the glycated peptide with that of its unmodified counterparts. Accuracy of this method depends on the unmodified and modified peptides having similar ionization efficiencies. We propose the use of non-glycated SILAC-labeled proteins to provide both the relative and absolute level of glycation in human IgG1 (Adalimumab) by measuring the peak area of the unmodified peptides relative to that of the SILAC labeled protein. We expect this quantitation approach will be applicable to other PTMs.

References

[1] Videira, P. Q.; Castro-Caldas, M., Linking Glycation and Glycosylation With Inflammation and Mitochondrial Dysfunction in Parkinson’s Disease. Front. Neurosci. 2018, 12, 381.

[2] Kuzan, A.,Toxicity of advanced glycation end products (Review). Biomedical Reports 2021, 46.

[3] Wei, B. et. al., Glycation of antibodies: Modification, methods and potential effects on biological functions. mAbs 2017, 9, 4, 586-594.

[4] Badgett, M. J.; Boyes, B.; Orlando, R., Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry. J. Chromatogr. A 2018, 1537, 58-65.

[5] Li, W. et. al., State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 2. Biopharmaceutical Characterization: The NISTmAb Case Study., 2015, 119-183.