Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach

• Published in: Analytical chemistry (Washington) 2023-09, Vol.95 (37), p.13813-13821
• Main Authors: Liu, Gao-Yuan, Zhang, Zhengqi, Yan, Yuetian, Wang, Shunhai, Li, Ning
• Format: Article
• Language: English
• Subjects: Chemistry; Covalence; Cross-linking; Crosslinking; Lysine; Mass spectrometry; Mass spectroscopy; Molecular weight; Monoclonal antibodies; Risk assessment; Serine
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c01602

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.