Analytical Chemistry, Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6707, United States.
Anal Chem. 2023 Sep 19;95(37):13813-13821. doi: 10.1021/acs.analchem.3c01602. Epub 2023 Sep 7.
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 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.
表征导致治疗性单克隆抗体 (mAb) 中共价高分子量 (HMW) 物质的交联对于风险评估具有重要意义，因为它不仅提供了一个框架，还为工艺改进提供了思路。然而，由于其复杂性和低丰度，鉴定 mAb 产品中的新型和未知交联可能极具挑战性。在这里，我们应用了一种多方面的基于 MS 的方法，报告了在赖氨酸和丝氨酸残基之间形成亚胺键的新型共价交联的发现。特别是，发现这种 Ser-Lys 交联是酸不稳定的，并且很容易被低 pH 下运行的常规 LC-MS 技术所忽略。值得注意的是，尽管在胶原蛋白交联中已经报道了基于亚胺的交联，但这是首次在导致共价 HMW 物质形成的 mAb 产物中发现 Ser-Lys 交联。
