Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

中文翻译：

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精确定量治疗性单克隆抗体中未加工的C端赖氨酸的18O-Labeling辅助LC-MS方法。

未加工的C端赖氨酸（C端Lys）是在治疗性单克隆抗体（mAb）中形成基本变异的最常见原因之一。尽管通常使用基于LC-MS的肽图分析方法对C末端Lys变体进行定量，使用来自两个C端肽段（含Lys和不含Lys）的相对MS响应，但这种方法通常会导致高估含Lys的肽电离效率的内在差异。本文中，我们报道了一种18O标记辅助LC-MS方法，该方法利用了羧肽酶B催化的Lys去除和18O标记来提高C端Lys定量的准确性。该方法的保真度首先使用模拟大范围C末端Lys水平的合成肽混合物标准品来证明。最后，新开发的方法用于案例研究中，其中准确量化和比较了用不同方法生产的mAb样品中的C末端Lys变体。这种新方法为需要准确的C术语Lys水平以协助决策和根本原因研究的研究提供了有价值的解决方案。