Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.

中文翻译：

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高分辨率 IMS-MS 可在 IgG4 单克隆抗体的互补决定区域分配额外的二硫键配对。

单克隆抗体 (mAb) 在治疗各种疾病（包括癌症和免疫系统疾病）方面发挥着越来越重要的作用。二硫键在治疗性抗体结构和活性关系中起关键作用。二硫键连接和半胱氨酸相关变体被认为是在 mAb 制造和储存期间必须监测的关键质量属性，因为非天然二硫键和聚集体可能导致生物功能和免疫原性丧失。互补决定区 (CDR) 中半胱氨酸残基的存在在人类抗体中很少见，但可能对抗原结合至关重要或对治疗性抗体的开发有害。因此，对其二硫键网络的深入表征是 mAb 可开发性评估的先决条件。质谱 (MS) 技术代表了准确识别二硫键连接性的强大工具。我们在此报告基于 MS 的 IgG4 表征，该 IgG4 在其轻链的 CDR 中包含两个额外的半胱氨酸残基。胰蛋白酶消化后的经典自下而上方法首先允许鉴定含有两个二硫键的二肽。为了进一步研究二硫键桥联二肽的构象异质性，我们进行了离子迁移谱-质谱 (IMS-MS) 实验。我们的研究结果突出了高分辨率 IMS-MS 在处理正在开发的人源化 IgG4 mAb 胰蛋白酶消化后产生的二硫肽构象方面的优势。通过比较 mAb 收集和合成肽的到达时间分布，环状 IMS 提供了对二硫键的明确评估。除了经典的肽图分析外，定性高分辨率 IMS-MS 对鉴定治疗性 mAb 中的二硫键也很有意义。