The size variant, which can be measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), is a critical quality attribute of monoclonal antibodies (mAbs). The CE-SDS size heterogeneity can hardly be identified by tandem mass spectrometry, which is an intractable obstacle of mAb development and quality control across the industry. We analyzed the purity of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) mAb, an antagonist of the human VEGFR-2, through reduced CE-SDS and observed glycosylated heavy chain heterogeneity. The heterogeneity has potential impact on safety, efficacy, and stability of drugs for clinical use. Therefore, it should be characterized so as to evaluate its potential risk. In order to identify the heterogeneity, we used mass spectrometry to confirm that the molecular size heterogeneity was not due to peptide bond cleavage in the heavy chain. Subsequently, we employed mass-spectrometry–glycosylation profiling and CE-SDS analysis of various glycosidase-treated samples, in addition to the preparation of mAb references with different glycoforms. Ultimately, we demonstrated that the heavy chain heterogeneity was induced by different levels of galactosylation modifications which will potentially impact the efficacy of antibody drugs (i.e., complement-dependent cytotoxicity). In this study, potential risk caused by heavy chain size heterogeneity was evaluated, which addressed the obstacle of mAb development and quality control. Therefore, this study offers a feasible approach for the investigation and identification of heavy chain heterogeneity in reduced CE-SDS, providing a novel strategy for mAb quality control and evaluation.

中文翻译：

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使用毛细管电泳十二烷基硫酸钠 (CE-SDS) 和液相色谱质谱 (LC-MS) 鉴定抗 VEGFR-2 单克隆抗体中的糖基化重链异质性

尺寸变异可通过毛细管电泳十二烷基硫酸钠 (CE-SDS) 进行测量，是单克隆抗体 (mAb) 的关键质量属性。CE-SDS尺寸异质性很难通过串联质谱法识别，这是整个行业mAb开发和质量控制的棘手障碍。我们通过还原 CE-SDS 分析了抗血管内皮生长因子受体 2 (VEGFR-2) mAb（人 VEGFR-2 的拮抗剂）的纯度，并观察了糖基化重链异质性。异质性对临床使用药物的安全性、有效性和稳定性具有潜在影响。因此，应对其进行表征，以评估其潜在风险。为了鉴定异质性，我们使用质谱法来确认分子大小异质性不是由于重链中的肽键断裂造成的。随后，除了制备具有不同糖型的 mAb 参考品外，我们还对各种糖苷酶处理的样品进行了质谱-糖基化分析和 CE-SDS 分析。最终，我们证明重链异质性是由不同水平的半乳糖基化修饰引起的，这可能会影响抗体药物的功效（即补体依赖性细胞毒性）。本研究评估了重链大小异质性带来的潜在风险，解决了单克隆抗体开发和质量控制的障碍。因此，本研究为还原CE-SDS中重链异质性的调查和鉴定提供了可行的方法，为mAb质量控制和评估提供了新的策略。