Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

中文翻译：

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通过SEC-MS对治疗性单克隆抗体的构象评估：阐明分析偏差，以应用于质量控制。

与治疗性单克隆抗体（mAbs）降解有关的免疫原性仍是其治疗功效和安全性的主要问题。因此，必须使用能够表征和检测mAb降解的分析方法。在这项研究中，建议同时进行体积排阻色谱（SEC）与天然质谱（MS）和荧光检测（FLD）的耦合，以检测降解的治疗性单克隆抗体和天然过程中可能发生的结构变化（例如二聚化，变性）的偏差。多发性硬化症。在不同的流动相条件（例如组成，pH，有机溶剂等）和质谱参数（例如气体温度，CID能量等）下，已对英夫利昔单抗的行为进行了全面研究。已经定义了避免人工变性和/或二聚化的实验条件。我们还证明了在开发条件下英夫利昔单抗对其生物学靶标TNFα的亲和力得以保留。此外，使用这种方法的二聚体，可以在长期保存后的曲妥珠单抗样品中检测到变性的单体和片段。通过将SEC与离子迁移质谱联用作为检测变性单体的正交方法，可以证实这些结果。