Sequence variants (SV) in protein bio therapeutics can be categorized as unwanted impurities and may raise serious concerns in efficacy and safety of the product. Early detection of specific sequence modifications, that can result in altered physicochemical and or biological properties, is therefore desirable in product manufacturing. Because of their low abundance, and finite resolving power of conventional analytical techniques, they are often overlooked in early drug development. Here, we present a case study where trace amount of a sequence variant is identified in a monoclonal antibody (mAb) based therapeutic protein by LC–MS/MS and the structural and functional features of the SV containing mAb is assessed using appropriate analytical techniques. Further, a very sensitive selected reaction monitoring (SRM) technique is developed to quantify the SV which revealed both prominent and inconspicuous nature of the variant in process chromatography. We present the extensive characterization of a sequence variant in protein biopharmaceutical and first report on control of sequence variants to < 0.05% in final drug product by utilizing SRM based mass spectrometry method during the purification steps.

蛋白质生物疗法中的序列变异 (SV) 可归类为不需要的杂质，并可能引起对产品功效和安全性的严重关注。因此，在产品制造中需要早期检测可能导致物理化学和/或生物学特性改变的特定序列修饰。由于它们的低丰度和传统分析技术的有限分辨率，它们在早期药物开发中经常被忽视。在这里，我们提出了一个案例研究，其中通过 LC–MS/MS 在基于单克隆抗体 (mAb) 的治疗性蛋白质中鉴定出痕量序列变体，并使用适当的分析技术评估了含有 mAb 的 SV 的结构和功能特征。更远，开发了一种非常灵敏的选择反应监测 (SRM) 技术来量化 SV，该 SV 揭示了过程色谱中变异的突出和不显眼的性质。我们展示了蛋白质生物制药中序列变异的广泛表征，并首次报告了在纯化步骤中利用基于 SRM 的质谱法将最终药物产品中的序列变异控制在 < 0.05%。