Analytical Chemistry Group , Regeneron Pharmaceuticals Inc. , 777 Old Saw Mill River Road , Tarrytown , New York 10591-6707 , United States.
Anal Chem. 2019 Sep 3;91(17):11417-11424. doi: 10.1021/acs.analchem.9b02793. Epub 2019 Aug 15.
Detection and quantitation of homodimer impurities in therapeutic bispecific antibody (bsAb) drug products is essential to support development and quality control (QC) release. LC-MS-based techniques have been frequently applied for this analysis. However, sensitive detection of low-abundance homodimer impurities can still be challenging for regular workflows, which is largely due to the lack of chromatographic resolution between the impurities and the main bsAb species. Here, we report the development of a novel analytical method, which couples mixed-mode size exclusion chromatography (mmSEC) with online native MS detection (mmSEC-MS) for highly sensitive detection and quantitation of homodimer impurities in bsAb samples. Secondary interactions between the protein analytes and the column matrix, which are typically unwanted in SEC applications, are utilized to separate mAb species with similar hydrodynamic volume but different surface characteristics. Using four different bsAbs as testing standards, we demonstrated the versatility of this method in separating homodimer species from bsAb based on either electrostatic interaction or hydrophobic interaction, which was easily achieved by utilizing SEC columns with different properties as well as modulating the salt concentrations. The chromatographic separation between homodimer impurities and bsAb, as achieved by the mmSEC method, was demonstrated to be critical for the improved sensitivity in detecting low-abundance homodimer impurities (LOD from 0.01% to 0.1%). To the best of our knowledge, this newly developed mmSEC-MS method represents the most sensitive MS-based technique in both detection and quantitation of homodimer impurities in bsAb samples.
检测和定量治疗性双特异性抗体 (bsAb) 药物产品中的同二聚体杂质对于支持开发和质量控制 (QC) 放行至关重要。基于 LC-MS 的技术已被频繁用于该分析。然而，对于常规工作流程，仍然难以敏感地检测低丰度同二聚体杂质，这主要是由于杂质和主要 bsAb 物种之间缺乏色谱分辨率。在这里，我们报告了一种新的分析方法的开发，该方法将混合模式尺寸排阻色谱 (mmSEC) 与在线天然 MS 检测 (mmSEC-MS) 相结合，用于高度敏感地检测和定量 bsAb 样品中的同二聚体杂质。蛋白质分析物与柱基质之间的次级相互作用，在 SEC 应用中通常是不希望出现的，用于分离具有相似流体力学体积但表面特性不同的 mAb 物种。使用四种不同的 bsAbs 作为测试标准，我们证明了该方法在根据静电相互作用或疏水相互作用分离同二聚体物种方面的多功能性，这可以通过使用具有不同特性的 SEC 柱以及调节盐浓度来轻松实现。mmSEC 方法实现的同二聚体杂质与 bsAb 之间的色谱分离对于提高检测低丰度同二聚体杂质的灵敏度（LOD 从 0.01%到 0.1%）至关重要。据我们所知，这种新开发的 mmSEC-MS 方法是在 bsAb 样品中检测和定量同二聚体杂质方面最灵敏的 MS 技术。
