Department of Chemical Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
J Am Soc Mass Spectrom. 2023 Dec 6;34(12):2801-2810. doi: 10.1021/jasms.3c00308. Epub 2023 Nov 23.
Assessment of product quality attributes such as charge heterogeneity is an upmost requisite for the release of a monoclonal antibody (mAb). Analytical techniques, such as cation-exchange chromatography (CEX), accomplish this, causing the mAb to separate into acidic, main species, and basic variants. Here, an online volatile-salt-containing two-dimensional liquid chromatography (2D-LC) method coupled with mass spectrometry (MS) was performed to characterize the charge heterogeneity of mAbs using CEX chromatography in the first dimension (D) and anion-exchange chromatography (AEX) in the second dimension (D). The main peak of the CEX profile of D was transferred through a 2D heart-cut method to D for further analysis by the AEX-MS method. In the CEX method, mAb A showed 10 distinct variants, while the AEX method resulted in eight variants. However, a total of 13 variants were successfully resolved for mAb A in the 2D method. Similarly, mAb B exhibited seven variants in the CEX method and four variants in the AEX method, but the 2D-LC method revealed a total of nine variants for mAb B. Likewise, mAb C displayed seven variants in CEX and seven variants in AEX, whereas the 2D-LC method unveiled a total of 11 variants for mAb C. Additionally, native MS analysis revealed that the resolved charge variants were identified as amidation, oxidation, and isomerization of Asp variants in the main peak, which were not resolved in stand-alone methods. The present study demonstrates how 2D-LC can assist in identifying minor variations in charge distribution or conformation of mAb variants that would otherwise not be picked up by a single analytical method alone.
评估产品质量属性，如荷质比不均一性，是放行单克隆抗体（mAb）的首要条件。分析技术，如阳离子交换色谱（CEX），可实现这一目的，使 mAb 分离为酸性、主要物种和碱性变体。在此，采用在线含挥发性盐二维液相色谱（2D-LC）与质谱（MS）联用技术，用 CEX 色谱在第一维（D）和阴离子交换色谱（AEX）在第二维（D）对 mAb 的荷质比不均一性进行了表征。D 中 CEX 图谱的主峰通过 2D 中心切割方法转移到 D 中，用 AEX-MS 方法进一步分析。在 CEX 方法中，mAb A 显示出 10 个明显的变体，而 AEX 方法则产生 8 个变体。然而，在 2D 方法中，成功解析了 mAb A 的总共 13 个变体。同样，mAb B 在 CEX 方法中显示出 7 个变体，在 AEX 方法中显示出 4 个变体，但 2D-LC 方法总共揭示了 mAb B 的 9 个变体。同样，mAb C 在 CEX 中显示出 7 个变体，在 AEX 中显示出 7 个变体，而 2D-LC 方法则揭示了 mAb C 的总共 11 个变体。此外，天然 MS 分析表明，解析出的荷质比变体被鉴定为主要峰中 Asp 变体的酰胺化、氧化和异构化，而这些在单独的方法中无法解析。本研究表明，2D-LC 如何有助于识别 mAb 变体电荷分布或构象的微小变化，否则仅凭单一分析方法无法检测到。
