Characterization of charge heterogeneity in monoclonal antibodies (mAbs) is needed during developability assessment and downstream development of drug candidates. Charge heterogeneity can come from post-translational modifications like deamidation, isomerization, and sialylation. Elucidation of charge variants with mass spectrometry (MS) has historically been challenging. Due to the nonvolatility and high ionic strength of conventional buffer systems, labor-intensive offline fractionation followed by MS analysis is routinely used. Here, we describe an alternative strategy that directly couples strong cation exchange (SCX) chromatography to high-resolution Orbitrap MS for online native MS analysis (SCX-MS). A combined pH and salt gradient was used for universal separation of mAbs from a wide range of pI values (6.38 ~ 9.2), including infliximab (Remicade®, chimeric IgG1/kappa), NISTmab (humanized IgG1/kappa) and trastuzumab (Herceptin®, humanized IgG1/kappa), without tailoring of chromatographic profiles. Liquid chromatography and MS parameters were optimized to achieve high-quality spectra and enhanced detection of low abundant species under high flow rate conditions. Genedata Expressionist, a vendor agnostic software, was used for data processing. This integrated strategy allows unbiased characterization of numerous charge variant species and low molecular weight fragments (<0.05%) without post-column flow splitting. The application was further expanded with middle-up approaches for subdomain analysis, which demonstrated the versatility of the strategy for analysis of various construct types. With our analysis of mAbs during developability assessment and forced degradation studies, which aimed at assessing potential critical quality attributes in antibody drug molecules, we provide, for the first time, direct visualization of molecular alterations of mAbs at intact level. Furthermore, strong correlation was observed between this novel MS approach and analysis by capillary isoelectric focusing.

中文翻译：

---

将强阳离子交换色谱与自然质谱联用，以高通量在线表征治疗性单克隆抗体的电荷异质性。

在可开发性评估和候选药物的下游开发过程中，需要鉴定单克隆抗体（mAb）中电荷异质性。电荷异质性可能来自翻译后修饰，例如脱酰胺，异构化和唾液酸化。从历史上讲，用质谱（MS）阐明电荷变体一直是一项挑战。由于常规缓冲系统的不挥发性和高离子强度，通常使用劳动密集型离线分馏，然后进行MS分析。在这里，我们描述了将强阳离子交换（SCX）色谱直接耦合到高分辨率Orbitrap MS的在线本机MS分析（SCX-MS）的替代策略。pH和盐梯度的组合可用于从广泛的pI值（6.38〜9.2）中通用分离mAb，包括英夫利昔单抗（Remicade®，嵌合IgG1 / kappa），NISTmab（人源化IgG1 / kappa）和曲妥珠单抗（人源化IgG1 / kappa），而无需调整色谱图。优化了液相色谱和MS参数，以实现高质量的光谱并在高流速条件下增强了对低丰度物种的检测。供应商不可知软件Genedata Expressionist用于数据处理。这种集成策略可以无偏地表征许多电荷变体和低分子量片段（<0.05％），而无需进行柱后流分离。该应用程序使用中上方法进行了子域分析，从而进一步扩展了该应用程序，这证明了该策略可用于分析各种构造类型的多功能性。通过在可开发性评估和强制降解研究过程中对mAb的分析，旨在评估抗体药物分子中潜在的关键质量属性，我们首次提供了完整水平下mAb分子变化的直接可视化。此外，在这种新颖的质谱方法与毛细管等电聚焦分析之间观察到很强的相关性。