Rapid, direct identification and quantitation of protein charge variants, and assessment of critical quality attributes with high sensitivity are important drivers required to accelerate the development of biotherapeutics. We describe the use of an enhanced microfluidic chip-based integrated imaged capillary isoelectric focusing-mass spectrometry (icIEF-MS) technology to assess multiple quality attributes of intact antibodies in a single run. Results demonstrate comprehensive detection of multiple charge variants of an aglycosylated knob-into-hole bispecific antibody. Upfront, on-chip separation by icIEF coupled to MS provides the orthogonal separation required to resolve and identify acidic posttranslational modifications including difficult-to-detect deamidation and glycation events at the intact protein level. In addition, on-chip UV detection enables pI determination and relative quantitation of charge isoforms. Six charge variant peaks were resolved by icIEF, mobilized toward the on-chip electrospray tip and directly identified by in-line icIEF-MS using a connected quadrupole time-of-flight mass spectrometer. In addition to acidic charge variants, basic variants were identified as C-terminal lysine, N-terminal cyclization, proline amidation, and the combination of modifications (not typically identified by other intact methods), including lysine and one or two hexose additions. Nonspecific chain cleavages were also resolved, along with their acidic charge variants, demonstrating highly sensitive and comprehensive intact antibody multi-attribute characterization within a 15-min run time.

中文翻译：

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通过基于微流控芯片的集成 icIEF-MS 技术对完整双特异性抗体进行快速多属性表征

蛋白质电荷变异体的快速、直接识别和定量，以及对关键质量属性的高灵敏度评估，是加速生物治疗药物开发所需的重要驱动力。我们描述了使用增强的基于微流控芯片的集成成像毛细管等电聚焦质谱 (icIEF-MS) 技术在单次运行中评估完整抗体的多种质量属性。结果表明，可以全面检测非糖基化的旋钮入孔双特异性抗体的多个电荷变体。通过 icIEF 与 MS 联用进行的前期片上分离提供了解决和识别酸性翻译后修饰所需的正交分离，包括完整蛋白质水平上难以检测的脱酰胺和糖基化事件。此外，电荷异构体的测定和相对定量。六个电荷变体峰由 icIEF 解析，移向片上电喷雾尖端，并使用连接的四极杆飞行时间质谱仪通过在线 icIEF-MS 直接识别。除了酸性电荷变体外，基本变体被鉴定为 C 末端赖氨酸、N 末端环化、脯氨酸酰胺化和修饰组合（通常未通过其他完整方法鉴定），包括赖氨酸和一个或两个己糖添加。非特异性链断裂及其酸性电荷变体也得到解决，证明了在 15 分钟的运行时间内高度灵敏和全面的完整抗体多属性表征。