Imaged capillary isoelectric focusing (iCIEF) has emerged as an important technique for therapeutic monoclonal antibody (mAb) charge heterogeneity analysis in the biopharmaceutical context, providing imaged detection and quantitation by UV without a mobilization step. Besides quantitation, the characterization of separated charge variants ideally directly by online electrospray ionization–mass spectrometry (ESI–MS) is crucial to ensure product quality, safety, and efficacy. Straightforward direct iCIEF–MS coupling combining high separation efficiency and quantitative results of iCIEF with the characterization power of MS enables deep characterization of mAb charge variants. A short technical setup and optimized methodical parameters (30 nl/min mobilization rate, 2%–4% ampholyte concentration, 0.5–2 mg/ml sample concentration) allow successful mAb charge variant peak assignment from iCIEF to MS. Despite a loss of separation resolution during the transfer, separated intact mAb charge variants, including deamidation as well as major and minor glycoforms even from low abundant charge variants, could be characterized by online ESI–MS with high precision. The presented setup provides a large potential for mAb charge heterogeneity characterization in biopharmaceutical applications.

中文翻译：

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通过 iCIEF 与质谱联用 (iCIEF–MS) 表征治疗性 mAb 电荷异质性

成像毛细管等电聚焦 (iCIEF) 已成为生物制药领域治疗性单克隆抗体 (mAb) 电荷异质性分析的一项重要技术，无需动员步骤即可通过紫外线进行成像检测和定量。除了定量之外，最好直接通过在线电喷雾电离-质谱 (ESI-MS) 对分离的电荷变体进行表征对于确保产品质量、安全性和有效性至关重要。直接的 iCIEF-MS 耦合将 iCIEF 的高分离效率和定量结果与 MS 的表征能力相结合，可以对 mAb 电荷变体进行深入表征。简短的技术设置和优化的方法参数（30 nl/min 流动率，2%–4% 两性电解质浓度，0. 5–2 mg/ml 样品浓度）允许从 iCIEF 到 MS 的 mAb 电荷变体峰分配成功。尽管在转移过程中分离分辨率有所下降，但分离的完整 mAb 电荷变体，包括脱酰胺以及主要和次要糖型，甚至来自低丰度电荷变体，都可以通过在线 ESI-MS 进行高精度表征。所提出的设置为 mAb 电荷异质性表征在生物制药应用中提供了巨大的潜力。