We report a new online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. To develop a successful, reliable CIEF-MS method for mAb, we have selected and optimized many critical, interrelating reagents and parameters that include (1) MS-friendly anolyte and catholyte; (2) a glycerol enhanced sample mixture that reduced non-CIEF electrophoretic mobility and band broadening; (3) ampholyte selected for balancing resolution and MS sensitivity; (4) sheath liquid composition optimized for efficient focusing, mobilization, and electrospray ionization; (5) judiciously selected CIEF running parameters including injection amount, field strength, and applied pressure. The fundamental premise of CIEF was well maintained as verified by the linear correlation (R² = 0.99) between pI values and migration time using a mixture of pI markers. In addition, the charge variant profiles of trastuzumab, bevacizumab, infliximab, and cetuximab, obtained using this CIEF-MS method, were corroborated by imaged CIEF-UV (iCIEF-UV) analyses. The relative standard deviations (RSD) of absolute migration time of pI markers were all less than 5% (n = 4). Triplicate analyses of bevacizumab showed RSD less than 1% for relative migration time to an internal standard and RSD of 7% for absolute MS peak area. Moreover, the antibody charge variants were characterized using the online intact MS data. To the best of our knowledge, this is the first time that direct online MS detection and characterization were achieved for mAb charge variants resolved by CIEF as indicated by a well-established linear pH gradient and correlated CIEF-UV charge variant profiles.

中文翻译：

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毛细管等电聚焦质谱法用于完整单克隆抗体电荷变体的分离和在线表征

我们报告了一种新的在线毛细管等电聚焦质谱（CIEF-MS）方法，用于使用电动泵鞘流纳米喷雾离子源和飞行时间MS以及压力辅助化学动员的单克隆抗体（mAb）电荷变异分析。为了开发成功，可靠的单抗CIEF-MS方法，我们选择并优化了许多关键的，相互关联的试剂和参数，其中包括：（1）MS友好型阳极电解液和阴极电解液；（2）甘油增强的样品混合物，降低了非CIEF的电泳迁移率和谱带展宽；（3）选择两性电解质以平衡分离度和MS灵敏度；（4）为有效聚焦，动员和电喷雾电离而优化的鞘液组合物；（5）明智地选择CIEF运行参数，包括注入量，场强，和施加压力。线性相关性证明，CIEF的基本前提得到了很好的维护（使用p I标记的混合物在p I值和迁移时间之间的R ² = 0.99）。此外，通过成像CIEF-UV（iCIEF-UV）分析证实了使用此CIEF-MS方法获得的曲妥珠单抗，贝伐单抗，英夫利昔单抗和西妥昔单抗的电荷变异特征。p I标记的绝对迁移时间的相对标准偏差（RSD）均小于5％（n= 4）。贝伐单抗一式三份的分析显示，相对于内标物的相对迁移时间，RSD小于1％，绝对MS峰面积的RSD小于7％。此外，使用在线完整MS数据对抗体电荷变体进行了表征。据我们所知，这是首次通过CIEF解析的mAb电荷变体实现直接在线MS检测和表征，这已通过完善的线性pH梯度和相关的CIEF-UV电荷变体图谱表明。