等电聚焦
化学
色谱法
分辨率(逻辑)
质谱法
分析化学(期刊)
等电点
电泳
电喷雾电离
计算机科学
生物化学
人工智能
酶
作者
Jun Dai,Qiangwei Xia,Chengjie Ji
出处
期刊:Methods in molecular biology
日期:2022-01-01
卷期号:: 55-65
被引量:3
标识
DOI:10.1007/978-1-0716-2325-1_6
摘要
Monoclonal antibodies (mAbs) are one of the most widely used types of protein therapeutics. Charge variants are important quality attributes for evaluating developability, activity, and safety for mAb therapeutics. Here, we report a novel online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for mAb charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. Key factors that enable online CIEF-MS include effective capillary electrophoresis-MS (CE-MS) interface with enhanced sensitivity, utilization of MS-friendly electrolytes, beneficial effects of glycerol that reduces non-CIEF electrophoretic mobility and limits band broadening, appropriate ampholyte type and concentration selection for balanced separation resolution and MS detection sensitivity, optimized sheath liquid composition to realize high-resolution CIEF separation and effective MS electrospray ionization, as well as judiciously selected CIEF running parameters. The fundamental premise of CIEF has been verified by the linear correlation between isoelectric point (pI) values and migration time using a mixture of pI markers. By achieving high separation resolutions that are similar as those obtained from imaged CIEF (iCIEF), this method successfully provides highly sensitive MS identification for intact mAb charge variants. Furthermore, a middle-up sample treatment workflow can be adopted to provide in-depth charge variant analysis at subunit level for mAbs with complex charge heterogeneity. The mAb subunit CIEF-MS reveals the source of charge variant with enhanced resolution on both CIEF separation and MS spectra. This novel CIEF-MS method is a valuable tool with distinct advantage for objective and accurate assessment of charge heterogeneity of protein therapeutics.
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