Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017, USA.
Anal Chem. 2010 Apr 15;82(8):3222-30. doi: 10.1021/ac9028856.
A set of related capillary zone electrophoresis (CZE) methods have been developed for the analysis of identity, charge variants, and disulfide isoforms of IgG monoclonal antibodies (mAbs). These methods utilize an uncoated capillary column. The combined use of concentrated zwitterionic (e-amino-caproic acid) buffer and acid flushing was effective in minimizing the adsorption of protein to the inner wall of a bare capillary. Under these conditions, a selective and reproducible separation of multiple IgG1 and IgG2 monoclonal antibodies (mAbs) was obtained with a long capillary column (40 cm effective length), allowing the reliable identification of different mAbs by migration time. A rapid ( approximately 10 min) and selective separation of charged variants of IgG mAbs was attained using a short capillary column (10 cm effective length). Finally, the addition of urea in the separation buffer resulted in the separation of disulfide isoforms of IgG2 mAbs by CZE. CZE methods using an uncoated capillary column offer a versatile, generic, and economical approach to the evaluation of identity, charge heterogeneity, and disulfide isoforms of IgG antibodies.
已经开发出了一套相关的毛细管区带电泳（CZE）方法，用于分析 IgG 单克隆抗体（mAb）的身份、电荷变异体和二硫键异构体。这些方法使用未涂层的毛细管柱。浓缩两性离子（e-氨基己酸）缓冲液和酸冲洗的联合使用可有效减少蛋白质对内壁的吸附。在这些条件下，使用长毛细管柱（有效长度 40 厘米）可实现多种 IgG1 和 IgG2 单克隆抗体（mAb）的选择性和重现性分离，允许通过迁移时间可靠地鉴定不同的 mAb。使用短毛细管柱（有效长度 10 厘米）可快速（约 10 分钟）和选择性地分离 IgG mAb 的电荷变异体。最后，在分离缓冲液中加入脲可导致 IgG2 mAb 的二硫键异构体通过 CZE 分离。使用未涂层毛细管柱的 CZE 方法为评估 IgG 抗体的身份、电荷异质性和二硫键异构体提供了一种通用、经济的方法。
