Jooß Kevin, Hühner Jens, Kiessig Steffen, Moritz Bernd, Neusüß Christian
Faculty of Chemistry, Aalen University, Beethovenstr. 1, 73430, Aalen, Germany.
Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg, Germany.
Anal Bioanal Chem. 2017 Oct;409(26):6057-6067. doi: 10.1007/s00216-017-0542-0. Epub 2017 Aug 12.
Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.
毛细管区带电泳（CZE）是一种功能强大的工具，正逐渐应用于单克隆抗体（mAb）电荷变体的分离。质谱（MS）是用于鉴定mAb变体的理想检测方法。在生物制药应用中，存在用于mAb变体定量的优化且经过验证的电解质系统。然而，这些电解质会极大地干扰电喷雾电离（ESI）过程。在此，开发了一种采用机械四通阀接口的中心切割CZE-CZE-MS装置，该装置在第一维使用基于ε-氨基己酸的通用背景电解质，在第二维使用乙酸。以曲妥珠单抗作为模型mAb系统，获得了其电荷变体的无干扰、高精度质谱数据（偏差小于1 Da）。讨论了所获得的质量准确度（百万分之几的低范围）与测量质量和计算质量的关系。在完整的模型抗体中检测到脱酰胺作用，并且在去糖基化水平上显著证实了相关的质量差异。预计CZE-CZE-MS装置适用于多种抗体和电解质系统。因此，它有可能成为用于表征在ESI干扰电解质中分离的抗体变体的有力工具。图形摘要二维毛细管区带电泳质谱用于表征完整单克隆抗体（mAb）电荷变体。一种通用但高度干扰电喷雾的电解质系统用作mAb电荷变体分离的第一维，并通过四通纳升阀与挥发性电解质系统耦合作为第二维。通过这种方式，获得了分离的mAb电荷变体（包括脱酰胺产物）的无干扰且精确的质谱数据。
