Intact NIST monoclonal antibody characterization—Proteoforms, glycoforms—Using CE-MS and CE-LIF

Chien-Hsun Chen, Hua‐tao Feng, R. Guo, Pingjing Li, A. K. Laserna, Ya Ji, Bao Hui Ng, Sam F. Y. Li, S. Khan, A. Paulus, Shiaw-Min Chen, A. Karger, M. Wenz, Daniel Lopez Ferrer, A. Huhmer, A. Krupke
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引用次数: 20

Abstract

Abstract Determining and linking the structural heterogeneity of recombinant antibodies to function is critical in the biopharmaceutical industry. We introduce a new microfluidic capillary electrophoresis—mass spectrometry (μCE-MS) approach to characterize intact monoclonal antibody (mAb) and simultaneously quantifying distinct variants. Our MS analysis of intact NIST mAb (RM8671) shows 18 variants identified amongst proteolytic and glycolytic modifications with a range of relative abundances between 0.1% and 100%. In order to verify our quantitative MS results, we used an established system based on capillary electrophoresis—with laser induced fluorescence (CE-LIF) for profiling the N-glycans. All major glycans were identified and further substantiated by exoglycosidase digestion. Interestingly, the µCE-MS analysis of intact NIST mAb consistently yielded higher amounts of G2FG2F-Hex glycoform (~3.4%), whereas the CE-LIF analysis indicates that only 1.4% of G2F-Gal is present. Therefore, the additional hexose adduct observed in µCE-MS may have been the glycation product of the mAb. Further analysis of deglycosylated mAb with µCE-MS made it possible to reveal the glycation with 10.5% of one hexose product and 4.9% of two hexose product in the intact deglycosylated antibody. An integrated solution using two orthogonal and complementary techniques for characterizing antibody glycosylation is provided here.
完整的NIST单克隆抗体表征——蛋白质、糖型——使用CE-MS和CE-LIF
摘要在生物制药行业中,确定重组抗体的结构异质性并将其与功能联系起来至关重要。我们介绍了一种新的微流体毛细管电泳-质谱法(μCE-MS)来表征完整的单克隆抗体(mAb),同时量化不同的变体。我们对完整NIST mAb(RM8671)的MS分析显示,在蛋白水解和糖酵解修饰中鉴定出18种变体,相对丰度范围在0.1%至100%之间。为了验证我们的定量MS结果,我们使用了一个基于毛细管电泳的既定系统——激光诱导荧光(CE-LIF)来分析N-聚糖。所有主要聚糖均已鉴定,并通过糖苷酶外切进一步证实。有趣的是,对完整NIST mAb的µCE-MS分析始终产生更高量的G2FG2F Hex糖型(~3.4%),而CE-LIF分析表明仅存在1.4%的G2F-Gal。因此,在µCE-MS中观察到的额外己糖加合物可能是mAb的糖基化产物。用µCE-MS对脱糖基化mAb进行进一步分析,可以揭示完整脱糖基抗体中10.5%的一种己糖产物和4.9%的两种己糖产品的糖基化。本文提供了一种使用两种正交和互补技术表征抗体糖基化的整合溶液。
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Cogent Chemistry
Cogent Chemistry CHEMISTRY, MULTIDISCIPLINARY-
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