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
{"title":"Intact NIST monoclonal antibody characterization—Proteoforms, glycoforms—Using CE-MS and CE-LIF","authors":"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","doi":"10.1080/23312009.2018.1480455","DOIUrl":null,"url":null,"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.","PeriodicalId":10640,"journal":{"name":"Cogent Chemistry","volume":" ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/23312009.2018.1480455","citationCount":"20","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cogent Chemistry","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1080/23312009.2018.1480455","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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.