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

Autor: Pingjing Li, Michael H. Wenz, Aran Paulus, Shaheer H. Khan, Andreas Krupke, Ya Ji, Daniel Lopez Ferrer, Huatao Feng, Andreas Huhmer, Chien-Hsun Chen, Sam Fong Yau Li, Achim E. Karger, Anna Karen Carrasco Laserna, Shiaw-Min Chen, Rui Guo, Bao Hui Ng
Jazyk: angličtina
Rok vydání: 2018
Předmět:
Zdroj: Cogent Chemistry, Vol 4, Iss 1 (2018)
ISSN: 2331-2009
Popis: 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.
Databáze: OpenAIRE
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