title: Comparison of Traditional 2-AB Fluorescence LC-MS/MS and Automated LC-MS for the Comparative Glycan Analysis of Monoclonal Antibodies.
abstract: Monoclonal antibody therapeutics are a heterogeneous mixture of glycoforms. Multiple methods exist for defining the glycan composition and relative abundance of species present. In the current report, two MS-based methods were compared for their ability to both identify glycans and monitor differences in the glycoprofile. Gross changes in the glycoprofile can be identified either by visual inspection of fluorescence profiles and correlated to glycan identities when coupled with online MS/MS (LC-F-MS/MS) or through extracted ion chromatograms using LC-MS. In the present study, both an LC-F-MS/MS method and an automated LC-MS label free approach were able to identify minor differences in low abundance glycoforms, and data indicate a disparity in glycosylation between the analyzed batches of US and foreign-sourced mAb X. Thus, either method may be useful in characterizing monoclonal antibody therapeutics products and could serve as a potential screening test for understanding process, comparability, similarity, and possibly detecting counterfeit agents.
authors.affiliation: National Institute of Standards and Technology, Biomolecular Measurement Division, Gaithersburg, Maryland, 20899.
authors.forename: John E
authors.initials: JE
authors.lastname: Schiel
authors.affiliation: Division of Pharmaceutical Research, Office of Testing and Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, 20993.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Rogstad
authors.affiliation: Division of Pharmaceutical Research, Office of Testing and Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, 20993.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Boyne
mesh_terms: D053000:Analytic Sample Preparation Methods; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D057205:Automation, Laboratory; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D058110:Counterfeit Drugs / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011786:Quality Control; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D062367:ortho-Aminobenzoates / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D058110:Counterfeit Drugs; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D006023:Glycoproteins; D004364:Pharmaceutical Preparations; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide
keywords: Fluorescence spectroscopy; Glycosylation; LC-MS; Mass spectrometry; Monoclonal antibody