title: Fast, robust and high-resolution glycosylation profiling of intact monoclonal IgG antibodies using nanoLC-chip-QTOF.
abstract: Optimal glycosylation of immunoglobulins is essential in the generation of therapeutic biologicals with respect to efficacy, pharmacokinetics and immunogenic properties. This challenge in the field of biopharmaceuticals requires technologies for fast, robust and quantitative analysis of glycosylation. Current analyses of monoclonal antibody glycosylation are proteolysis-based mass spectrometry methods, which provide detailed structural information, but suffer a number of drawbacks such as lengthy sample preparation with the possibility to introduce artifacts. Here, we describe a fast, robust and high-resolution nanoLC-chip-QTOF method for quantitative analysis of intact monoclonal IgG glycosylation profiling. The method is able to detect hypoglycosylation, i.e. the lack of whole glycans, which is an important advantage over the well-established methods for free N-glycan or glycopeptide analysis. Moreover, the method is highly amenable to automation and because no digestion steps are involved, it provides direct relative quantitative information of both glycans on each IgG attachment site. We demonstrate that the ease and robustness make this technique ideally suited for quality control of the production process of mAb biopharmaceuticals, and provides new opportunities to study the clinical impact of mAb-glycosylation in patients with monoclonal gammopathies.
authors.affiliation: Laboratory Medical Immunology, Department of Laboratory Medicine, Radboudumc, Nijmegen, The Netherlands.
authors.forename: Joannes F M
authors.initials: JF
authors.lastname: Jacobs
authors.affiliation: Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboudumc, Nijmegen, The Netherlands.
authors.forename: Ron A
authors.initials: RA
authors.lastname: Wevers
authors.affiliation: Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboudumc, Nijmegen, The Netherlands; Department of Neurology, Radboudumc, Nijmegen, The Netherlands.
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Lefeber
authors.affiliation: Translational Metabolic Laboratory, Department of Laboratory Medicine, Radboudumc, Nijmegen, The Netherlands. Electronic address: monique.vanscherpenzeel@radboudumc.nl.
authors.forename: Monique
authors.initials: M
authors.lastname: van Scherpenzeel
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry*; D013058:Mass Spectrometry; D036103:Nanotechnology
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G
keywords: Cryoglobulin; Intact protein analysis; Monoclonal Gammopathy; QTOF high resolution mass spectrometry; Therapeutic monoclonal antibodies