title: Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry.
abstract: A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical Abstract Flowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass spectrometry (MS).
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, 27100, Pavia, Italy.|Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Sara
authors.initials: S
authors.lastname: Tengattini
authors.affiliation: Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands. e.dominguezvega@vu.nl.
authors.forename: Elena
authors.initials: E
authors.lastname: Domínguez-Vega
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, 27100, Pavia, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Temporini
authors.affiliation: Department of Drug Sciences, University of Pavia, via Taramelli 12, 27100, Pavia, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Terreni
authors.affiliation: Division of BioAnalytical Chemistry, Department of Chemistry and Pharmaceutical Sciences, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
authors.forename: Govert W
authors.initials: GW
authors.lastname: Somsen
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000276:immunology; D000327:Adsorption; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D019075:Electrophoresis, Capillary / Q000379:methods*; D006001:Glycoconjugates / Q000737:chemistry* / Q000276:immunology; D006031:Glycosylation; D006583:Hexadimethrine Bromide / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000276:immunology; D014376:Tuberculosis / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D032581:Tuberculosis Vaccines / Q000737:chemistry* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D006001:Glycoconjugates; C501710:TB10.4 antigen, Mycobacterium tuberculosis; D032581:Tuberculosis Vaccines; D006583:Hexadimethrine Bromide; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis
keywords: Antigenic proteins; CE-MS; Glycoconjugate vaccines; Intact protein analysis; Neo-glycoproteins; Protein characterization