title: Rapid characterization of N-linked glycans from secreted and gel-purified monoclonal antibodies using MALDI-ToF mass spectrometry.
abstract: Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell- and antibody class-dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel-purified MAbs produced in NS0 and CHO cells, in which small-scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI-ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi-well formats.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Hansen
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Dickson
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
authors.forename: Gill M
authors.initials: GM
authors.lastname: Stephens
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Sellick
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D011134:Polysaccharides / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D011134:Polysaccharides