title: Development and qualification of an antibody rapid deglycosylation method.
abstract: N-linked glycosylation can influence the biological activity and safety of an antibody as well as be a measure of the consistency of the production process. The released N-glycans is an important part of the development of a therapeutic antibody. The traditional method for N-glycan analysis requires complex and laborious sample preparation and lengthy analysis time. Two preparation steps with limited control are removal of the antibody backbone by ice-cold ethanol precipitation and water removal before 2-AB fluorescent dye labeling. Simplification of the sample preparation and better control of key steps that allows for the characterization/quantitation of glycans during all stages of development of a therapeutic antibody is desired. Recently Prozyme introduced a rapid deglycosylation kit and a rapid tagging kit that address some of these issues. The deglycosylation kit immobilizes the antibody on a membrane, thereby eliminating the precipitation step. An instant fluorescent tag kit eliminates the water removal before the 2-AB labeling step. In addition use of a new chromatography column can improve the glycan resolution and shorten the analysis time. The evaluation and qualification of the Rapid Deglycosylation Kit (RDK) and instant 2-AB tagging with the improved chromatography are highlighted.
authors.affiliation: Analytical R&D, Pfizer BioTherapeutics R&D Pharmaceutical Sciences, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA. Kenneth.s.cook@pfizer.com
authors.forename: K Steven
authors.initials: KS
authors.lastname: Cook
authors.forename: Kevin
authors.initials: K
authors.lastname: Bullock
authors.forename: Timothy
authors.initials: T
authors.lastname: Sullivan
mesh_terms: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006031:Glycosylation; D006801:Humans; D007158:Immunologic Techniques; D011134:Polysaccharides / Q000302:isolation & purification; D011489:Protein Denaturation
chemical_list: D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D011134:Polysaccharides