title: On-line CE-LIF-MS technology for the direct characterization of N-linked glycans from therapeutic antibodies.
abstract: Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.
authors.affiliation: Protein Analytical Chemistry, Genentech Inc., South San Francisco, California 94080, USA. gennaro@gene.com
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Gennaro
authors.forename: Oscar
authors.initials: O
authors.lastname: Salas-Solano
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*
chemical_list: D000911:Antibodies, Monoclonal; D011134:Polysaccharides