title: HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals.
abstract: In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.
authors.affiliation: Sandoz GmbH, Biochemiestrasse 10, 6250, Kundl, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Melmer
authors.forename: Thomas
authors.initials: T
authors.lastname: Stangler
authors.forename: Mark
authors.initials: M
authors.lastname: Schiefermeier
authors.forename: Werner
authors.initials: W
authors.lastname: Brunner
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Toll
authors.forename: Alfred
authors.initials: A
authors.lastname: Rupprechter
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lindner
authors.forename: Andreas
authors.initials: A
authors.lastname: Premstaller
mesh_terms: D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D062367:ortho-Aminobenzoates / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide