title: Fully Automated Sample Preparation for Ultrafast N-Glycosylation Analysis of Antibody Therapeutics.
abstract: There is a growing demand in the biopharmaceutical industry for high-throughput, large-scale N-glycosylation profiling of therapeutic antibodies in all phases of product development, but especially during clone selection when hundreds of samples should be analyzed in a short period of time to assure their glycosylation-based biological activity. Our group has recently developed a magnetic bead-based protocol for N-glycosylation analysis of glycoproteins to alleviate the hard-to-automate centrifugation and vacuum-centrifugation steps of the currently used protocols. Glycan release, fluorophore labeling, and cleanup were all optimized, resulting in a <4 h magnetic bead-based process with excellent yield and good repeatability. This article demonstrates the next level of this work by automating all steps of the optimized magnetic bead-based protocol from endoglycosidase digestion, through fluorophore labeling and cleanup with high-throughput sample processing in 96-well plate format, using an automated laboratory workstation. Capillary electrophoresis analysis of the fluorophore-labeled glycans was also optimized for rapid (<3 min) separation to accommodate the high-throughput processing of the automated sample preparation workflow. Ultrafast N-glycosylation analyses of several commercially relevant antibody therapeutics are also shown and compared to their biosimilar counterparts, addressing the biological significance of the differences.
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary.
authors.forename: Marton
authors.initials: M
authors.lastname: Szigeti
authors.affiliation: Sciex Separations, Brea, CA, USA.
authors.forename: Clarence
authors.initials: C
authors.lastname: Lew
authors.affiliation: Sciex Separations, Brea, CA, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Roby
authors.affiliation: Horváth Csaba Laboratory of Bioseparation Sciences, University of Debrecen, Debrecen, Hungary MTA-PE Translational Glycomics Research Group, University of Pannonia, Veszprem, Hungary a.guttman@neu.edu.
authors.forename: Andras
authors.initials: A
authors.lastname: Guttman
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D057205:Automation, Laboratory / Q000379:methods*; D001688:Biological Products / Q000737:chemistry*; D001702:Biopharmaceutics / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D006023:Glycoproteins / Q000737:chemistry*; D008280:Magnetics; D008863:Microspheres; D011134:Polysaccharides / Q000032:analysis*; D013048:Specimen Handling / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
chemical_list: D000906:Antibodies; D001688:Biological Products; D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides
keywords: N-glycosylation; antibody therapeutics; automation; capillary electrophoresis; magnetic beads