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.