Rapid Magnetic Bead Based Sample Preparation for Automated and High Throughput N-Glycan Analysis of Therapeutic Antibodies

摘要

Full automation to enable high throughput N-glycosylation profiling and sequencing with good reproducibility is vital to fulfill the contemporary needs of the biopharmaceutical industry and requirements of national regulatory agencies. The most prevalently used glycoanalytical methods of capillary electrophoresis and hydrophilic interaction liquid chromatography, while very efficient, both necessitate extensive sample preparation and cleanup, including glycoprotein capture, N-glycan release, fluorescent derivatization, purification, and preconcentration steps during the process. Currently used protocols to fulfill these tasks require multiple centrifugation and vacuum-centrifugation steps, making liquid handling robot mediated automated sample preparation difficult and expensive. In this paper we report on a rapid magnetic bead based sample preparation approach that enables full automation including all the process phases just in a couple of hours without requiring any centrifugation and/or vacuum centrifugation steps. This novel protocol has been compared to conventional glycan sample preparation strategies using standard glycoproteins (IgG, fetuin, and RNase B) and featured rapid processing time, high release and labeling efficiency, good reproducibility, and the potential of easy automation.