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

Abstract

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.

Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

“…For these linkage position studies the substrate was labeled with a chromophore detected by fluorescence. The fluorescently conjugated oligosaccharides and glycans were labeled as previously described 34 with slight modifications. Glycan labeling was accomplished using 100 mM 8-aminopyrene-1,3,6-trisulfonic acid in 20% acetic acid for a reaction of 7 nmol glycan:250 nmol dye in a total reaction volume of 5 μL.…”

Evaporative fluorophore labeling of carbohydrates via reductive amination

“…Procedures reported in the literature apply comparable but not unified reaction conditions and reagent volumes for fluorescent sugar labeling [10], most of them suggesting overnight incubation at 37°C [29,33] or several hours of reaction times at higher temperatures (50°C) [34].…”

Advances in mass spectrometry‐based glycomics

“…Conventionally, lectin‐based enrichment , hydrophilic interaction chromatography (HILIC) , porous graphitized carbon (PGC) and hydrazide based methods , are extensively used for the purification and enrichment of glycans. Over the past 5 years, several studies reported the use of a variety of materials to facilitate high‐throughput sample preparation .…”