Acquisition of a ‘Xevo G2-XS QTof’ Mass Spectrometer and Measurement of Monoclonal Antibody Glycan Heterogeneity by Orthogonal Methodologies

NIPTE will procure a 'Xevo G2-XS QTof' mass spectrometer (Waters) coupled to an Acquity UPLC H-Class PLUS Bio system (Waters) with a fluorescence detector for released glycan analysis. In addition, NIPTE will purchase an Acquity UPLC Glycan BEH Amide column (Waters), access to UNIFI data processing software, and a GlycoWorks RapiFluor MS N-Glycan kit (Waters).  

For this project, samples will be fluorescently labeled with the reagents provided in the RapiFluor MS N-Glycan kit and data will be acquired, processed, and reported via the Waters system. This approach will be used to measure and quantitatively evaluate the glycan profile of a reference monoclonal antibody using glycan standards as well as the glycan profile of a reference monoclonal antibody where the release was performed at a central laboratory.  

An orthogonal method for glycan release will also be conducted using GlykoPrep N-glycan kits (Agilent) designed to be completed on the automated Agilent AssayMAP Bravo liquid handling platform. Two GlykoPrep kits (GlykoPrep 2-AB and GlykoPrep InstantPC) will be procured.  

The data will be acquired, processed, and reported using the same Waters system. Additionally, we will perform orthogonal analyses of glycosylation using intact mass analysis on a Q Exactive mass spectrometer (ThermoFisher), and by LC-MS/MS following trypsin digest of mAb samples with an AccuMAP Low pH protein digestion kit (Promega). A Fusion Lumos Orbitrap (ThermoFisher) will be used for data acquisition. The data will be processed using the Protein Metrics Byos software suites for intact mass and peptide mapping analyses.