Novel Affinity Chromatography Ligand Utilizing the Biological Interaction Between the N‐linked glycans on Monoclonal Antibodies and the FcγIIIA Receptor
Authors: Melideo Scott L. , Vitrac Heidi , Evangelisto James , Garrett Charley , Hevner Abbie , Stewart Jordan , Evans William E. , Kervinen Jukka 
Published: May 2022

Glycosylation is one of the major post‐translational modifications (PTMs) in proteins, with significant effects on protein folding, conformation, stability, and activity. Glycosylation is particularly important for therapeutic protein drugs, such as monoclonal antibodies (mAbs), as it can profoundly impact their pharmacological properties, stability, and safety. Therefore, understanding the inherent heterogeneity of mAb glycoforms is an integral part of their manufacturing processes and quality control.Most marketed mAbs belong to the immunoglobulin G1 (IgG1), IgG2, or IgG4 subclass, carrying N‐glycosylation at the conserved asparagine position 297 (Asn‐297) in the CH2 domain of the Fc region. N‐glycosylation is critical for the Fc receptor binding and consequently affects key antibody effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC). However, assessing ADCC activity using a cell‐based assay remains a complicated, time‐consuming, and expensive task for laboratories.Here, we present a novel workflow leveraging FcγR‐IIIA biology to identify N‐glycan compositions and how to chromatographically determine changes in glycosylation in a time and cost‐effective manner. The TSKgel® FcR‐IIIA analytical and semi‐preparative columns separate mAbs into multiple subsets based on their affinity to the FcγR‐IIIA ligand. Utilizing an increasingly acidic pH gradient, mAb glycoforms are eluted from the column in an order that correlates to their ADCC activity. The analytical TSKgel FcR‐IIIA‐NPR column provides rapid assessment of a mAb's glycoform distribution, whereas the semipreparative TSKgel FcR‐IIIA‐5PW column enables collection of the peaks for more complex orthogonal released N‐glycan analyses and N‐glycosylation profiling using peptide mapping technique.With several mAbs containing various glycosylation patterns, we directly correlated the N‐glycan nature, structure, content, and elution profile on the FcR‐IIIA columns. Furthermore, we present a direct correlation between higher ADCC activity and binding affinity on the FcR‐IIIA columns, indicating that the complexity of the mAb glycosylation pattern is reflected in the observed FcR‐IIIA elution profile. Finally, employing both qualitative and quantitative assessments, we quantified the impact of individual N‐glycan structure and composition on mAb binding to the FcR ligand, providing an additional dimension to our understanding of the FcR‐IIIA columns.This in‐depth correlation analysis provides improved tools to predict, understand, and analyze changes in FcR‐IIIA elution profiles and how changes in the N‐glycan composition influence them. Altogether, this study describes a new framework for utilizing the TSKgel FcR‐IIIA columns for glycosylation profiling, providing a powerful way to simultaneously capture, profile, and characterize the glycosylation state of a given mAb of interest in a comprehensive manner.