Benchmarking glycoform-resolved affinity separation – mass spectrometry assays for studying FcγRIIIa binding

• DOI: 10.3389/fimmu.2024.1347871
• Publication Date: 2024-02-26T04:42:41Z
• Authors: Christoph Gstöttner; Steffen Lippold; Michaela Hook; Feng Yang; Markus Haberger; Manfred Wuhrer; David Falck; Tilman Schlothauer; Elena Domínguez-Vega
• Keywords: FcγRIIIA receptor; 0303 health sciences; glycosylation; Fc gamma RIIIA receptor; Immunology; Receptors, IgG; Antibodies, Monoclonal; RC581-607; affinity capillary electrophoresis; Mass Spectrometry; affinity chromatography; Benchmarking; 03 medical and health sciences; monoclonal antibody; Polysaccharides; affinity interaction; Immunoglobulin G; Immunologic diseases. Allergy; Mannose; mass spectrometry

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan with receptor, differences in glycosylation can drastically influence binding affinity. Understanding differential of mAb glycoforms is a very important, yet challenging task due co-existence multiple sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated mass spectrometry (MS) provide glycoform-resolved profiles proteins based on their either dissociation or equilibrium constants. To cross-validate ranking provided by these complementary novel approaches, both techniques were benchmarked using same constructs. Both approaches able assess - glycoform selective manner showed clear increase fully versus hemi-fucosylated Also, other features, increasing elevated galactosylation high mannose consistent. We further applied towards F158 allotype FcγRIIIa, which was not reported before. similar profile compared V158 receptor strongest afucosylation only slight additional galactosylation. decrease variant. Overall, comparable results line orthogonal methods proving capabilities separation-based study FcγR glycoforms.