Fc gamma receptor IIIb binding of individual antibody proteoforms resolved by affinity chromatography-mass spectrometry

Lippold, S.; Knaupp, A.; Ru, A.H. de; Tjokrodirijo, R.T.N.; Veelen, P.A. van; Puijenbroek, E. van; ... ; Falck, D. (2021)

Article / Letter to editor

The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (Fc gamma R). Fc gamma RIIIb contributes to neutrophil activation and is involved in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These processes present important mechanisms-of-actions of therapeutic antibodies. The very low affinity of IgG toward Fc gamma RIIIb (K-D similar to 10 mu M) is a technical challenge for interaction studies. Additionally, the interaction is strongly dependent on IgG glycosylation, a major contributor to proteoform heterogeneity. We developed an affinity chromatography-mass spectrometry (AC-MS) assay for analyzing IgG-Fc gamma RIIIb interactions in a proteoform-resolved manner. This proved to be well suited to study low-affinity interactions. The applicability and selectivity of the method were demonstrated on a panel of nine different IgG monoclonal antibodies (mAbs), including no-affinity, low-affinity and high-affinity Fc-engineered or glycoengineered mAbs. Thereby, we could reproduce reported affinity rankings of different IgG glycosylation features and IgG subclasses. Additional post-translational modifications (IgG1 Met252 oxidation, IgG3 hinge-region O-glycosylation) showed no effect on Fc gamma RIIIb binding. Interestingly, we observed indications of an effect of the variable domain sequence on the Fc-binding that deserves further attention. Our new AC-MS method is a powerful tool for expanding knowledge on structure-function relationships of the IgG-Fc gamma RIIIb interaction. Hence, this assay may substantially improve the efficiency of assessing critical quality attributes of therapeutic mAbs with respect to an important aspect of neutrophil activation.

• Monoclonal antibody (mAb) characterization
• Fc gamma receptor IIIb
• affinity chromatography
• IgG Fc glycosylation
• glycoproteomics
• mass spectrometry
• structure-function relationship

• All authors: Lippold, S.; Knaupp, A.; Ru, A.H. de; Tjokrodirijo, R.T.N.; Veelen, P.A. van; Puijenbroek, E. van; Taeye, S.W. de; Reusch, D.; Vidarsson, G.; Wuhrer, M.; Schlothauer, T.; Falck, D.
• Date: 2021-01-01
• Journal: mAbs
• Volume: 13
• Issue: 1
• DOI: doi:10.1080/19420862.2021.1982847
• Link: https://www.tandfonline.com/doi/pdf/10.1080/19420862.2021.1982847?needAc...