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

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.Proteomic

Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of individual healthy donors

Fc gamma receptors (Fc gamma Rs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and Fc gamma R impacts their interaction dramatically. Regarding Fc gamma R glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map Fc gamma RIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for Fc gamma RIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying Fc gamma RIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous Fc gamma RIIIa on natural killer cells. This method will allow assessment of differences in Fc gamma RIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions

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

The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb 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 FcɣRIIIb (KD ~ 10 µM) 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-FcɣRIIIb 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 FcɣRIIIb 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-FcɣRIIIb 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

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors

Fc gamma receptors (Fc gamma Rs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and Fc gamma R impacts their interaction dramatically. Regarding Fc gamma R glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map Fc gamma RIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for Fc gamma RIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying Fc gamma RIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous Fc gamma RIIIa on natural killer cells. This method will allow assessment of differences in Fc gamma RIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.Proteomic

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics

Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications

Immunoglobulin (Ig) glycosylation is recognized for its influence on Ig turnover and effector functions. However, the large-scale profiling of Ig glycosylation in a biomedical setting is challenged by the existence of different Ig isotypes and subclasses, their varying serum concentrations, and the presence of multiple glycosylation sites per Ig. Here, a high-throughput nanoliquid chromatography (LC)- mass spectrometry (MS)-based method for simultaneous analysis of IgG and IgA glycopeptides was developed and applied on a serum sample set from 185 healthy donors. Sample preparation from minute amounts of serum was performed in 96-well plate format. Prior to trypsin digestion, IgG and IgA were enriched simultaneously, followed by a one-step denaturation, reduction, and alkylation. The obtained nanoLC-MS data were subjected to semiautomated, targeted feature integration and quality control. The combined and simplified protocol displayed high overall method repeatability, as assessed using pooled plasma and serum standards. Taking all samples together, 143 individual N- and O-glycopeptides were reliably quantified. These glycopeptides were attributable to 11 different peptide backbones, derived from IgG1, IgG2/3, IgG4, IgA1, IgA2, and the joining chain from dimeric IgA. Using this method, novel associations were found between IgA N- and O-glycosylation and age. Furthermore, previously reported associations of IgG Fc glycosylation with age in healthy individuals were confirmed. In conclusion, the new method paves the way for high-throughput multiprotein plasma glycoproteomics