Receptors for human IgG (FcγR) are transmembrane glycoproteins consisting of three families designated I to III. Each family consists of several genes, each encoding for a separate protein with most of them inducing an activating response via IgG immune complexes. Of these receptors, FcγRIIa has the widest expression, as it is present on all myeloid cell types, such as neutrophils, basophils, mast cells, eosinophils, monocytes, macrophages, dendritic cells but also platelets (1–3). Antibody binding and signaling via FcγRIIa receptor can induce immunological responses such as maturation of dendritic cells, cytokine and chemokine release leading further to T cell activation or platelet aggregation (2, 4). FcγRIIa contributes to the removal of immune complexes by macrophages via antibody-dependent cell-mediated phagocytosis (ADCP) (4). These aspects are also very important for monoclonal antibody therapies (5). In the population two different allotypes of the FcγRIIa gene can be observed, namely FcγRIIa H131 and FcγRIIa R131 (6, 7) with allotype expression influencing the clinical outcome of immunotherapy (8). The extracellular domain of FcγRIIa binds IgGs in their Fc region at the second domain of the heavy chain (CH2). Next to this binding site in the CH2 domain, the CH3 domain has been reported to be additionally involved in FcγRIIa binding (9), whereas another study on IgG1 indicated that the solely the CH2 domain interacted with FcγRIIa (10). In particular two amino acids in the lower hinge region in position L234, L235 (11, 12) and P329 in the upper hinge region appear to be critical for the interaction, and their mutation leads to a nearly complete loss of affinity (13, 14). This is especially of interest for some antibody therapeutic applications where no activation of the immune system via Fc receptor functions is wanted (14).

Antibodies contain a conserved N-glycosylation site in the Fc domain, close to the FcγRIIa binding interface. The influence of the Fc glycan on binding has been widely discussed. Complete deglycosylation of IgG1 monoclonal antibodies (mAbs) has been demonstrated to lead to severe decrease of affinity due to a decrease in antibody conformational stability (15–17). To study the effect of different IgG1 glycoforms, glycoengineered IgG variants (i.e. with higher populations of certain glycan moieties) have been explored, yet with controversial results. In a recent publication employing hypergalactosylated variants no difference in FcγRIIa binding compared to the reference material was observed (18), while Subedi et al. using more enriched glycoforms reported an approximately 1.5 times higher affinity for antibodies containing high amounts of galactosylation (19). A similar effect of an increased binding of galactosylated variants was found by Thomann et al. (20). Notably, results on the effect of sialylation were again divergent: Whereas Subedi et al. hardly observed any increase on affinity for sialylated glycans (19), the study of Thomann et al. found that increased sialylation resulted in increased binding to the FcγRIIa (20). Functionally, enhanced galactosylated IgG also seem to elevate platelet activation upon encounter of anti-SARS-CoV-2 IgG1 complexed with S proteins (21). These differences between studies most probably arise by the fact that glycoengineering still provides a mixture of glycoforms and that different glycoengineering strategies can lead to different glycoform mixtures (22). Up to now techniques to study the interaction between FcγRIIa (e.g. ELISA or SPR assays) cannot distinguish between co-existing glycoforms making the assessment of glycoform-specific binding challenging (18, 23). A recent alternative approach used an in-house produced FcγRIIa column combined with UV detection but still required samples enriched in specific glycoforms (20). Therefore, a glycoform-resolved, multiplexed approach would be of tremendous benefit to answer these questions. Furthermore, the influence of other common glycoforms in IgG1 therapeutics, such as high mannose structures has hitherto not been addressed and would benefit from such strategy facilitating direct analysis without the need of specific glycoforms.

We have recently developed a novel approach based on affinity capillary electrophoresis – mass spectrometry (CE-MS) that allows monitoring the specific binding of antibody proteoform mixtures (including glycosylated variants) to the FcRn receptor (24). Mobility shift affinity capillary electrophoresis is an approach able to determine binding affinities of specific proteoforms by monitoring the change of their electrophoretic mobility after addition of the interacting partner to the background electrolyte (25). The complex formed between the antibody and receptor presents a different electrophoretic mobility than the free species indicating binding. The schematic shown in Figure 1 describes the behavior of species with different affinities to the Fc receptor. Whereas a species without affinity shows no change on the mobility shift, a species with high affinity to the Fc receptor shows a large mobility shift. Another species with a slightly lower affinity will also have a slightly lower mobility shift compared to the high affinity species. This way affinity CE allows to define the affinity of different species in a mixture due to their change in electrophoretic mobility. Coupling to mass spectrometric detection permits the multiplexed, parallel analysis and structural assessment of the specific species responsible of the binding. This approach opens new possibilities as multiple species (e.g glycoforms) can be monitored simultaneously. A mayor benefit of this platform is the ability to determine also medium and low affinity interactions as often observed for Fc receptors and antibodies (high nM-µM range) which can be challenging for SPR approaches.

Here, we explore the approach to study the binding affinity between FcγRIIa and different IgG1 glycoforms. Mass spectrometric detection allowed the parallel, integrated analysis of different glycoforms without the need of tedious glycoengineering for separately producing different glycoforms or the IgG molecules. IgGs with both complex-type and high mannose-type glycosylation were explored. Furthermore, we studied the impact of different glycan core structure units in respect to their affinity to the FcγRIIa, which allowed us to see an increase in the affinity with increasing size of the N-glycan core structure.

Binding assessment of specific protein variants or proteoforms remains a big technical challenge. In the antibody field, developments in glycoengineering strategies have permitted to study the influence of glycosylation in binding with various FcRs in more in detail (18, 20, 23). Yet, glycoengineered antibodies are not pure glycoforms, as also shown in Table S1 and often bring ambiguities due to the presence of other glycoforms and PTMs that can potentially influence the binding. Monitoring relative binding of individual proteoforms or glycoforms from a complex mixture provides unique opportunities to discern their differences. Affinity CE-MS strategy allows to assign the relative affinities not only of specific glycoforms, but also of truncated glycoforms, hemi-glycosylated and aglycosylated antibodies without the need of tedious production via glycoengineering or fractionation of different species. Individual glycoforms are resolved by MS at the composition level, allowing to assess dozens – and potentially even more – proteoforms and glycosylation variants in a single experiment independently of their relative abundance (26, 27). While structural information is obtained in the gas phase, biomolecular interactions are probed in-solution allowing affinity assessment. Furthermore, due to the high selectivity of the approach small differences in affinity (often unresolved by standard binding techniques) can be monitored. Another benefit of the approach is that minimal amounts of antibody and receptor (only few µg) are necessary to determine the affinity allowing to study the interaction of IgGs with receptors where only limited amounts of receptor are available.

In line with earlier results, we observed only very minor binding of aglycosylated IgG for FcγRIIa (14, 28), whereas the LALA-PG showed no binding to the FcγRIIa receptor (14). Interestingly, for the antibody containing only GlcNAc either on one or on both sides a reduction in binding to the FcγRIIa could be observed however stronger than compared to the non-glycosylated antibody binding. Such an effect has previously been reported for FcγRIIIa (29), however was never studied for the FcγRIIa receptor. A more recent study on IgG confirmed this finding and showed that the binding affinity for only one GlcNAc to the FcγRIIIa receptor is lower compared to an entire complex type glycan, however not completely abolished (30). They also demonstrated the importance of the GlcNAc residue to stabilize the C’E loop of the IgG molecule, which is critical for the FcγR interaction (31). Also, a report by Krapp et al. shows that partial removal of sugar moieties lead to a more and more closing of the antibody structure, which will also affect FcγR binding (32). We also found an increased affinity with only two GlcNAc per IgG molecule compared to the completely deglycosylated IgG molecule. This is the first time, that this was observed for the interaction with the FcγRIIa and furthermore we could observe an increase in affinity for 4 GlcNAc or an additional mannose, which might be due to an additional stabilization of the C’E loop and therefore enhanced accessibility for the FcγRIIa.

Similarly, we found that the hemi-glycosylated antibodies have a lower binding affinity to the FcγRIIa compared to the fully glycosylated antibody. Even though the binding of the FcγRIIa and the antibody is either in a 1:1 (33, 34) or an asymmetric 2:1 (one FcγR dimer binds one mAb) (35) ratio both glycans are necessary to obtain a high affinity. This was also shown in a report of Ha et al. where they isolated hemi-glycosylated antibodies using cation exchange chromatography and found in SPR measurements that the affinity to both FcγRIIa allotypes is decreased by half (K_D approximately 2 times higher) (36). Most likely this decreased binding affinity is connected to the decrease in thermal stability of hemi-glycosylated antibodies, which also might impact the structure of the CH2 domain.

Next to glycan occupancy, we could study the influence of specific glycans on binding. Certain glycan features, such as afucosylation are known to have a strong influence on the binding to the FcγRIII receptor. However, we observed no binding difference between afucosylated variants for neither FcγRIIa allotype, which is in agreement with other studies (15). Also, in the context of higher galactosylation no influence of afucosylation on the binding to the FcγRIIa could be found. Galactosylation of complex type glycans was reported to show a slight increase in binding to the FcγRIIa with the highest affinity for the antibody with two G2F glycans (20, 23, 37). In our analysis we confirmed that increase of galactosylation enhances the affinity to the FcγRIIa. For sialylation the literature is less conclusive, which might be due to the fact that sialic acids can be linked differently to the terminal galactose (2,3 vs 2,6 linkage depending of expression system) and thereby show a different binding. Whereas some reports suggest an increase in binding to the FcγRIIa (23) or a slight increase (20) other reports see either an increase or a decrease depending on the presence of core fucose and bisection (18). This example shows already the urge to look at each glycoform specifically, as glycoengineering still result in a mixture of different glycoforms. For sialylation we also observed an increase in affinity to the FcγRIIa. Interestingly, based on our data it seems that the IgG with only one sialic acid has a slightly stronger shift suggesting a higher affinity than compared to the IgGs with two or three sialic acids. A similar effect was also observed by SPR and FcgRIIa using glycoengineered IgG variants (20, 23). Contrary, using a range of 1-72% sialylation in differently engineered antibodies with regard to bisection, fucosylation and galactosylation, Dekkers et al. found no effect of sialic acid content of IgG1 on FcγRIIa binding, while a slight decrease of FcγRIIIa/b binding was found for the afucosylated and bisected variants. A minor effect with this regards was also found comparing binding of FcγRIIa-H131 to fucosylated, non-bisected and sialylated IgG1 with afucosylated, bisected and non-sialylated IgG1 (18). However the differences observed in these reports, including our study, are very minor and might be due to experimental errors. Furthermore different sialic acid linkages (2,3 versus 2,6) might have a different binding behavior. While 2,3 linked sialic acids are often present in biopharmaceuticals sialic acids are 2,6 linked in naturally occurring human antibodies and their behavior will be addressed in future studies. Overall can be noted that all glycoengineered samples are never pure species showing the great advantage of our assay to determine the affinity of each specific glycoform in a mixture without the need of any prefractionation or glycoengineering.

High mannose glycoforms showed a decrease in affinity, which was so far never reported for the FcγRIIa. The effect was the strongest when two high mannose glycans were combined on the Fc portion and less, but still lower compared to G0F/G0F when one Man5 structure was in combined with a complex type glycan. This decrease in affinity might be a result of a decrease in the antibody stability (38). This might also affect the structure of the CH2 domain and therefore explain the decrease in affinity. Most likely is the structure of the Man5/G0F glycan not so dramatically influenced as the one with Man5/Man5. This finding is of utmost importance especially of therapeutic IgG1 samples, which can contain higher amounts of Man5 structures. Similarly to our finding, Man5 glycoforms were recently shown to have a reduced binding to FcγRIIIa compared to the afucosylated complex type glycans (39). Therefore the expansion of our platform to other receptors such as FcγRIIIa is warranted to support this finding.

The original contributions presented in the study are included in the article/Supplementary materials, further inquiries can be directed to the corresponding author/s.

CG performed the measurements and processed the data. ED-V conceived the idea and designed the experiments. AK and TS provided the Fc receptors, DR and GV provided the mAb samples. CG and ED-V drafted the manuscript. CG, GV, AK, TS, DR, MW, and ED-V reviewed this manuscript equally.

This work was supported by the Analytics for Biologics project (Grant agreement ID 765502) of the European Commission and the LUMC Fellowship 2020 to ED-V.

Authors DR, TS, AK are employed by the company Roche Diagnostics GmbH, Penzberg, Germany.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.