One of today's key challenges is the ability to decode the functions of complex carbohydrates in various biological contexts. To generate high-quality glycomics data in a high-throughput fashion, we developed a robotized and lowcost N-glycan analysis platform for glycoprofiling of immunoglobulin G antibodies (IgG), which are central players of the immune system and of vital importance in the biopharmaceutical industry. The key features include (a) rapid IgG affinity purification and sample concentration, (b) protein denaturation and glycan release on a multiwell filtration device, (c) glycan purification on solid-supported hydrazide, and (d) glycan quantification by ultra performance liquid chromatography. The sample preparation workflow was automated using a robotic liquid-handling workstation, allowing the preparation of 96 samples (or multiples thereof) in 22 h with excellent reproducibility and, thus, should greatly facilitate biomarker discovery and glycosylation monitoring of therapeutic IgGs.

FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.

Immunoglobulins and Fc receptors are critical glycoprotein components of the immune system. Fc receptors bind the Fc (effector) region of antibody molecules and communicate information within the innate and adaptive immune systems. Glycosylation of antibodies, particularly in the Fc region of IgG, has been extensively studied in health and disease. The N-glycans in the identical heavy chains have been shown to be critical for maintaining structural integrity, communication with the Fc receptor and the downstream immunological response. Less is known about glycosylation of the Fc receptor in either healthy or disease states, however, recent studies have implicated an active role for receptor associated oligosaccharides in the antibody-receptor interaction. Research into Fc receptor glycosylation is increasing rapidly, where Fc receptors are routinely used to analyze the binding of therapeutic monoclonal antibodies and where glycosylation of receptors expressed by cells of the immune system could potentially be used to mediate and control the differential binding of immunoglobulins. Here we discuss the glycosylation of immunoglobulin antibodies (IgA, IgE, IgG) and the Fc receptors (FcαR, FcεR, FcγR, FcRn) that bind them, the function of carbohydrates in the immune response and recent advances in our understanding of these critical glycoproteins.

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Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody–drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Immunoglobulin (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addition of conserved glycans to the antibody Fc region, which is critical for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addition, FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided.