Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.

Twoplex 12/13 C6 aniline stable isotope and linkage-specific sialic acid labeling 2D-LC-MS workflow for quantitative N-glycomics.

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease-associated glycan alterations to the quantitative characterization of N-glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI-TOF-MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run-to-run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC-MSE workflow for the fractionation and relative quantitation of twoplex isotopically labeled N-linked oligosaccharides using neutral 12 C6 and 13 C6 aniline (Δmass = 6 Da). Additional linkage-specific derivatization of sialic acids using 4-(4,6-dimethoxy-1,3,5-trizain-2-yl)-4-methylmorpholinium chloride offered simultaneous and advanced in-depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N-glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex 12 C6 /13 C6 aniline 2D LC-MS platform is ideally suited for differential glycomic analysis of structurally complex N-glycan pools due to combination and analysis of samples in a single LC-MS injection and the associated minimization in technical variation.

N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality.

Etanercept is a highly glycosylated therapeutic Fc-fusion protein that contains multiple N- and O-glycosylation sites. An in-depth characterization of the glycosylation of etanercept was carried out using liquid chromatography/mass spectrometry (LC/MS) methods in a systematic approach in which we analyzed the N- and O-linked glycans and located the occupied O-glycosylation sites. Etanercept was first treated with peptide N-glycosidase F to release the N-glycans. The N-glycan pool was labeled with a 2-aminobenzamide (2-AB) fluorescence tag and separated using ultraperformance liquid chromatography-hydrophilic interaction liquid chromatography (UPLC-HILIC). Preliminary structures were assigned using Glycobase. These assignments, which included monosaccharide sequence and linkage information, were confirmed by exoglycosidase array digestions of aliquots of the N-glycan pool. The removal of the N-glycans from etanercept facilitated the selective characterization of O-glycopeptides and enabled the O-glycans to be identified. These were predominantly of the core 1 subtype (HexHexNAc O-structure) attached to Ser/Thr residues. α2→3,6,8,9 sialidase was used to remove the sialic acid residues on the O-glycans allowing the use of an automated LC/MS(E) protocol to identify the O-glycopeptides. Electron-transfer dissociation (ETD) was then used to pinpoint the 12 occupied O-glycosylation sites. The determination of N- and O-glycans and O-glycosylation sites in etanercept provides a basis for future studies addressing the biological importance of specific protein glycosylations in the production of safe and efficacious biotherapeutics.