Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1

Abstract

Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor’s quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor’s properties, most notably its binding to Fcγ receptors.

Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain

“…The Fc Asn 297 is glycosylated but is within the core of the Fc dimer. Non-glycosylated Fc remains stable and folds properly, but the dimer formed by Fc domains lacking Asn 297 glycosylation has a tighter conformation, and this can reduce FcRn binding by ~10 fold 56 . Plant and metazoan glycosylation differ 57 , leading to possibly diminished FcRn binding and absorption.…”

Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte‐Derived Dendritic Cell Activation via FcγRIIa

“…47 Interestingly, Kannicht et al showed that the Fc domain of rFVIII-Fc contains a remarkably low level of sialylation (less than 1%). 48 In contrast, the sialylation degree of the Fc domain of plasma-derived human IgG's is around 13%. 49 Thus, it can be speculated, that the very low sialic acid content of the fusion construct (and the abolishment of binding to sialic acid-specific receptors) in combination with the binding to FVIII-specific receptors and a co-ligation with FcgRIIa supports signaling pathways that drive DC activation.…”

“…For example, terminal sialylation of the Fc domain has been shown to be important for the anti‐inflammatory activity of IgG's, 45,46 probably by promoting an anti‐inflammatory milieu through the interaction with sialic acid recognizing receptors 47 . Interestingly, Kannicht et al showed that the Fc domain of rFVIII‐Fc contains a remarkably low level of sialylation (less than 1%) 48 . In contrast, the sialylation degree of the Fc domain of plasma‐derived human IgG's is around 13% 49 .…”

Patient-derived cancer modeling for precision medicine in colorectal cancer: beyond the cancer cell line

“…72 Despite recent rapid progress in the organon-a-chip model, a lot of devices are still complex to fabricate and integrated genetic quantification is quite difficult in these systems. 73,74 Through continued development and advancement, this approach will lead to powerful in-vitro pre-clinical cancer models that can facilitate the discovery of anti-cancer drugs.…”