Isolation and characterization of IgG1 with asymmetrical Fc glycosylation

• Published in: Glycobiology (Oxford) Vol. 21; no. 8; pp. 1087 - 1096
• Main Authors: Ha, Sha, Ou, Yangsi, Vlasak, Josef, Li, Yuan, Wang, Shiyi, Vo, Kim, Du, Yi, Mach, Anna, Fang, Yulin, Zhang, Ningyan
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.2011
• Subjects: Antibody-Dependent Cell Cytotoxicity; Calorimetry, Differential Scanning; Chromatography, Liquid; Glycosylation; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - immunology; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Immunoglobulin G - isolation & purification; Mass Spectrometry; Temperature; asymmetrical glycosylation; therapeutic antibody; Fc R binding; Fc effector function
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwr047

N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemi-glycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease (∼20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (Fc Rs) including the high-affinity Fc RI, and the low-affinity Fc RIIA, Fc RIIB, Fc RIIIA and Fc RIIIB. The decreased binding affinities to Fc Rs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.