Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column

Abstract

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

“…Affinity chromatography analysis used ligand without glycosylation and with eight amino acid mutations. 53 FcγRIIIa has five putative N-glycosylation sites, and those N-glycosylations at Asn-162 and Asn-45 have been reported to be key regulators of IgG-Fc binding. 54,55 To the best of our knowledge, however, no reports have indicated that each one of those amino acid mutations modulate IgG-Fc binding.…”

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

“…13 More recently, AC-UV and subsequent glycan analysis were used to assess the heterogeneity of Fc-glycosylation of a therapeutic antibody, using a non-glycosylated FcɣRIIIa. 19 However, glycosylation has been shown to be critical for differential recognition of IgGglycoforms by wild-type FcɣRIIIa. 20 Thus, no significant differences for individual glycoforms, except for galactosylation levels, were observed.…”

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology

“…However, the effect of galactosylation on FcγRIIIa affinity is less clear [16]. A structure study suggested that the terminal galactose moiety could decrease the conformational entropy of CH2 and facilitate the engagement of Fc with FcγRIIIa [21]. In this study, the later-elution peaks in FcγRIIIa affinity chromatograms represent highly galactosylated antibody, reflecting an increasing affinity for FcγRIIIa.…”

“…Recently, a new technology, FcγRIIIa affinity chromatography [19,20], was developed to enable quantitative comparison of glycan effects on effector function [21]. By using an FcγRIIIa-immobilized TSKgel FcR-IIIA-NPR column, the novel chromatography is able to separate antibodies according to their affinity to the FcγRIIIa receptor, which conventional surface plasmon resonance (SPR) methods cannot distinguish [22].…”