Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based Chromatography Technique

Abstract

Most clinically approved large biotherapeutics are monoclonal antibodies (mAbs), primarily belonging to immunoglobulin G subclass-1 (IgG1) and, to a lesser extent, IgG2 and IgG4. Glycosylation is the main source of post-translational heterogeneity of mAbs, impacting their drug therapeutic mechanism of action (MOA). Glycosylation is also one of the critical factors in drug product solubility, kinetics, stability and efficacy. Thus, monitoring glycan critical quality attributes (CQAs) is an essential part of any biopharmaceutical development. The binding affinity of an IgG to its cellular Fc receptor (FcR) depends on both its IgG subclass and Fc domain glycosylation pattern. Since composition of the N-glycans also correlates to the Antibody-Dependent Cellular Cytotoxicity (ADCC), the glycosylation pattern needs to be monitored for consistency in potency and efficacy. This applies for the original mAb biologics as well as biosimilars. In this chapter, we present a truly novel way to assess the variances in mAb glycoforms using FcγRIIIa-based affinity chromatography. First, a brief overview of the Fc receptor function is presented. Then, the principle of FcR-based affinity chromatography is explained including how this column’s potential to analyze a variety of mAbs according to their N-glycan content is highly selective and robust. Finally, we provide examples of the FcR column’s potential to improve analytical characterization of mAbs with practical applications such as effective cell line screening, monitoring of glycoengineering, process development and process control in manufacturing.

FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab

“…Each peak represents a mixture of glycoforms, with variable amounts of galactose and other sugar moieties. The increased elution time of the peaks with respect to each other corroborates with the increased number of terminal galactose moieties 24 . The dynamic and conformational assembly of the Fc region of the antibody is modulated by galactose and thus, exhibits a more rigid conformation.…”

“…tose moieties. 24 The dynamic and conformational assembly of the Fc region of the antibody is modulated by galactose and thus, exhibits a more rigid conformation. This conformational variation governed by the galactose content affects the binding affinity of Rituximab to FcγRIIIA.…”

Chromatographic Analysis of the N-Glycan Profile on Therapeutic Antibodies Using FcγRIIIa Affinity Column Chromatography

“…Therapeutic IgGs recognize specific cell surface-expressed antigens and elicit immune effector functions representative of ADCC activity via the interaction between the Fc region and Fcγ receptors (FcγRs). The N -glycan structure can also affect the dynamic behavior of the CH2 domain on Fc. , Since there is a relationship between the binding ability of IgG to FcγRIIIa and ADCC activity, FcγRIIIa-immobilized column chromatography has been used to evaluate ADCC activity without the cleavage of a glycan from IgGs. , …”

“…4,5 Since there is a relationship between the binding ability of IgG to FcγRIIIa and ADCC activity, FcγRIIIa-immobilized column chromatography has been used to evaluate ADCC activity without the cleavage of a glycan from IgGs. 6,7 In general, therapeutic antibodies show three peaks in FcγRIIIa-immobilized affinity column chromatography because of the microheterogeneity of the Fc-glycan, and a longer retention time exhibits higher ADCC activity. The increase in retention time correlates with an increased number of terminal galactose.…”

“… 4 , 5 Since there is a relationship between the binding ability of IgG to FcγRIIIa and ADCC activity, FcγRIIIa-immobilized column chromatography has been used to evaluate ADCC activity without the cleavage of a glycan from IgGs. 6 , 7 …”