RESUMO
Quality control of monoclonal antibodies is challenging due in part to the diversity of post-translational modifications present. The regulation of the N-glycans of IgG-Fc domain is one of the key factors to maintain the safety and efficacy of antibody drugs. The FcγRIIIa affinity column is an attractive tool for the precise analysis of the N-glycans in IgG-Fc domain. We used the mutant FcγRIIIa, which is produced in Escherichia coli and is therefore not glycosylated, as an affinity reagent to analyze the N-glycans of monoclonal antibodies expressed in Expi293 and ExpiCHO cells. The monoclonal antibodies expressed in these cells showed very different chromatograms, because of differences in terminal galactose residues on the IgG-Fc domains. We also carried out kinetic and thermodynamic analyses to understand the interaction between monoclonal antibodies and the mutant FcγRIIIa. Expi293 cell-derived monoclonal antibodies had higher affinity for the mutant FcγRIIIa than those derived from ExpiCHO cells, due to slower off rates and lower binding entropy loss. Collectively, our results suggest that the FcγRIIIa column can be used to analyze the glycosylation of antibodies rapidly and specifically.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Polissacarídeos/isolamento & purificação , Receptores de IgG/genética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Galactose/genética , Glicosilação , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Ligantes , Polissacarídeos/química , Polissacarídeos/genética , Processamento de Proteína Pós-Traducional/genética , Receptores de IgG/imunologiaRESUMO
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.