Characterization of Fc?RIa (CD64) as a ligand molecule for site-specific IgG1 capture: A side-by-side comparison with protein a

Abstract

Fc ?receptors (Fc?Rs) are one of the structures that can initiate effector function for monoclonal antibodies. Fc?RIa has the highest affinity toward IgG1-type monoclonal antibodies among all Fc?Rs. In this study, a comprehensive characterization was performed for Fc?RIa as a potential affinity ligand for IgG1-type monoclonal antibody binding. The binding interactions were assessed with the SPR technique using different immobilization techniques such as EDC-NHS coupling, streptavidin-biotin interaction, and His-tagged Fc?RIa capture. The His-tagged Fc?RIa capture was the most convenient method based on assay repeatability. Next, a crude IgG1 sample and its fractions with different monomer contents obtained from protein A affinity chromatography were used to evaluate Fc?RIa protein in terms of monoclonal antibody binding capacity. The samples were also compared with a protein A-immobilized chip (a frequently used affinity ligand) for IgG1 binding responses. The antibody binding capacity of the protein A-immobilized chip surface was significantly better than that of the Fc?RIa-immobilized chip surface due to its 5 Ig binding domains. The antibody binding responses changed similarly with protein A depending on the monomer content of the sample. Finally, a different configuration was used to assess the binding affinity of free Fc?Rs (Fc?RIa, Fc?RIIa, and Fc?RIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the Fc?RIa was utilized as a ligand, nonimmobilized or free Fc?RIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of Fc?RI revealed that the association rate (ka 50-80 × 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 × 105 M-1 s-1). In addition, the dissociation rate (kd 10-5 s-1) seemed slower over the His capture method (10-4 s-1) and provided stability on the chip surface during the dissociation phase. The KD values for Fc?RIa were found in the picomolar range (2.1-10.33 pM from steady-state affinity analysis and 37.5-46.2 pM from kinetic analysis) for IgG1-type antibodies. Fc?RIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the Fc?RIa-captured surface, Fc?RIa presented a significant antibody binding capacity in protein L configuration. The results suggest Fc?RIa protein as a potential ligand for site-oriented immobilization of IgG1-type monoclonal antibodies, and it needs further performance investigation on different surfaces and interfaces for applications such as sensing and antibody purification.