Abstract
Fc γ receptors (FcγRs) are one of the structures that can initiate effector function for monoclonal antibodies. FcγRIa has the highest affinity toward IgG1-type monoclonal antibodies among all FcγRs. In this study, a comprehensive characterization was performed for FcγRIa 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 FcγRIa capture. The His-tagged FcγRIa 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 FcγRIa 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 FcγRIa-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 FcγRs (FcγRIa, FcγRIIa, and FcγRIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the FcγRIa was utilized as a ligand, nonimmobilized or free FcγRIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of FcγRI 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 FcγRIa 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. FcγRIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the FcγRIa-captured surface, FcγRIa presented a significant antibody binding capacity in protein L configuration. The results suggest FcγRIa 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.