Characterization of FcγRIa (CD64) as a Ligand Molecule for Site- Specific IgG1 Capture: A Side-By-Side Comparison with Protein A

Fc gamma receptors (Fc gamma Rs) are one of the structures that can initiate effector function for monoclonal antibodies. Fc gamma RIa has the highest affinity toward IgG1-type monoclonal antibodies among all Fc gamma Rs. In this study, a comprehensive characterization was performed for Fc gamma 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 gamma RIa capture. The His-tagged Fc gamma 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 gamma 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 gamma 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 gamma Rs (Fc gamma RIa, Fc gamma RIIa, and Fc gamma RIIIa) to three different immobilized IgGs by immobilizing protein L to the chip surface. Unlike previous immobilization techniques tested where the Fc gamma RIa was utilized as a ligand, nonimmobilized or free Fc gamma RIa resulted in a significantly higher antibody binding response than free protein A. In this configuration, kinetics data of Fc gamma RI revealed that the association rate (ka 50-80 x 105 M-1 s-1) increased in comparison to His capture method (1.9-2.4 x 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 gamma 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 gamma RIa possesses comparable ligand potential as well as protein A. Even though the protein A-immobilized surface bound more antibodies than the Fc gamma RIa-captured surface, Fc gamma RIa presented a significant antibody binding capacity in protein L configuration. The results suggest Fc gamma 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.