Real-time monitoring of papain digestion of antibodies immobilized with various strategies by optical interferometry

Abstract

Antigen binding fragments (Fabs) employed in research are typically generated by the papain digestion of monoclonal antibodies. However, the interaction between papain and antibodies at the interface remains unclear. Herein, we developed ordered porous layer interferometry for the label-free monitoring of the interaction between the antibody and papain at liquid-solid interfaces. Human immunoglobulin G (hIgG) was used as the model antibody, and different strategies were employed to immobilize it on the surface of silica colloidal crystal (SCC) films which are optical interferometric substrates. It was observed that different immobilization strategies induced different changes in the optical thickness (OT) of SCCs. The order of rate of the changes of OT from largest to smallest was IgG immobilized by protein A orientation, glutaraldehyde coupling, and physical adsorption. This phenomenon can be explained by the varied orientations of the antibodies created at the interface by the different modification procedures. The Fab-up orientation maximized the exposure of the hinge region sulfhydryl group and easily underwent conformational transitions because hIgG was immobilized by protein A. This process stimulates papain to produce the highest degree of activity, resulting in the greatest decrease in OT. This study provides insights into the catalysis of papain on antibodies.