Vibration-Induced Disruption of Antibody-Antigen Bonds
Authors:   , Rosario Ruben , Mutharasan Rajakkannu 
Published: July 2021

The primary objective of the research was to develop a new methodology for disruption of antibody bonds. Specifically, the goal was to experimentally demonstrate that specifically-bound proteins can be released from a vibrating surface at physiological conditions. The second objective was to examine how vibration induced release using computational methods. There is a need to develop effective methods for disrupting antibody bonds that do not damage the antibody or binding target. Controlled antibody binding and release is frequently used in industrial bioseparation methods, but the traditional chromatography unit operations have their drawbacks. For instance, consider one of the most prevalent separation unit operations used in the purification of therapeutic antibodies, namely, Protein A chromatography. The Protein A chromatography column contains a resin with surface-immobilized Protein A, which binds tightly to the Fc region of immunoglobulins. In this type of chromatography, the column is operated in a bind-and-elute mode with the following steps: (1) the impure suspension of valuable antibodies is introduced to the column; (2) the antibodies bind and saturate the resin; (3) the suspension is flushed from the column; (3) and, finally, an elution buffer is used to release the antibodies from the resin. Low-pH citrate and glycine-HCl buffers are commonly used to elute the antibodies from the column. While these low-pH buffers are effective at disrupting the electrostatic and hydrophobic interactions in the antibody-Protein A bond, they result in degradation of the antibody product. Thus, an alternative elution method that releases specifically-bound proteins at physiological pH is preferable. Vibration-induced disruption of antibody bonds occurs when the resulting physical forces overwhelm the electrostatic and hydrophobic interactions between the antibodies and their binding partners. Previously, the internally-driven millimeter scale cantilever was observed to inhibit weak, nonspecific interactions between proteins and the vibrating surface. In this work, specifically-bound species were released from a resonating self-driven millimeter-sized cantilever. As a proof of concept, experiments were designed to demonstrate release of the model cellular target, E. coli O157:H7, at physiological pH. Electrochemical measurements and micrographs confirmed that the resonating cantilever released more than 80% (n=11) of antibody-bound E. coli cells from the vibrating cantilever surface. The research then moved to the more challenging task of demonstrating release of specifically-bound antibodies from surface-immobilized binding partners. For the first time, multiple methods demonstrated that vibration could induce release of specifically-bound antibodies: electrochemical impedance and enzyme assays confirmed that the vibrating cantilever released 76±25% (n=6) of anti-Bovine Serum Albumin from its surface-immobilized antigen. Hence, when the cantilever vibrated with sufficient intensity, most of the high-avidity antibody bonds were disrupted. This is a fascinating discovery that could be useful in industry, especially when considering that the Protein A-antibody interaction is weaker than most antibody-antigen bonds. Three-dimensional finite element models indicated that the surface acceleration of a resonating cantilever reached approximately 2.68x105 m/s2 during the E. coli release experiments. Given that the exerted force is the product of the bacterium's mass (~1 pg) and acceleration, the force would reach a maximum of 268 pN. This magnitude of force is well beyond the 50-200 pN range of force required to effect antibody-antigen bond rupture. Hence, inertial force can conceivably result in the release of cellular targets. The release of specifically-bound proteins, however is another matter. The mass of the average antibody is many orders of magnitude lower than that of any cell. Hence, the cantilever fails to generate sufficient inertial force to affect the bond. However, a computational fluid dynamic model, indicated that the resonating cantilever induced a steady fluid flow called acoustic streaming. The resulting steady, high shear rates were thus the likely agent behind protein release.