Detecting binding activity of a therapeutic monoclonal antibody targeting vascular endothelial growth factor using surface plasmon resonance

Abstract

The use of biopharmaceuticals, particularly monoclonal antibodies (mAbs), has become a major application following advancements in recombinant DNA technology. The treatment of a wide range of diseases, among them cancer, asthma, cardiovascular conditions, infections, inflammatory and autoimmune disorders, including allergies, has become possible with the development of mAbs. Surface plasmon resonance (SPR) is a well-known and reliable method that can evaluate biomolecular interactions in real-time in terms of binding affinity and kinetics without requiring labeling procedures. Using SPR technology to determine the kinetic parameters of a mAb molecule resulting from its binding with its target receptor is a common approach to characterize the binding activity of the final product. To characterize the binding kinetics between two molecules, one molecule is first stabilized on the sensor chip surface by immobilization or capturing steps. Then, the other molecule is flowed over the sensor chip surface at variety concentrations in a running buffer to observe the interaction between the two molecules and to determine the resulting kinetic parameters. In this study, after an IgG monoclonal antibody was captured on a Protein A chip surface, the target VEGF121 growth factor was flowed over the surface at five different concentrations. By employing single-cycle kinetic analysis, the kinetic parameters were measured, providing information about the interaction between the two molecules. To develop a robust kinetic assay, it was necessary to optimize several parameters, including the capture molecule (mAb) concentration, the concentration range of the VEGF121 molecule, dissociation time, regeneration time, and the established method sequence. While evaluating the robustness and reliability of the method, important factors considered included quality control parameters from the evaluation software, plotted residuals, and the consistency of Rmax and KD values between replicates. As a result of the method development studies, a capture concentration of 13.4 nM, a dissociation time of 240 seconds and a regeneration time of 30 seconds were decided upon, ensuring that the deviation in KD values between repeated studies was less than 20 %. After the KD value was obtained with low deviation between replicate studies, qualification studies including specificity, system precision, repeatability and intermediate precision parameters were initiated. As a result of 6 replicate studies conducted by two different analysts, the percentage difference between the analysts was determined to be 0.14%. Additionally, kinetic binding studies were performed with mAb molecules from 12 different production batches to demonstrate the effectiveness of the developed method in determining the binding activity of mAb molecules in different lots. These studies showed quite similar kinetic parameters for different production batches.