title: Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex.
abstract: To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb. The results revealed that as the negative charge on the biosensor surface decreased, the binding kinetics and K(D) approached the accurate binding parameters more closely when measured in solution. Two potential causes for the artifactual Biacore surface-based measurements are (i) steric hindrance of antigen binding arising from an interaction of the negatively charged carboxymethyldextran matrix with the mAb, which is a highly basic protein with a pI of 9.4, and (ii) an electrostatic repulsion between the negatively charged antigen and the carboxymethyldextran matrix. Importantly, simple diagnostic tests can be performed early in the measurement process to identify these types of matrix-mediated artifacts.
authors: Takeda San Francisco, 285 E. Grand Ave. South San Francisco, CA 94080, USA.
authors: Andrew W
authors: AW
authors: Drake
authors: Margaret L
authors: ML
authors: Tang
authors: Giuseppe A
authors: GA
authors: Papalia
authors: Gregory
authors: G
authors: Landes
authors: Mary
authors: M
authors: Haak-Frendscho
authors: Scott L
authors: SL
authors: Klakamp
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D016477:Artifacts; D003911:Dextrans / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D020349:Surface Plasmon Resonance / Q000379:methods*; D013499:Surface Properties
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D003911:Dextrans; C014392:carboxymethyl dextran