Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions

The Octet biosensors provide a high-throughput alternative to the well-established surface plasmon resonance (SPR) and SPR imaging (SPRi) biosensors to characterize antibody-antigen interactions. However, the utility of the Octet biosensors for accurate and reproducible measurement of binding rate constants of monoclonal antibodies (mAbs) is limited due to challenges such as analyte rebinding, and mass transport limitation (MTL). This study focuses on addressing these challenges and provides experimental conditions to reliably measure kinetics of mAb-antigen interactions. The mAb capture density of less than 0.6 nm was found to be optimal to measure a wide range of binding affinities on Octet HTX biosensor. The titration kinetic and single cycle kinetic assays performed on Octet HTX generated reproducible binding kinetic parameters and correlated with the values measured on Biacore 4000 and MASS-1. Kinetic assays performed on 0.1 nm density mAb surfaces significantly reduced MTL and enabled characterization of picomolar affinity mAbs. Finally, kinetic analysis performed on 150 antibodies to 10 antigens with molecular weights ranging from 21kD to 105kD showed concordance between Octet HTX, Biacore 4000 and MASS-1 (R2 > 0.90). The data presented in this study suggest that under optimal experimental conditions, Octet biosensor is capable of generating kinetic values comparable to SPR/SPRi biosensors. •High density antibody capture surface exhibited analyte rebinding and mass transport limitation (MTL).•Kinetic assays performed on 0.1 nm density antibody surface significantly reduced MTL and enabled characterization of picomolar affinity interactions.•A total of 165 fully human antibodies binding to 14 different antigens with molecular weight ranging from 14kD to 105kD were characterized on Octet HTX, Biacore 4000 and MASS-1 and the results revealed that measured binding kinetic values were comparable across different biosensors.