(D) Reproducibility studies of 3 antibodies by MSD-SET. the throughput formerly only achievable with surface-based methods. Keywords:high throughput, affinity, antibody, epitope, KD, MSD, kinetic exclusion, BLI == Introduction == Binding affinity and epitopic coverage are crucial biophysical characteristics for identification of therapeutically relevant antibodies.1Generation of 10003000 unique lead antibodies per week necessitates a high throughput methodology to measure affinity and determine epitopic diversity. When measuring affinities based on currently available methods, one must compromise either on throughput or accuracy. Surface-based methods such as biolayer interferometry (BLI) and surface plasmon resonance (SPR) are widely used for affinity measurements by monitoring dissociation and association events in real time, with affinity (KD) determined by the ratio of dissociation to association rate constants (kd/ka).2,3BLI based devices, such as ForteBios Octet RED384, allow quick evaluation of binding kinetics in a 96- or 384-well format. Antigen solutions can be re-used multiple occasions, resulting in a negligible amount of antigen consumption.4In addition, epitopic diversity can be assessed by interrogating the simultaneous binding of specific antibody pairs to the same target.5In contrast, while advancements in SPR instrumentation allow for high throughput analysis, antigen consumption remains a challenge when limited material is available.6For accurate surface-based KDmeasurements, Z-YVAD-FMK it is often recommended to use the lowest practical sensor loading density and multiple concentrations of analyte to minimize mass transport related effects and rebinding.7-11For high affinity interactions, even the most optimized conditions may not yield an Rabbit Polyclonal to PPIF accurate KD, due to limitations in instrumentation sensitivity for slow Z-YVAD-FMK dissociation kinetics. Within these limitations, BLI methodology is useful for ranking the affinity of antibodies down to the single digit nM range. Because of our need to assess hundreds of antibodies per week beyond the practical affinity limitation of BLI-based methods in high throughput mode, we developed a solution-based method for the rapid determination of KDs for hundreds of antibodies in the sub-nanomolar affinity range. The kinetic exclusion assay (KinExA, Sapidyne) provides an assessment of free ligand at equilibrium, rather than measuring real-time association and dissociation rates to determine affinity. In this assay, we prefer to hold the ligand concentration constant while varying the receptor concentration (reverse orientation); following the establishment of an equilibrium, the percent of free ligand is determined to calculate an equilibrium KD.12,13Kinetic exclusion can also directly measure association rate constants using the same principle. The sensitivity of the KinExA instrument allows for accurate and reproducible measurements. Affinities in the several hundred femtomolar range and on-rates as high as 107s1M1have been measured,14,15but one of the main limitations is usually that only a handful of KDmeasurements can Z-YVAD-FMK be made per day without accounting for time for samples to come to equilibrium. To overcome this speed limitation, a magnetic beads-based method compatible with high throughput answer phase equilibrium KDmeasurement was first report by Naenel et al.26A newer technique employing the kinetic exclusion principle on Gyrolab discs allows up to 20 affinity measurements to be made in duplicate within 4 h.16 In this paper, we describe the development of a high throughput solution-equilibrium KDbased method that allows for the accurate determination of affinities into the femtomolar range. Combining solution-based KDmeasurements with a BLI based approach for epitope binning allows for rapid identification of therapeutically relevant leads. == Results == == Epitope binning by BLI == Octet RED384 offers the ability to quickly evaluate epitope distribution among a panel of discovered antibodies.5A bin is related to the epitope of the antigen and can be viewed as an antibodys footprint on an antigens surface. In a typical epitope binning assay, the control antibody is usually first loaded to the sensor through its Fc-region, followed by blocking of remaining Fc-binding sites around the sensor. Next, the sensor is usually exposed to antigen, shown by the response increase in the first binding step ofFigure 1, followed by exposure to a second antibody. When the antibody around the sensor competes with itself.