Rate Constant of Dissociation (koff)
IgG Capture Assay
HBS-EP buffer; 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.005% P-20
Kinetic rate constants (kon and koff) for binding of the MEDI4893 to WT-AT and each mutant were measured by employing an IgG capture assay on a Biacore T200 instrument. Protein A was immobilized on a CM5 sensor chip with a final surface density of ∼2,000 resonance units (RUs). MEDI4893 was prepared at 10 nM in instrument buffer (HBS-EP buffer; 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% P-20), along with 2-fold serial dilutions of AT (0.048 nM to 50 nM). A sequential approach was utilized for kinetic measurements. MEDI4893 was first injected over the capture surface at a flow rate of 10 μL/min. Once the binding of the captured IgG stabilized, a single concentration of the AT protein was injected over both capture and reference surfaces at a flow rate of 50 μL/min. The resulting binding response curves yielded the association phase data. Following the injection of AT, the flow was then switched back to instrument buffer for 10 min to permit the collection of dissociation phase data, followed by a 1-min pulse of 10 mM glycine, pH 1.7, to regenerate the protein A surfaces on the chip. Binding responses from duplicate injections of each concentration of AT were recorded against anti-AT MAb MEDI4893. In addition, several buffer injections were interspersed throughout the injection series. Select buffer injections were used along with the reference cell responses to correct the raw data sets for injection artifacts and/or nonspecific binding interactions, commonly referred to as double referencing. Fully corrected binding data were then globally fit to a 1:1 binding model (BIAevaluation 4.1 software; BIAcore, Inc.) that included a term to correct for possible mass transport-limited binding. These analyses determined the kinetic rate constants kon and koff, from which the apparent dissociation constant (KD) was calculated as koff/kon.