FcRn Interaction Analyses By Surface Plasmon Resonance (SPR) And ELISA
The binding interaction of the BsAbs with recombinant cynomolgus monkey FcRn was monitored by SPR detection using a BIAcore 3000 instrument (Biacore Inc.) as described previously.24, 25 Briefly, cynomolgus monkey FcRn was immobilized directly to a CM5 chip using the standard amine-coupling kit methodology. BsAbs samples were prepared in running buffer (PBS, pH 6.0, 0.0005% Tween 20) and evaluated in duplicate. Samples were injected for 30 seconds at a flow rate of 100 µL/min and a dissociation time of 300 seconds at 25C. BsAbs were dissociated from FcRn using 1x PBS (pH 7.4) dissociation buffer. Kinetic binding constants were determined through global fits of the average of two data sets collected on separate days using Biacore T200 Evaluation, version 1.0. BsAb binding to FcRn was also evaluated at pH 7.4 (PBS, pH pH 7.4, 0.0005% Tween 20) at single concentration (5 µM diluted PBS, pH pH 7.4, 0.0005% Tween 20). Data collected at pH 7.4 was not fit since there was no observable signal.
The evaluation of the pH-dependent dissociation of BsAbs from FcRn was conducted using an ELISA as previously reported.24, 25Briefly, biotinylated cynomolgus monkey FcRn was produced by reacting each purified soluble protein with EZ-Link® Sulfo-NHS-Biotin (Pierce) using the conditions supplied by the vendor, and the FcRn:biotin ratio was measured as 1.0 and 1.0, respectively, using the EZ™ biotin quantitation kit (Pierce). The pH-dependent ELISA for the BsAbs was performed as described in earlier studies at pH 6.0 and pH 7.4.24, 25 Optical density (OD) data at pH 6.0 and pH 7.4 were analyzed and expressed as the total BsAb that remained bound to FcRn at pH 7.4 [(ODpH7.4/ODpH6.0) x 100%)]. The mean and standard deviation of three independent experiments were determined.