FVIII TAb Assay

Antibodies against hFVIII were measured in cynomolgus monkey plasma using a bridging ECL assay on the MSD QuickPlex Imager. B domain-deleted (BDD) recombinant hFVIII-SQ (Xyntha) was either conjugated with an LC-LC biotin tag (FVIII-Bio) or a Sulfo-NHS tag (FVIII-Ru). TQCs were prepared using a mouse monoclonal anti- hFVIII (Green Mountain Antibodies, catalog number GMA-8012) spiked into pooled cynomolgus CC plasma at 8,000 ng/mL and serially diluted 1:4 a total of six times to 1.95 ng/mL. LQCs at 40.0 ng/mL, a separate naive pool used as a blank negative QC (NQC), and the CC plasma were tested on all plates. Samples and QCs were heat-treated for 30 min to dissociate any antibody complexes with endogenous plasma FVIII and then placed on ice. Next, samples and QCs were diluted to the MRD of 1:20 in diluent buffer (TBS with 1% casein). The conjugated reagents were combined at equimolar ratios (0.5 μg/mL each) and incubated 1:1 with diluted samples and QCs. During the incubation, anti-hFVIII antibodies in plasma formed complexes with the conjugated FVIII reagents. The mixture was transferred to a streptavidin-coated polypropylene MSD plate to capture the hFVIII-antibody complexes. Plates were washed with Dulbecco’s PBS (DPBS) containing 0.1% Tween 20 and 0.05% Proclin300 before addition of MSD read buffer T (1× concentration) containing the substrate TPA, which reacts chemically with ruthenium (FVIII-Ru) in the presence of voltage applied to each well by the MSD QuickPlex Imager plate reader. Samples that contained human anti-FVIII antibodies bound to both the FVIII-Bio and FVIII-Ru generated ECL signals that were detected by the MSD Imager. All plasma samples were analyzed in a screening assay to identify the presence or absence of anti-FVIII antibodies in the samples. Mean sample ECLU values were normalized as S/N values by dividing them by the mean ECLU value of the CC plasma that was run on the same plate. If the normalized ECLU signal for a given plasma sample was greater or equal to the plate-specific SCP (mean CC signal × SCP factor) (SCP factor [SCPF] = 1.05), the sample was considered positive; otherwise, the sample was considered negative. The limit of detection (LOD) for the assay was 1.93 ng/mL of the monoclonal positive-control anti-FVIII antibody.