Functional Characterization of Baxter's Recombinant Human ADAMTS13 Drug Candidate.

Abstract 2235

Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their functional properties.

The ability of rADAMTS13 to degrade human full-length rVWF under moderate denaturing conditions was analyzed by two functional assays (VWF:CB and VWF:RCo activity) and by gel electrophoresis (multimer analysis and immunoblot analysis). In all assays, the extent of rVWF degradation served as a direct measure for ADAMTS13 activity.

VWF:CB activity of rVWF was reduced upon incubation with rADAMTS13, with only minimal differences between the individual batches. VWF:RCo activity of rVWF also declined after exposure to rADAMTS13, with no relevant differences between rADAMTS13 batches. Likewise, VWF multimer analysis demonstrated the disappearance of high molecular weight multimers upon incubation with rADAMTS13. Non-reducing SDS-PAGE followed by immunostaining using a polyclonal anti-human VWF antibody clearly showed the appearance of the C-terminal and N-terminal VWF fragments, with no visible differences in the intensity of these bands between preclinical and clinical Phase I rADAMTS13 batches.

Proteolytic activity of rADAMTS13 towards its multimeric VWF substrate was confirmed in two flow-based assays using full-length rVWF as substrate. One assay was based on the VenaFlux platform technology and determined the rADAMTS13 concentration-dependent decrease of the surface coverage of fluorescently-labeled platelets that had been perfused together with red blood cells and rVWF over an adhesive surface of a microcapillary. The other assay measured the extent of VWF cleavage product generation after subjecting a reaction mixture consisting of multimeric rVWF, lyophilized platelets and rADAMTS13 to fluid shear stress on a vortex mixer. Both assays revealed a high comparability between preclinical and clinical batches.

All assays thus demonstrated consistency between rADAMTS13 batches over different production stages. Importantly, comparability was also shown between the ADAMTS13 activity measured by the assays using full-length VWF as substrate and FRETS-VWF73 assay used for potency assignment of rADAMTS13.