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

Abstract 2236

Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. The protein is produced in a CHO cell-line using serum and protein-free fermentation technology. The purification process does not use immune-affinity chromatography, and includes two viral reduction steps. The final drug product is formulated without proteins of animal or human origin.

Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their structural properties. The molecular weight of rADAMTS13 was determined by MALDI TOF as ∼173 kDa. SDS-PAGE showed one band at ∼190 kDa; no additional distinct protein bands corresponding to other proteins or product related degradation products were visible in the gels. Investigation of the primary structure of rADAMTS13 by peptide mapping revealed a sequence coverage of 98% compared to the theoretical amino acid sequence, and no peptides from the signal or propeptide. Peptide mapping was also used to determine posttranslational modifications of rADAMTS13. Seven O-glycosylation sites were identified in addition to the predicted N-glycosylation, O-fucosylation and C-mannosylation sites. The relative monosaccharide and N-linked oligosaccharides composition was consistent in all preclinical and clinical rADAMTS13 batches. The main N-glycan structure found on rADAMTS13 is a biantennary, core fucosylated complex type oligosaccharide with one or two sialic acids. Quantification of sialic acid determination showed consistent amounts of sialic acid on the terminal ends of the glycans in preclinical and clinical rADAMTS13 samples. N-glycolylneuraminic acid, a potentially immunogenic variant of sialic acid, was <1% the amount of total sialic acids.

Higher order structure analysis using dynamic light scattering showed a similar hydrodynamic diameter for all preclinical and clinical lots. Finally, FT-IR and CD analysis showed comparable amounts of α-helix and β-sheet in preclinical and clinical lots.

Taken together, similarity was shown between the preclinical and clinical lots of rADAMTS13 in all assays. A phase I clinical trial using rADAMTS13 for the treatment of hereditary TTP patients is expected to start soon.