Modification of rVWF with Polysialic Acid: Biochemical and Functional Characterization in Mice with VWD.

Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we tried to introduce additional sialic acids on rVWF to obtain the opposite: prolonged survival of rVWF in the circulation. Additional sialic acid residues were introduced to rVWF by chemically attaching polysialic acid (PSA, colominic acid) to lysines on recombinant VWF. rVWF was purified from the cell culture supernatant of CHO cells co-expressing rFVIII and rVWF. Coupling of PSA was performed by terminal oxidation of the vicinal diols in PSA to aldehydes and binding to the primary amino groups in the VWF molecule by Schiff-base formation. Schiff-bases were stabilized by reduction with NaCNBH₃ under formation of a secondary amine.

PSA-rVWF conjugates retained their multimeric structure but had slightly increased molecular sizes of the VWF sub-units due to bound PSA. The attachment of PSA to rVWF resulted in a decrease of RCo activity and a decrease in FVIII binding capacity to approximately 60%.

Administration of PSA-rVWF at a dose of 100 units rVWF antigen per kg to VWF-deficient mice resulted in a 2-fold increase in the survival of rVWF as compared to non-modified rVWF. A secondary rise in endogenous murine FVIII after substitution of VWD mice with human VWF occurred elevating FVIII levels from 0.1 (baseline) to 0.4 IU FVIII/mL at 3 hours after application of both non-modified and polysialylated rVWF. Maximum levels of ~ 0.4 IU FVIII/mL were maintained for more than 20 hours in the group treated with PSA-rVWF despite the reduced FVIII binding capacity observed in vitro, while FVIII levels in mice treated with non-modified rVWF steadily declined and returned to their baseline level after 21 hours. The FVIII AUC was 5.3 and 3.3 IU/mL/hr for mice treated with PSA-rVWF and non-modified rVWF, respectively.

We conclude that polysialylation of rVWF represents a method to prolong the survival of rVWF in the circulation.