Michael E. Hrinda

Removal of viral contaminants by monoclonal antibody purification of plasma proteins.

The transmittance of pathogenic viruses by the widespread administration of protein fractions such as F VIII prepared on a large scale from pooled human plasma has been of growing concern. We have now demonstrated that significant amounts of pathogenic viruses including LAV/HTLVIII may be removed by a new large scale fractionation process for the preparation of human F VIII (Monoclate) which employs immunoaffinity chromatography. Model viruses representative of different virus families and the LAV strain of HIV were added to cryoprecipitate and then the mixture was processed as for Monoclate manufacturing. Virus titers were determined at each step of the fractionation procedures. An overall reduction of at least 6 logs was obtained for the model viruses and the HIV due to the purification process. An added heating step further increased the safety margin for the product resulting in at least an overall reduction of 7-9 logs for HIV. Clinical experience with Monoclate in virgin hemophiliacs has confirmed its viral safety. Our laboratories are exploiting a similar strategy of immunoaffinity chromatography to ensure the viral safety of FIX and protein C preparations derived from plasma.

Production and functional characterization of a recombinant fragment of von Willebrand factor (vWF): an antagonist to platelet receptor GP Ib.

We expressed a recombinant peptide fragment (Ser445–Val733) of human von Wille-brand factor (vWF), containing the binding domain for the platelet receptor of GP Ib, in E. coli. This 33 kD peptide blocks binding of the intact vWF molecule to GP Ib in the presence of modulators. Thus, it offers potential as an antithrombotic agent. High level expression was achieved in a plasmid construct driven by the bacteriophage T7 promoter. The peptide was solubilized from inclusion bodies in strong chaotrope, then reduced and alkylated. Following purification, formulation at pH 3.5, and lyophilization, the reconstituted experimental product (RG 12986) exists as an equilibrium of monomer and dimer species. When formulated above pH 5.0, soluble aggregates are formed; these solutions have less bioactivity than RG 12986. Interestingly, the non-aggregated state of RG 12986 remains conserved following dilution and incubation with platelet-poor plasma. The overall purification/low pH formulation strategies may be applicable to other E. coli recombinant proteins having a tendency to aggregate following removal of chaotrope near physiologic pH when in a concentrated format.