Von Willebrand factor (VWF) has been proposed to reduce the immunogenicity of therapeutic factor VIII (FVIII) in patients with hemophilia A. Using FVIII-deficient mice, we compared the immunogenicity of different preparations of plasma-derived (pd) and recombinant (r) FVIII. Treatment of mice with pdFVIII induced significantly lower titers of FVIII inhibitors, as measured by ELISA and in vitro coagulation assays, compared with rFVIII. Furthermore, pre-incubation of rFVIII with excess VWF significantly reduced rFVIII immunogenicity. Our data confirm that pdFVIII induces lower levels of inhibitors than rFVIII, and that VWF is an immuno-chaperone molecule for FVIII. ABSTRACTH emophilia A is a rare genetic hemorrhagic disorder that affects 1 in 5,000-10,000 males. It results from the absence of endogenous pro-coagulant factor VIII (FVIII).1 The management of bleeding episodes involves the intravenous administration of therapeutic FVIII to restore normal hemostasis. Sources of therapeutic FVIII are either pools of plasma from healthy donors or recombinant molecules produced by genetic engineering. In up to 35% of cases, the administration of exogenous FVIII to patients with hemophilia A leads to the development of anti-FVIII alloantibodies that inhibit the pro-coagulant activity of FVIII.2 The occurrence of anti-FVIII antibodies, therefore, prevents further use of FVIII and is a major therapeutic challenge. Several risk factors associated with the development of FVIII inhibitors have been identified. 3,4 In particular, VWF has been proposed as a key chaperon molecule in reducing the immunogenicity of therapeutic FVIII in patients with hemophilia A. There is some evidence to suggest that the incidence of inhibitor development is lower when patients are treated with VWF-containing plasma-derived FVIII (pdFVIII) than when recombinant products without VWF are used. 5 In fact, studies in a murine model of hemophilia A have indicated that the immunogenicity of FVIII is reduced in the presence of VWF. 6We used the well-established model of FVIII-deficient mice to compare the immunogenicity of therapeutic preparations of pdFVIII and recombinant FVIII (rFVIII) that

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as gamma-carboxylation, N- and O-glycosylation, beta-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC-ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn(322) compared to Asn(145). Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser(60) and Ser(52) which are modified by oligosaccharide structures such as fucose and Glc(Xyl)(0-1-2), respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.

We have perfected a large-scale manufacturing process to produce a human plasma-derived VWF concentrate that boasts high specific activity and is very safe for the treatment of patients with von Willebrand disease.

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