Product case study Bene Fix

BeneFix (tradename, also known as nonacog alfa) is a recombinant human blood factor IX approved for general medical use in the USA and EU since 1997. It is indicated for the control and prevention of haemorrhagic episodes in patients with haemophilia B (Christmas disease), including use in a surgical setting. The 55 kDa, 415 amino acid, single-chain protein displays identical amino acid sequence and a relatively similar post-translational modification profile to that of native, serum-derived factor IX. Post-translational modifications present include both N- and O-linked glycosylation, y-carboxylation, P-hydroxylation, sulfation and phosphorylation. The recombinant blood factor IX is produced in an engineered CHO cell line, and downstream processing entails multiple chromatographic steps, ultrafiltration and diafiltration. The final product is presented in lyophilized form in single-use vials and contains histidine, sucrose, glycine and polysorbate 80 as excipients. Vials contain 250, 500 or 1000 IU of factor IX (as determined by a one-stage clotting assay against a World Health Organization international standard). The product is intended for i.v. administration and exact dosage/administration regimes depend upon the severity of the factor IX deficiency, the location and extent of bleeding, and the patient's clinical condition.

The product displays a mean serum half-life of 18.8 h in humans. It has been evaluated in four clinical trials involving a total of 128 subjects and in the context of both spontaneous bleeding and surgery. Some 88 per cent of the total infusions administered for bleeding were rated as providing a 'good' or 'excellent' response. Reported side effects, although uncommon, included hypersensitivity, as well as headache, fever and nausea. BeneFix is marketed by Wyeth.

recombinant products. At least one monoclonal antibody has been raised which specifically binds only to factor IX which contains pre-bound Ca2+ (i.e. the Ca2+-dependent conformation of factor IX). Immobilization of this antibody allowed the development of an immunoaffinity system in which factor IX binds to the column in the presence of a Ca2+-containing buffer. Subsequent elu-tion is promoted simply by inclusion of a chelating agent (e.g. EDTA) in the elution buffer.

Some 5-25 per cent of individuals suffering from haemophilia A develop anti-factor VIII antibodies, and 3-6 per cent of haemophilia B sufferers develop anti-factor IX antibodies. This complicates treatment of these conditions and, as mentioned previously, one approach to their treatment is direct administration of factor VIIa. The therapeutic rationale is that factor VIIa could directly activate the final common steps of the coagulation cascade, independently of either factor VIII or IX (Figure 12.1). Factor VIIa forms a complex with tissue factor that, in the presence of phospholipids and Ca2+, activates factor X.

A recombinant form of factor VIIa (called 'NovoSeven' or 'eptacog alfa-activated') is marketed by Novo-Nordisk (Table 12.2). The recombinant molecule is produced in a BHK cell line, and the final product differs only slightly (in its glycosylation pattern only) from the native molecule.

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor VIIa complex. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried.

A (very rare) genetic deficiency in the production of factor XIII also results in impaired clotting efficacy in affected persons. In this case, covalent links that normally characterize transformation of a soft clot into a hard clot are not formed. Factor XIII preparations, partially purified from human blood, are used to treat individuals with this condition; to date, no recombinant version of the product has been commercialized.

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