Nonclinical Safety Assessment of a Long-Acting Recombinant PEGylated Factor Eight (BAY 94-9027) With a 60 kDa PEG.

BAY 94-9027 (Jivi) is a site-specifically PEGylated human B-domain-deleted (BDD) recombinant factor VIII (FVIII), with a 60 kDa branched PEG molecule attached. The nonclinical safety of BAY 94-9027 was evaluated in a toxicology program that included 2 weeks intravenous (IV) toxicity studies in rats and rabbits, a juvenile toxicity study in rats as well as a 26-week chronic study in rats. Doses of 75, 750, or 2250 IU/kg given every other day for 2 weeks did not elicit any findings related to BAY 94-9027. Specifically, no thrombus formation or histological changes such as cellular vacuolation were seen. In the chronic toxicity study, 40, 400, and 1200 IU/kg of BAY 94-9027 given twice weekly did not induce adverse effects related to BAY 94-9027, and no tissue vacuolation was observed. There was no PEG detected in choroid plexus or other areas of the brain, cerebrospinal fluid or in spleen or kidneys. These results were supported by toxicity studies in rats and rabbits treated with PEG 60 kDa attached to the maleimide linker (PEG-60-Mal-Cys). No findings related to PEG-60-Mal-Cys were seen. These results demonstrate the safety of BAY 94-9027 for long-term use.

Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.

Patients with haemophilia (PWH) are usually monitored by the one-stage activated partial thromboplastin time (aPTT) factor VIII (FVIII) assay. Different aPTT activators may affect clotting time (CT) and FVIII:C levels in patients treated with PEGylated FVIII. To evaluate the characteristics of PEGylated FVIII (BAY 94-9027) in various aPTT clotting assays, and to identify suitable aPTT reagents for monitoring BAY 94-9027 during the treatment of PWH, BAY 94-9027 and World Health Organization (WHO) 8th FVIII standards (WHO-8) were spiked into pooled and individual severe haemophilia A plasma at 1.0, 0.25 and 0.05 IU mL(-1) . Five commercial aPTT reagents widely used in clinical laboratories were compared and evaluated for BAY 94-9027 activity in plasma from PWH. BAY 94-9027 and WHO-8 bestowed similar CT and excellent precision when ellagic acid (SynthAFax, Dade Actin, and Cephascreen) aPTT reagents were used. In contrast, BAY 94-9027 showed significantly prolonged CT and poor precision compared with WHO-8 using silica aPTT reagents (APTT-SP and STA PTT 5). Furthermore, free 60-kDa polyethylene glycol (PEG), used for the conjugation of FVIII, showed a dose-dependent prolongation of CT in the APTT-SP assay. There was no effect on the SynthAFax-APTT, prothrombin time, or FXIa-initiated thrombin generation assay, demonstrating that the PEG moiety on FVIII has no general effect on the coagulation cascade. In summary, ellagic aPTT reagents (SynthAFax, Dade Actin, and Cephascreen) are most suitable for evaluating potency of BAY 94-9027 and should be the preferred aPTT reagents used in clinical laboratories for monitoring FVIII activity after infusion of BAY 94-9027 to PWH.

Development of long-acting recombinant FVIII and FIX Fc fusion proteins for the management of hemophilia.

INTRODUCTION: Prophylactic treatment with replacement clotting factor is the recommended regimen for patients with severe hemophilia to prevent bleeding episodes. However, currently available replacement clotting factors are limited by their relatively short half-lives and require intravenous injections up to three times weekly to maintain protective levels, which can impact compliance and, thus, patient outcomes. AREAS COVERED: The potential advantages of long-acting coagulation factors, including reduced injection frequency, increased treatment adherence, and improved clinical outcomes, are discussed. Fragment crystallizable (Fc) fusion technology is introduced and the development of long-acting recombinant factor VIII Fc (rFVIIIFc) and recombinant factor IX Fc (rFIXFc) fusion proteins for the treatment of hemophilia A and B, respectively, are described. Preclinical and clinical studies of rFVIIIFc and rFIXFc showing improved pharmacokinetics over currently available products are reviewed. EXPERT OPINION: Long-acting coagulation factors, including rFVIIIFc and rFIXFc, have the potential to change current paradigms of care for hemophilia A and B, respectively. Less frequent infusions may provide prolonged protection from bleeding and bleed resolution with fewer injections. In addition, long-acting coagulation factors provide an opportunity for improved individualized treatment for hemophilia.

A solvent/detergent-treated and 15-nm filtered factor VIII: a new safety standard for plasma-derived coagulation factor concentrates.

BACKGROUND: Since the early 1990 s the Committee for Proprietary Medicinal Products has set the mandatory requirement that all manufacturing processes for blood products include two virus removal/inactivation steps that are complementary in their action. OBJECTIVES: The objective was to develop a manufacturing process for factor VIII (FVIII) including two complementary steps of viral inactivation/elimination. METHODS: A 35-15 nm nanofiltration step was added to a former FVIII manufacturing process that included solvent/detergent (S/D) treatment to generate a new FVIII concentrate called Factane. The impact of nanofiltration on the structural and functional characteristics of FVIII, as well as virus/transmissible spongiform encephalopathy reduction factors were assessed. RESULTS: Using an innovative approach, FVIII was successfully nanofiltered at 35-15 nm, while the biological properties of the active substance were unmodified. FVIII coagulant and antigen content for Factane and previous S/D-treated FVIII (FVIII-LFB, commercialized as Facteur VIII-LFB) were comparable. The FVIII one-stage chromogenic and coagulant/antigen ratios confirmed that nanofiltered FVIII was not activated. After nanofiltration, the copurified von Willebrand factor (vWF) was reduced but vWF/FVIII binding properties were unaffected. Phospholipid binding and thrombin proteolysis studies displayed no differences between Factane and FVIII-LFB. The rate of factor Xa generation was slightly lower for Factane when compared to FVIII-LFB. Viral validation studies with different viruses showed no detectable virus in the filtrate. CONCLUSIONS: Nanofiltration of FVIII at 15 nm is feasible despite the large molecular weight of FVIII and vWF. Nanofiltration has been proven to be highly effective at removing infectious agents while preserving the structural and functional integrity of FVIII.

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+.

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.

Defective discoidin domain structure, subunit assembly, and endoplasmic reticulum processing of retinoschisin are primary mechanisms responsible for X-linked retinoschisis.

Retinoschisin is a 24-kDa discoidin domain-containing protein that is secreted from photoreceptor and bipolar cells as a large disulfide-linked multisubunit complex. It functions as a cell adhesion protein to maintain the cellular organization and synaptic structure of the retina. Over 125 different mutations in the RS1 gene are associated with X-linked juvenile retinoschisis, the most common form of early onset macular degeneration in males. To identify molecular determinants important for retinoschisin structure and function and elucidate molecular and cellular mechanisms responsible for X-linked juvenile retinoschisis, we have analyzed the expression, protein folding, disulfide-linked subunit assembly, intracellular localization, and secretion of wild-type retinoschisin, 15 Cys-to-Ser variants and 12 disease-linked mutants. Our studies, together with molecular modeling of the discoidin domain, identify Cys residues involved in intramolecular and intermolecular disulfide bonds essential for protein folding and subunit assembly. We show that misfolding of the discoidin domain, defective disulfide-linked subunit assembly, and inability of retinoschisin to insert into the endoplasmic reticulum membrane as part of the protein secretion process are three primary mechanisms responsible for the loss in the function of retinoschisin as a cell adhesion protein and the pathogenesis of X-linked juvenile retinoschisis.

Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity.

Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).

In vitro stability of recombinant human factor VIII (Recombinate).

Factor VIII (FVIII) is currently administered in diverse settings and by a range of methods, and it is important that the stability of specific FVIII preparations be documented for these varying uses. This study of Recombinate recombinant human FVIII (rhFVIII) evaluated: (i) thermostability; (ii) photostability; (iii) stability during simulated continuous infusion; and (iv) stability after dilution. This evaluation was conducted over a range of initial rhFVIII potencies and under differing conditions of temperature, light exposure, dilution and heparin usage. FVIII biological activity was measured by one-stage and chromogenic substrate assays. Microbiological assessment was also performed. Lyophilized rhFVIII was found to be highly thermostable, as evidenced by an energy of activation (Ea) of 16.2 kcal mol-1 and recovery of 99.3% of initial activity after incubation for 6 months at 40 degrees C and 93.8% at 60 degrees C for 2 months. No significant loss of activity could be detected after accelerated simulated natural daylight exposure of lyophilized rhFVIII, although partial activity loss was observed after similar exposure of reconstituted rhFVIII. Shielding in foil wrap effectively prevented such photodegradation of reconstituted rhFVIII. Based upon these results, exposure of lyophilized rhFVIII to sunlight is unlikely to affect stability adversely. Activity of reconstituted rhFVIII (22-106 IU mL-1) remained stable during simulated continuous infusion for 96 h at ambient (20-25 degrees C) and elevated (28-32 degrees C) temperature, and in the presence or absence of 1 U mL-1 heparin. After dilution of reconstituted rhFVIII, an immediate 14-42% loss of expected rhFVIII activity was observed depending upon diluent composition. Accordingly, potential partial loss of rhFVIII activity should be taken into account when dilution is being considered. rhFVIII remained sterile at least 96 h during simulated continuous infusion. rhFVIII is a robust preparation exhibiting biological stability under a wide array of clinically relevant conditions.

Structure and topography of the membrane-binding C2 domain of factor VIII in the presence of dodecylphosphocholine micelles.

A 21 residue peptide from the C2 domain of the antihaemophilic factor VIII competes with factor VIII for membrane-binding sites in vitro. Here, we provide the structure and topography of the peptide in solution, on dodecylphosphocholine (DPC) micelles, determined using 1H-NMR spectroscopy. The peptide assumes an amphipathic structure comprising an extended N-terminal region and a C-terminal helix. The average root-mean-square deviation is 0.7+/-0.2 A for the superimposition of the backbone atoms of Ile6 to Arg18 on the lowest energy structure. Whereas the backbone conformation is similar to that in SDS micelles, the Trp11 side-chain orientation is dramatically changed. The indole ring is nearly parallel to the peptide backbone in SDS micelles but perpendicular in DPC micelles. Further, pKa values of the two histidines change by more than 1 pH unit in SDS relative to DPC, which localizes the imidazole rings to the interfacial region. Line-broadening induced by spin-labelled phosphatidylcholine shows that most of the amino acid side-chains that penetrate the DPC micelle are hydrophobic. Thus, the long axis of the peptide lies parallel to the micelle surface and the hydrophobic face of the alpha-helix provides hydrophobic membrane interaction. The large chemical shift changes shown by Trp11 and N-terminal amino acid residues in SDS relative to DPC indicate that this region may be involved in membrane phospholipid recognition. 1H-NMR assignments, CD spectra, one-dimensional 1H-NMR spectra, chemical-shift analysis and nuclear Overhauser effect information are reported in Supplementary Publication SUP 50184 (11 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K, from whom copies can be obtained according to the terms indicated in Biochem. J. (1997) 321, 8.

A 110-amino acid region within the A1-domain of coagulation factor VIII inhibits secretion from mammalian cells.

Factor VIII is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A. Factor VIII is homologous to blood coagulation factor V, both having a domain structure of A1-A2-B-A3-C1-C2. Previous transfection studies demonstrated that factor VIII is 10-fold less efficiently expressed than the homologous coagulation factor, factor V. The inefficient expression correlated with interaction of the factor VIII primary translation product with the protein chaperonin BiP in the lumen of the endoplasmic reticulum. In contrast, factor V was not detected in association with BiP and was secreted efficiently. To determine whether specific amino acid sequences within factor VIII inhibit secretion, we have studied the secretion of factor VIII deletion and factor VIII/factor V chimeric proteins upon transient transfection of COS-1 monkey cells. A chimeric factor VIII protein that contained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera having the A1- and A2-domains of factor VIII was secreted with low efficiency, similar to wild-type factor VIII. These results suggested that sequences within the A1- and A2-domains were responsible for the low secretion efficiency of factor VIII. Secretion of A1-domain-deleted factor VIII was increased approximately 10-fold compared to wild-type factor VIII or A2-domain-deleted factor VIII. Expression of the factor VIII A1-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domain alone directed synthesis of secreted protein. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild-type factor VIII, however, the secreted protein was not functional and the heavy and light chains were not associated. These results localize a 110-amino acid region within the A1-domain that inhibits factor VIII secretion. This region is clustered with multiple short peptide sequences that have potential to bind BiP.