Physicochemical characterisation of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

rVIII-SingleChain is a novel recombinant single-chain factor VIII (FVIII) construct, comprising covalently bonded heavy and light chains. Post-translational modifications of FVIII affect physicochemical parameters, including hydrophobicity and charge. The most relevant post-translational modifications of FVIII products are N-glycosylation of asparagine residues and tyrosine sulphations. Here, the physicochemical properties, thrombin cleavage products and post-translational modifications of rVIII-SingleChain were investigated and compared against commercially available recombinant FVIII (rFVIII) products with a predominant two-chain structure (B-domain deleted rFVIII and full-length rFVIII). rVIII-SingleChain was expressed in Chinese hamster ovary (CHO) cells and purified by chromatographic methods. Physicochemical properties of rVIII-SingleChain or thrombin-derived cleavage products were assessed using size-exclusion chromatography, reversed-phase chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Analysis of the respective carbohydrate structures was performed after release of N-glycans by PNGase F followed by fluorescence labelling and high-performance liquid chromatography. Proteolysis by trypsin generated the corresponding peptides, which were analysed for sulphated tyrosines by liquid chromatography-electrospray ionisation time of flight-mass spectrometry. rVIII-SingleChain was shown to be of high purity and homogeneity, and presented a well-defined single-chain molecule with predominant ß-sheet conformation. The coagulation-relevant thrombin-activation products of rVIII-SingleChain were comparable with those obtained by activation of commercially available rFVIII products. rVIII-SingleChain post-translational modifications were similar to other CHO cell-derived rFVIII products for N-glycopattern and tyrosine sulphation. In conclusion, rVIII-SingleChain is of high homogeneity and purity, and provides an expected cleavage pattern on activation, setting the basis for optimal efficacy in the patient.

Non-clinical pharmacokinetics and pharmacodynamics of rVIII-SingleChain, a novel recombinant single-chain factor VIII.

INTRODUCTION: rVIII-SingleChain (CSL627), a novel recombinant coagulation factor VIII (FVIII), is under investigation in a phase I/III clinical programme (AFFINITY) for the treatment of haemophilia A. Non-clinical studies were conducted to investigate the pharmacokinetic/pharmacodynamic profile of rVIII-SingleChain in comparison with full-length recombinant FVIII. MATERIALS AND METHODS: Binding affinity of rVIII-SingleChain for von Willebrand factor was investigated by surface plasmon resonance analysis. The pharmacokinetic profile of rVIII-SingleChain was compared with a marketed full-length recombinant FVIII concentrate (Advate(®)) in haemophilia A mice, von Willebrand factor knock-out mice, Crl:CD (SD) rats, rabbits and cynomolgus monkeys. Systemic FVIII activity or antigen levels were recorded. Procoagulant activity was measured in an FeCl3-induced arterial occlusion model and by recording thrombin generation activity (ex vivo) after administration of 200-250 IU/kg rVIII-SingleChain or full-length FVIII to haemophilia A mice. RESULTS: rVIII-SingleChain displayed a high affinity for von Willebrand factor (KD=44 pM vs. 139 pM for full-length recombinant FVIII). In all animal species tested, rVIII-SingleChain had more favourable pharmacokinetic properties than full-length recombinant FVIII: clearance was decreased and area under the curve and terminal half-life were enhanced vs. full-length recombinant FVIII, while in vivo recovery and volume of distribution were equivalent. rVIII-SingleChain showed a prolonged thrombin generation potential and prolonged procoagulant activity vs. full-length recombinant FVIII in an FeCl3-induced arterial occlusion model. CONCLUSIONS: rVIII-SingleChain had a higher affinity for von Willebrand factor than full-length recombinant FVIII and displayed favourable pharmacokinetic/pharmacodynamic properties in non-clinical models.

Preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII.

INTRODUCTION: The preclinical efficacy and safety of rVIII-SingleChain (CSL627), a novel recombinant single-chain factor VIII, was assessed in a series of animal studies. MATERIALS AND METHODS: In the tail-clip bleeding model, hemophilia A mice were injected with escalating doses (1-150 IU/kg) of rVIII-SingleChain, B-domain deleted (BDD) rFVIII (ReFacto AF(®)), or full-length rFVIII products (Advate(®), Helixate(®)). Total blood loss and the percentage of animals in which hemostasis occurred were assessed in this observer-blinded, randomized study. In a second non-randomized study in hemophilia A mice, thromboelastographic analysis, thrombin generation, and activated partial thromboplastin time assays were performed. General safety and toxicity were assessed in three animal species, including determination of the prothrombotic potential of rVIII-SingleChain in a rabbit venous thrombosis model. RESULTS: Under acute bleeding conditions, the effect of rVIII-SingleChain on total blood loss and hemostasis was indistinguishable from BDD and full-length rFVIII. rVIII-SingleChain and full-length rFVIII (both 20 IU/kg) corrected thromboelastographic parameters, activated partial thromboplastin time, and thrombin generation to a similar degree in hemophilia A mice. In a thrombosis model, the effect of rVIII-SingleChain on thrombus incidence was non-significant and comparable to BDD rFVIII at doses up to 500 IU/kg. Treatment with rVIII-SingleChain did not cause anaphylactic reaction or local intolerance in safety and toxicity studies, and demonstrated an excellent overall safety profile. CONCLUSIONS: rVIII-SingleChain showed convincing hemostatic efficacy and excellent tolerability in animal studies, warranting continued investigation in human Phase I/III trials (AFFINITY).

Genetic fusion to albumin improves the pharmacokinetic properties of factor IX.

Haemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates. To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX). In FIX deficient (FIX(-/-)) mice the in-vivo recovery and the AUC were also significantly increased. The efficacy in reducing bleeding time was shown in FIX(-/-) mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

Comparative analysis and classification of von Willebrand factor/factor VIII concentrates: impact on treatment of patients with von Willebrand disease.

von Willebrand disease (vWD) is a bleeding disorder that results from defects in the quality or quantity of von Willebrand factor (vWF), a glycoprotein essential for normal thrombus formation. vWF circulates in plasma as multimers in sizes ranging up to 20,000 kd. The high molecular weight vWF (HMWvWF) multimers are most essential for primary hemostasis, whereas the lower molecular weight multimers are less functionally active. For many patients, the treatment of choice is factor replacement with a vWF/FVIII concentrate, preferably one with a high content of HMWvWF multimers. Given that the commercially available vWF/FVIII concentrates seem to differ substantially in their biochemical properties as well as in their clinical efficacy, we did a comparative study with 12 vWF/FVIII concentrates to investigate content and activities of FVIII and vWF, as well as the content of HMWvWF multimers. The content of HMWvWF multimers varied considerably among the 12 concentrates. The specific vWF activities, as assessed by ristocetin cofactor activity (vWF:RCo) and collagen-binding activity (vWF:CB), correlated well with the HMWvWF content of the products. Of the products tested, Haemate P/Humate-P had the highest content of HMWvWF multimers (with a multimer pattern closest to that of normal human plasma), the highest specific vWF activities, and the highest values of vWF:RCo and vWF:CB per unit of FVIII:coagulant (C). The goal of bleeding prophylaxis and treatment in type 2, severe type 1, and type 3 vWD patients is to normalize vWF activities (vWF:RCo and vWF:CB) and FVIII:C preferentially by vWF/FVIII concentrates containing the high vWF multimers and a high vWF:RCo/FVIII ratio to achieve normal primary and secondary hemostasis. Based on the present study of a comparative analysis of currently available vWF/FVIII concentrates, a classification of vWF/FVIII products is proposed.

The importance of factor XIII as a component of fibrin sealants.

BACKGROUND: The need for factor XIII (FXIII) in fibrin sealant is subject to discussions. Some commercially available fibrin sealants (FS) contain high levels of FXIII (up to 70-80 U/mL) while others contain low levels or none. The objective of the present studies was to investigate the need for FXIII in FS. MATERIALS AND METHODS: Beriplast P, a commercial FS containing 40-80 U/mL FXIII, was compared with FS with different concentrations of FXIII or FXIII depleted FS. In Study 1, Beriplast P or FS with 4 U/mL FXIII was allowed to cross-link for 0.25, 1, and 2 min and the resulting fibrin gamma- and beta-chains as well as gamma-gamma-chain dimers were analyzed by SDS-PAGE and densitometry. In Study 2, a series of six FS was prepared from fibrinogen containing 0 (FS-FXIII), 17.5, 35, 70, 140, and 280 U/mL FXIII. The intrasealant strength was determined using a biomechanical test (twin-bottle test). In Study 3, 18 rabbits underwent bilateral partial kidney resection (acute model); right (n = 18) and left (n = 18) kidneys were treated with Beriplast P or FS with 4 U/mL FXIII (FS + 4U FXIII). In Study 4, rabbits were randomly assigned to receive FXIII depleted FS (FS-FXIII) (n = 15); FS + 3 U/mL FXIII (n = 10); FS + 10 U/mL FXIII (n = 10); or Beriplast P (n = 15), after partial rabbit kidney resection (chronic model). The primary endpoint for Studies 3 and 4 was hemostasis. RESULTS: In Study 1, Beriplast P produced significantly more fibrin gamma-chain cross-linking in the observed period than FS with 4 U/mL FXIII. In Study 2, fibrin clot strength increased as a function of FXIII concentration with a maximum of 70 U/mL. In Study 3, significantly more animals achieved hemostasis in the Beriplast P treated group than in the FS + 4U FXIII group (P = 0.03 at 15 s; P < 0.001 at 1 and 2 min). In Study 4, FS containing FXIII (all concentrations) achieved more efficient hemostasis and reduced the need for respray compared with FS-FXIII. Beriplast P was associated with a lower incidence of postoperative adhesions compared with the other treatment groups. At autopsy, the majority of clots formed from FXIII containing FS exhibited mild to moderate clot lysis, whereas the majority of clots in the FS-FXIII group showed severe lysis. CONCLUSIONS: FXIII, when added to fibrinogen concentrate in FS, improved fibrin cross-linking and clot strength. FXIII containing FS achieved more effective hemostasis than FXIII depleted FS. FXIII is an essential component of commercial FS and should be present in FS at an optimal concentration of about 40-80 U/mL.

Targeted inactivation of the mouse locus encoding coagulation factor XIII-A: hemostatic abnormalities in mutant mice and characterization of the coagulation deficit.

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin gamma-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.