Plasma-derived FVIII/VWF complex shows higher protection against inhibitors than isolated FVIII after infusion in haemophilic patients: A translational study.

INTRODUCTION: Presence of von Willebrand factor (VWF) in FVIII concentrates offers protection against neutralizing inhibitors in haemophilia A (HA). Whether this protection is more evident in plasma-derived (pd) FVIII/VWF or recombinant (r) FVIII concentrates remains controversial. AIM: We investigated the protection exerted by VWF against FVIII inhibitors in an in vivo mouse model of HA exposed to pdFVIII/VWF or to various rFVIII concentrates. METHODS: Haemophilia A mice received the different FVIII concentrates after administration of vehicle or an inhibitory IgG purified from a commercial pool of HA plasma with inhibitors and FVIII:C recoveries were measured. Furthermore, using a novel clinically oriented ex vivo approach, Bethesda inhibitory activities (BU) of a commercial pool of HA plasma with inhibitors were assessed using normal plasma, or plasma from severe HA patients, without inhibitors, after treatment with the same concentrates. RESULTS: in vivo studies showed that pdFVIII/VWF offers markedly higher protection against inhibitors when compared with any of the FVIII products without VWF. More importantly, in the ex vivo studies, plasma from patients treated with pdFVIII/VWF showed higher protection against inhibitors (P values ranging .05-.001) in comparison with that observed in plasma from patients who received FVIII products without VWF, regardless of the type of product evaluated. CONCLUSION: Data indicate that FVIII+VWF complexes assembled in the circulation after rFVIII infusion are not equivalent to the naturally formed complex in pdFVIII/VWF. Therefore, rFVIII infused into HA patients with inhibitors would be less protected by VWF than the FVIII in pdFVIII/VWF concentrates.

Conformation of the von Willebrand factor/factor VIII complex in quasi-static flow.

Von Willebrand factor (VWF) is a plasma glycoprotein that circulates noncovalently bound to blood coagulation factor VIII (fVIII). VWF is a population of multimers composed of a variable number of ∼280 kDa monomers that is activated in shear flow to bind collagen and platelet glycoprotein Ibα. Electron microscopy, atomic force microscopy, small-angle neutron scattering, and theoretical studies have produced a model in which the conformation of VWF under static conditions is a compact, globular "ball-of-yarn," implying strong, attractive forces between monomers. We performed sedimentation velocity (SV) analytical ultracentrifugation measurements on unfractionated VWF/fVIII complexes. There was a 20% per mg/ml decrease in the weight-average sedimentation coefficient, sw, in contrast to the ∼1% per mg/ml decrease observed for compact globular proteins. SV and dynamic light scattering measurements were performed on VWF/fVIII complexes fractionated by size-exclusion chromatography to obtain sw values and z-average diffusion coefficients, Dz. Molecular weights estimated using these values in the Svedberg equation ranged from 1.7 to 4.1 MDa. Frictional ratios calculated from Dz and molecular weights ranged from 2.9 to 3.4, in contrast to values of 1.1-1.3 observed for globular proteins. The Mark-Houwink-Kuhn-Sakurada scaling relationships between sw, Dz and molecular weight, [Formula: see text] and [Formula: see text] , yielded estimates of 0.51 and -0.49 for as and aD, respectively, consistent with a random coil, in contrast to the as value of 0.65 observed for globular proteins. These results indicate that interactions between monomers are weak or nonexistent and that activation of VWF is intramonomeric.

Characterization of the von Willebrand factor/factor VIII complex produced by a novel purification process.

Factor VIII (FVIII) is a blood coagulation protein that circulates as a complex with von Willebrand factor (vWF) in the plasma. In the survey of inhibitors in plasma product exposed toddlers (SIPPET) study, plasma-derived FVIII containing vWF was less immunogenic in hemophilia A patients than products with only high-purity FVIII only or recombinant FVIII. The  FVIII purified by the conventional purification process using anion-exchange (AEX) chromatography had a low vWF content. In this study, purified vWF was added to the purified FVIII to increase the vWF content. The purified vWF was recovered from the discarded washing solution of the AEX chromatography using cation-exchange (CEX) chromatography. The vWF/FVIII complex had an abundance of high molecular weight vWF similar to the normal plasma, and a low reactivity of FVIII inhibitors. Furthermore, its efficacy was observed in a mouse model of hemophilia A. Therefore, the vWF/FVIII complex produced by our new purification method could be an effective and less immunogenic therapeutic agent for the hemophilia A and von Willebrand disease.

How Full-Length FVIII Benefits from Its Heterogeneity - Insights into the Role of the B-Domain.

PURPOSE: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-ß sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

Purification Methods for Recombinant Factor VIII Expressed in Human Liver SK-Hep Cells.

Coagulation factor VIII is one of the largest proteins attempted to be expressed in recombinant form. A very complex and labile protein which has a very short half-live and need a fast and efficient purification chain. Here, we describe a simple purification sequence using multimodal Capto MMC, affinity FVIII select and ion exchange SP-Fastflow chromatography steps without subjecting the target molecule to mechanical and temperature stress, separating impurities from rFVIII using net charge, hydrophobicity, and affinity of the molecules.

Identification of aggregates in therapeutic formulations of recombinant full-length factor VIII products by sedimentation velocity analytical ultracentrifugation.

Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A. Aggregates in biopharmaceutical products are an immunogenic risk factor. Aggregates were identified in recombinant full-length factor VIII products. Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. SUMMARY: Background The development of inhibitory anti-factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full-length FVIII is more immunogenic than plasma-derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti-drug antibodies. Objective To evaluate recombinant full-length FVIII products for the presence of aggregates. Methods Advate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s20,w values and molecular weights. Results The major component of all three products was a population of ~7.5 S heterodimers with a weight-average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer-exchanged Advate and Kogenate. SV AUC analysis of a plasma-derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII. Conclusions Aggregate formation has been identified in recombinant full-length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.

Establishment of the 2nd Korean national biological reference standard for blood coagulation factor VIII:C concentrate.

Since the 1st Korean national biological reference standard for factor (F)VIII concentrate, established in 2001, has shown declining potency, we conducted this study to replace this standard with a 2nd Korean national biological reference standard for blood coagulation FVIII concentrate. The candidate materials for the 2nd standard were prepared in 8000 vials with 10 IU/ml of target potency, according to the approved manufacturing process of blood coagulation Factor VIII:C Monoclonal Antibody-purified, Freeze-dried Human Blood Coagulation Factor VIII:C. Potency was evaluated by one-stage clotting and chromogenic methods and the stability was confirmed to meet the specifications during a period of 73 months. Since the potencies obtained by the two methods differed significantly (P < 0.015), the values were determined separately according to the geometric means (8.9 and 7.4 IU/vial, respectively). The geometric coefficients of interlaboratory variability were 3.4% and 7.6% by the one-stage clotting and chromogenic assays, respectively.

Evaluation of the virus clearance capacity and robustness of the manufacturing process for the recombinant factor VIII protein, turoctocog alfa.

Turoctocog alfa is a B-domain-truncated recombinant factor VIII protein produced in a Chinese hamster ovary (CHO) cell line. The aim of this study was to evaluate the virus clearance capacity and robustness of the turoctocog alfa purification process. Virus clearance evaluation studies were conducted utilising a scaled-down version of the manufacturing process. Total virus clearance was evaluated using the ecotropic murine leukaemia virus (eMuLV) as a model for non-infectious retrovirus-like particles (RVLPs) and certain enveloped viruses. Other viruses utilised included: infectious bovine rhinotracheitis (IBRV), minute virus of mice (MVM), bovine enterovirus (BEV) and Reo-3 virus (Reo-3). Robust clearance of all model viruses was demonstrated with either new or reused resins. Overall, virus reduction factors were: >18.0 log10 (eMuLV); 11.0 log10 (MVM); >11.8 log10 (Reo-3; >5.0 log10 using nanofiltration); >15.3 log10 (BEV) and >12.7 log10 (IBRV). Taken together, these values demonstrate that the purification process for turoctocog alfa effectively removes a range of enveloped and non-enveloped viruses of different physicochemical properties and sizes.

A randomized study of very low-dose factor VIII prophylaxis in severe haemophilia - A success story from a resource limited country.

INTRODUCTION: Current factor prophylaxis strategy practised in developed countries is not feasible in resource constraint developing countries like India. AIM: The aim of this study was to investigate the efficacy and safety of very low-dose factor prophylaxis in India. METHODS: Children of 1-10 years of age with severe haemophilia were randomized to Prophylaxis group and Episodic (On demand) group. Children in prophylaxis group received very low-dose factor VIII (FVIII) concentrate, i.e. 10 units kg(-1) body weights on 2 days a week. Episodic group received factor concentrate in standard recommended doses. The study period was 11.5 months. RESULTS: In total 21 children were enrolled in this study, 11 assigned to prophylaxis and 10 to episodic group. Children on prophylaxis had 11 joint bleeds in comparison to 57 joint bleeds in episodic group. Mean number of haemarthrosis per patient per month were 0.08 (0.08 ± 0.13) in prophylaxis group compared to 0.48 (0.48 ± 0.34) in episodic group (P < 0.05). Total FVIII consumption was 87.51 and 56.32 units kg(-1) month(-1) in prophylaxis and episodic group respectively (P = ns). Overall median hospital emergency visits were 1 day in prophylaxis group and 9 days in episodic group (P ≤ 0.05). Median days of absenteeism from school were 25 days in episodic group and 3 days in prophylaxis group (P < 0.05). No significant complications were noted in prophylaxis group and compliance was 98%. CONCLUSION: To conclude, low-dose FVIII prophylaxis is cost effective, efficacious and a safe method of preventing joint bleeds and consequent joint damages.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

INTRODUCTION: Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. AIMS: To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. METHODS AND RESULTS: The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. CONCLUSIONS: Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin.

Manufacturing process used to produce long-acting recombinant factor VIII Fc fusion protein.

Recombinant factor VIII Fc fusion protein (rFVIIIFc) is a long-acting coagulation factor approved for the treatment of hemophilia A. Here, the rFVIIIFc manufacturing process and results of studies evaluating product quality and the capacity of the process to remove potential impurities and viruses are described. This manufacturing process utilized readily transferable and scalable unit operations and employed multi-step purification and viral clearance processing, including a novel affinity chromatography adsorbent and a 15 nm pore size virus removal nanofilter. A cell line derived from human embryonic kidney (HEK) 293H cells was used to produce rFVIIIFc. Validation studies evaluated identity, purity, activity, and safety. Process-related impurity clearance and viral clearance spiking studies demonstrate robust and reproducible removal of impurities and viruses, with total viral clearance >8-15 log10 for four model viruses (xenotropic murine leukemia virus, mice minute virus, reovirus type 3, and suid herpes virus 1). Terminal galactose-α-1,3-galactose and N-glycolylneuraminic acid, two non-human glycans, were undetectable in rFVIIIFc. Biochemical and in vitro biological analyses confirmed the purity, activity, and consistency of rFVIIIFc. In conclusion, this manufacturing process produces a highly pure product free of viruses, impurities, and non-human glycan structures, with scale capabilities to ensure a consistent and adequate supply of rFVIIIFc.

Danger signal-dependent activation of human dendritic cells by plasma-derived factor VIII products.

Treatment of haemophilia A by infusions of the clotting factor VIII (FVIII) results in the development of inhibitors/anti-drug antibodies in up to 25 % of patients. Mechanisms leading to immunogenicity of FVIII products are not yet fully understood. Amongst other factors, danger signals as elicited upon infection or surgery have been proposed to play a role. In the present study, we focused on effects of danger signals on maturation and activation of dendritic cells (DC) in the context of FVIII application. Human monocyte-derived DC were treated with FVIII alone, with a danger signal alone or a combination of both. By testing more than 60 different healthy donors, we show that FVIII and the bacterial danger signal lipopolysaccharide synergise in increasing DC activation, as characterised by increased expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. The degree and frequency of this synergistic activation correlate with CD86 expression levels on immature DC prior to stimulation. In our assay system, plasma-derived but not recombinant FVIII products activate human DC in a danger signal-dependent manner. Further tested danger signals, such as R848 also induced DC activation in combination with FVIII, albeit not in every tested donor. In our hands, human DC but not human B cells or macrophages could be activated by FVIII in a danger signal-dependent manner. Our results suggest that immunogenicity of FVIII is a result of multiple factors including the presence of danger, predisposition of the patient, and the choice of a FVIII product for treatment.

Purification of coagulation factor VIII by immobilized metal affinity chromatography.

Factor VIII (FVIII) is a glycoprotein that plays an essential role in blood coagulation cascade. Purification of plasma-derived coagulation FVIII by direct application of plasma to a chromatographic column is a method of choice. Anion exchange column is a very powerful method because FVIII is strongly adsorbed, resulting in good activity recovery and high purification factor. However, vitamin-K-dependent coagulation factors coelute with FVIII. In the present study, we report the separation of vitamin-K-dependent coagulation proteins from FVIII using immobilized metal affinity chromatography (IMAC) with Cu(2+) as the metal ligand. Plasma was directly loaded to a Q Sepharose Big Beads column, and FVIII was recovered with 65% activity and a purification factor of approximately 50 times. Then, the Q Sepharose eluate was applied to the IMAC-Cu(2+) column, and FVIII was eluted with 200 mM imidazole, with up to 85% recovery of activity. The mass recovery in this fraction was less than 10% of the applied mass of protein. Vitamin-K-dependent proteins elute with imidazole concentrations of lower than 60 mM. Because of the difference in affinity, FVIII could be completely separated from the vitamin-K-dependent proteins in the IMAC column.

Turoctocog alfa (NovoEight®)--from design to clinical proof of concept.

Turoctocog alfa (NovoEight®) is a recombinant factor VIII (rFVIII) with a truncated B-domain made from the sequence coding for 10 amino acids from the N-terminus and 11 amino acids from the C-terminus of the naturally occurring B-domain. Turoctocog alfa is produced in Chinese hamster ovary (CHO) cells without addition of any human- or animal-derived materials. During secretion, some rFVIII molecules are cleaved at the C-terminal of the heavy chain (HC) at amino acid 720, and a monoclonal antibody binding C-terminal to this position is used in the purification process allowing isolation of the intact rFVIII. Viral inactivation is ensured by a detergent inactivation step as well as a 20-nm nano-filtration step. Characterisation of the purified protein demonstrated that turoctocog alfa was fully sulphated at Tyr346 and Tyr1664, which is required for optimal proteolytic activation by thrombin. Kinetic assessments confirmed that turoctocog alfa was activated by thrombin at a similar rate as seen for other rFVIII products fully sulphated at these positions. Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd ) for binding to von Willebrand factor (VWF). Half-lives of 7.2 ± 0.9 h in F8-KO mice and 8.9 ± 1.8 h haemophilia A dogs supported that turoctocog alfa bound to VWF after infusion. Functional studies including thromboelastography analysis of human haemophilia A whole blood with added turoctocog alfa and effect studies in mice bleeding models demonstrated a dose-dependent effect of turoctocog alfa. The non-clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.

Innovative approach for improved rFVIII concentrate.

The development of a new recombinant factor VIII was designed and implemented to answer a number of unmet needs of patients affected by hemophilia A. Turoctocog alfa is bioengineered in a specific Chinese hamster ovary clone to present translational and posttranslational characteristics (sulphation, glycosylation) biosimilar to natural circulating forms of FVIII, with the aim to devoid any minimal change which may impact immunogenicity and antigenicity of recombinant protein. Both producer cell line and media are maintained free of any animal or human plasma derivative. Downstream processes of purification are performed by five steps (immunoaffinity chromatography, ion-exchange chromatography, virus inactivation by means of solvent-detergent treatment and nanofiltration, and to end with gel filtration), to provide the best possible margin of safety from known and unknown infectious agents. Large clinical trials seem to confirm the expectations placed in Turoctocog alfa in terms of high quality and safety of recombinant FVIII toward the goal of overcoming actual and future challenges of hemophilia therapy.

Expression, purification, and partial in vitro characterization of biologically active human coagulation factor VIII light chain (A3-C1-C2) in Pichia pastoris.

Recombinant coagulation factor VIII (FVIII) expressed in mammalian expression systems is used extensively in the treatment of hemophilia A. It is reported that the heavy (A1-A2) and light chains (A3-C1-C2) of factor VIII purified from plasma regained the coagulation activity by dimerization in vitro. In this work, cDNA coding for the light chain of human coagulation factor VIII (FVIII-LC) was cloned into pPICZα-A expression vector downstream of alcohol oxidase promoter and α-mating signal sequence from Saccharomyces cerevisiae in order to express the protein with a native N-terminus. The methylotrophic yeast, Pichia pastoris X-33, was transformed with this cassette, and transformants were selected for production of human factor VIII light chain into culture media. SDS-PAGE and Western blot analysis confirmed the expression of factor VIII light chain protein. The expressed protein was purified to near homogeneity using histidine ligand affinity chromatography (2.342 mg/L). The biological activity of FVIII-LC was confirmed by analyzing the interaction between FVIII-LC and phospholipid vesicles. The data presented here indicate the possibilities of exploring cost-effective systems to express complex proteins of therapeutic value.

Detection of intracellular Factor VIII protein in peripheral blood mononuclear cells by flow cytometry.

Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

von Willebrand factor contributes to longer half-life of PEGylated factor VIII in vivo.

PEGylation of B-domain deleted factor VIII (PEG-FVIII-BDD) prolongs the half-life of the molecule by approximately twofold in animals (Mei et al., Blood 2010; 116: 270). To investigate the role of von Willebrand factor (vWF) in the catabolism of PEG-FVIII-BDD in vivo, a FVIII-BDD mutant (F8V), which is incapable of binding vWF, was generated by deleting the vWF-binding region in the a3 domain of FVIIII-BDD. F8V was expressed, purified and PEGylated by site-specific conjugation. The biochemical and biological properties of F8V and PEGylated F8V (PEG-F8V) were evaluated in vitro and in vivo. The specific activity of purified F8V by a chromogenic assay was similar to FVIII-BDD and PEGylation had minimal impact on the specific activity of F8V in this assay. Analysis by Biacore indicated that both F8V and PEG-F8V display greatly reduced vWF binding in vitro. Pharmacokinetic studies in FVIII knockout (HaemA) mice showed that the terminal half-life (T1/2 ) of F8V was dramatically reduced relative to FVIII-BDD (0.6 h vs. 6.03 h). PEGylation of F8V promoted a significant increase in T1/2 , although PEGylation did not fully compensate for the loss in vWF binding. PEG-F8V showed a shorter T1/2 than PEG-FVIII-BDD both in HaemA mice (7.7 h vs. 14.3 h) and in Sprague-Dawley male rats (2.0 ± 0.3 h vs. 6.0 ± 0.5 h). These data demonstrated that vWF contributes to the longer T1/2 of PEG-FVIII-BDD. Furthermore, this suggests that the clearance of the FVIII:vWF complex, through vWF receptors, is not the sole factor which places an upper limit on the duration of PEG-FVIII circulation in plasma.

Development and characterization of recombinant ovine coagulation factor VIII.

Animal models of the bleeding disorder, hemophilia A, have been an integral component of the biopharmaceutical development process and have facilitated the development of recombinant coagulation factor VIII (fVIII) products capable of restoring median survival of persons with hemophilia A to that of the general population. However, there remain several limitations to recombinant fVIII as a biotherapeutic, including invasiveness of intravenous infusion, short half-life, immunogenicity, and lack of availability to the majority of the world's population. The recently described ovine model of hemophilia A is the largest and most accurate phenocopy. Affected sheep die prematurely due to bleeding-related pathogenesis and display robust adaptive humoral immunity to non-ovine fVIII. Herein, we describe the development and characterization of recombinant ovine fVIII (ofVIII) to support further the utility of the ovine hemophilia A model. Full-length and B-domain deleted (BDD) ofVIII cDNAs were generated and demonstrated to facilitate greater biosynthetic rates than their human fVIII counterparts while both BDD constructs showed greater expression rates than the same-species full-length versions. A top recombinant BDD ofVIII producing baby hamster kidney clone was identified and used to biosynthesize raw material for purification and biochemical characterization. Highly purified recombinant BDD ofVIII preparations possess a specific activity nearly 2-fold higher than recombinant BDD human fVIII and display a differential glycosylation pattern. However, binding to the carrier protein, von Willebrand factor, which is critical for stability of fVIII in circulation, is indistinguishable. Decay of thrombin-activated ofVIIIa is 2-fold slower than human fVIII indicating greater intrinsic stability. Furthermore, intravenous administration of ofVIII effectively reverses the bleeding phenotype in the murine model of hemophilia A. Recombinant ofVIII should facilitate the maintenance of the ovine hemophilia A herd and their utilization as a relevant large animal model for the research and development of novel nucleic acid and protein-based therapies for hemophilia A.