In vitro field study and worldwide survey assessing how clinical haemostasis laboratories analyse recombinant and plasma-derived von Willebrand factor products.

INTRODUCTION: Several well-established clinical laboratory methods are available to measure von Willebrand factor (VWF) in plasma samples, but few data are available on their use for analysing recombinant VWF (rVWF). AIM: To evaluate how clinical diagnostic laboratories analyse rVWF and plasma-derived VWF (pdVWF) spiked in vitro into VWF-deficient plasma using quantitative protein and functional assays of VWF. METHODS: Human VWF-deficient plasma samples were spiked with rVWF (vonicog alfa; Takeda) or pdVWF/factor VIII (pdVWF/FVIII; antihemophilic factor/VWF complex [human], CSL Behring), each at final concentrations of 1.0, 0.6, 0.2, 0.1 IU/mL VWF:ristocetin cofactor activity (VWF:RCo) according to labelled VWF activity. The ISTH SSC secondary coagulation standard was used as a control. Participating laboratories received three sets of these blinded aliquots. Mean results per assay were compared with the expected potency based on the labelled VWF:RCo activity. RESULTS: Among 39 laboratories, the most commonly established assay was VWF:RCo; 22 laboratories reported data from 2214 tests. Despite a trend to lower values, VWF:RCo activities for rVWF were in agreement with target concentrations (71%-109%), whereas VWF:platelet glycoprotein Ib (VWF:GpIb) and VWF collagen-binding activity (VWF:CB) assays gave high recoveries (up to 132% and 127%, respectively). In contrast, pdVWF/FVIII was substantially underestimated by VWF:GpIb and VWF:CB assays (56%-86% recoveries), whereas the VWF:RCo assay gave recoveries of 47%-112% for pdVWF/FVIII. CONCLUSION: The results of VWF assays used in clinical laboratories differ between rVWF and pdVWF, particularly for VWF:GpIb and VWF:CB assays. These differences may arise from the higher multimeric structure of rVWF compared to pdVWF.

Treatment with recombinant ADAMTS13, alleviates hypoxia/reoxygenation-induced pathologies in a mouse model of human sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is an inherited red blood cell disorder with a causative substitution in the beta-globin gene that encodes beta-globin in hemoglobin. Furthermore, the ensuing vasculopathy in the microvasculature involves heightened endothelial cell adhesion, inflammation, and coagulopathy, all of which contribute to vaso-occlusive crisis (VOC) and the sequelae of SCD. In particular, dysregulation of the von Willebrand factor (VWF) and a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in human SCD pathology. OBJECTIVES: To investigate the beneficial potential of treatment with recombinant ADAMTS13 (rADAMTS13) to alleviate VOC. METHODS: Pharmacologic treatment with rADAMTS13 in vitro or in vivo was performed in a humanized mouse model of SCD that was exposed to hypoxia/reoxygenation stress as a model of VOC. Then, pharmacokinetic, pharmacodynamic, and behavioral analyses were performed. RESULTS: Administration of rADAMTS13 to SCD mice dose-dependently increased plasma ADAMTS13 activity, reduced VWF activity/antigen ratios, and reduced baseline hemolysis (free hemoglobin and total bilirubin) within 24 hours. rADAMTS13 was administered in SCD mice, followed by hypoxia/reoxygenation stress, and reduced VWF activity/antigen ratios in parallel to significantly (p < .01) improved recovery during the reoxygenation phase. Consistent with the results in SCD mice, we demonstrate in a human in vitro system that treatment with rADAMTS13 counteracts the inhibitory activity of hemoglobin on the VWF/ADAMTS13-axis. CONCLUSION: Collectively, our data provide evidence that relative ADAMTS13 insufficiency in SCD mice is corrected by pharmacologic treatment with rADAMTS13 and provides an effective disease-modifying approach in a human SCD mouse model.

Structure and Function of Recombinant versus Plasma-Derived von Willebrand Factor and Impact on Multimer Pharmacokinetics in von Willebrand Disease.

Background: Recombinant von Willebrand factor (rVWF, vonicog alfa) is a purified VWF concentrate produced from Chinese hamster ovary cells. rVWF is not exposed to the VWF-cleaving protease ADAMTS13 and so is not subject to proteolytic degradation of large (L) and ultra-large (UL) VWF multimers by that enzyme. Purpose: To compare the structure and function of rVWF with the human plasma-derived VWF [pdVWF] concentrates Haemate P®/Humate-P®, Voncento®, Wilate®/Eqwilate®, and Wilfactin®/Willfact®; to investigate the relationship between VWF multimeric pattern and VWF:ristocetin cofactor (VWF:RCo) activity through population pharmacokinetic (PK) modeling in patients with severe von Willebrand disease (VWD) treated with rVWF. Methods: Analyses included VWF:RCo activity, VWF:collagen-binding activity, VWF:platelet glycoprotein Ib receptor binding, factor VIII (FVIII) binding capacity, and VWF-mediated platelet adhesion under flow conditions. VWF multimeric structure was determined by agarose gel electrophoresis. Population PK models describing the activity-time profile of small, medium, and L/UL multimers following intravenous administration of rVWF in patients with severe VWD were developed. Results: Findings demonstrate that rVWF contains a non-degraded VWF multimer pattern including the UL multimers not present in pdVWF concentrates. rVWF displayed higher specific platelet-binding activity, and faster mediation of platelet adhesion to collagen under shear stress versus pdVWF concentrates. rVWF also demonstrated higher FVIII binding capacity than Haemate P®, Voncento® and Wilate®. Modeling provided evidence that VWF:RCo activity in patients with severe VWD treated with rVWF is associated with L/UL VWF multimers in the circulation. Conclusions: Findings suggest that the L and UL multimers preserved in rVWF contribute to high biological activity and might be important for providing hemostatic efficacy.

Von Willebrand Factor Multimer Analysis by Low Resolution SDS-Agarose Gel Electrophoresis.

Von Willebrand factor (VWF) is a complex glycoprotein found in plasma, composed of disulfide-bond-linked multimers with apparent molecular weights between 500 kDa and 20,000 kDa. After release of VWF from storage granules, it is cleaved in flowing blood by the specific metalloproteinase ADAMTS13, resulting in a highly characteristic cleavage pattern and structure. As the structure of VWF multimers determines diagnosis of von Willebrand disease, which has different sub-types with different multimer- and cleavage patterns, VWF analysis is performed using low-resolution horizontal SDS-agarose gel electrophoresis. However, almost every laboratory uses a different protocol, and all experimental details are rarely, if at all, described. Therefore, the results from similar methods may be substantially different. Here, we present a detailed description of a validated VWF multimer method that we have developed. It has been successfully used for over more than 20 years in quality control of recombinant and plasma-derived VWF drug products, and in preclinical and clinical studies with VWF drug candidates. As most of the published methods, it enables visualization of VWF multimers separated by electrophoresis by immunostaining with a polyclonal anti-human VWF antibody followed by a secondary antibody coupled to alkaline phosphatase. VWF appears as a series of regularly spaced bands on the low and middle molecular weight range of the gel, with an unresolved zone in the high molecular weight (HMW) range, where ultra-large multimers are found. An example is shown below. This low-resolution agarose gel electrophoresis allows the determination of the number of VWF multimers with high reproducibility. Graphical abstract: Example of electrophoretic analysis of multimer structure of four batches of a recombinant VWF drug substance.

Selective human factor VIII activity measurement after analytical in-line purification.

Background: It is essential to measure the activity of factor VIII (FVIII) throughout the life cycle of a coagulation FVIII concentrate. Such measurement in nonclinical pharmacokinetic studies is potentially biased by the presence of endogenous nonhuman FVIII, and certain manufacturing process-related additives can also impact the assay performance. Finally, the presence of FVIII activity-mimicking antibodies poses challenges when measuring FVIII in samples. Therefore, we developed an antibody-based chromogenic FVIII assay, which facilitates the selective and sensitive activity measurement of human FVIII in the presence of animal plasma and interfering agents. Methods: Plate-bound monoclonal anti-FVIII antibody specifically captured human FVIII, which was then measured with a chromogenic activity assay. A human reference plasma preparation was used to construct the calibration curve. Spike recovery was carried out in a citrated cynomolgus monkey plasma-solvent/detergent mixture and in the presence of the bispecific antibody emicizumab. Results: The calibration curve ranged from 3.03 to 97.0 mIU FVIII/ml and was obtained repeatedly with good accuracy. B domain-deleted and full-length FVIII did not differ in their responses. Recovery of spiked human FVIII in citrated cynomolgus monkey plasma was 102.7%, while neither native monkey plasma nor the other animal specimen tested showed any activity. Solvent/detergent solution and the bispecific antibody emicizumab had no influence on the assay. Conclusion: Combining antibody-mediated specific capture of human FVIII and a chromogenic activity assay resulted in a selective and sensitive measurement of human FVIII with no interference by endogenous, nonhuman FVIII, manufacturing process additives, or an FVIII activity-mimicking antibody.

Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease.

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

Recombinant ADAMTS13 reduces abnormally up-regulated von Willebrand factor in plasma from patients with severe COVID-19.

Thrombosis affecting the pulmonary and systemic vasculature is common during severe COVID-19 and causes adverse outcomes. Although thrombosis likely results from inflammatory activation of vascular cells, the mediators of thrombosis remain unconfirmed. In a cross-sectional cohort of 36 severe COVID-19 patients, we show that markedly increased plasma von Willebrand factor (VWF) levels were accompanied by a partial reduction in the VWF regulatory protease ADAMTS13. In all patients we find this VWF/ADAMTS13 imbalance to be associated with persistence of ultra-high-molecular-weight (UHMW) VWF multimers that are highly thrombogenic in some disease settings. Incubation of plasma samples from patients with severe COVID-19 with recombinant ADAMTS13 (rADAMTS13) substantially reduced the abnormally high VWF activity, reduced overall multimer size and depleted UHMW VWF multimers in a time and concentration dependent manner. Our data implicate disruption of normal VWF/ADAMTS13 homeostasis in the pathogenesis of severe COVID-19 and indicate that this can be reversed ex vivo by correction of low plasma ADAMTS13 levels. These findings suggest a potential therapeutic role for rADAMTS13 in helping restore haemostatic balance in COVID-19 patients.

Evaluation of Factor VIII Polysialylation: Identification of a Longer-Acting Experimental Therapy in Mice and Monkeys.

Extended half-life (EHL) factor therapies are needed to reduce the burden of prophylaxis and improve treatment adherence in patients with hemophilia. BAX 826 is a novel polysialylated full-length recombinant factor VIII [polysialyic acid (PSA) rFVIII] with improved pharmacokinetics (PK), prolonged pharmacology, and maintained safety attributes to enable longer-acting rFVIII therapy. In factor VIII (FVIII)-deficient hemophilic mice, PSArFVIII showed a substantially higher mean residence time (>2-fold) and exposure (>3-fold), and prolonged efficacy in tail-bleeding experiments (48 vs. 30 hours) compared with unmodified recombinant FVIII (rFVIII), as well as a potentially favorable immunogenicity profile. Reduced binding to a scavenger receptor (low-density lipoprotein receptor-related protein 1) and von Willebrand factor (VWF) as well as a largely VWF-independent circulation time in mice provide a rationale for prolonged BAX 826 activity. The significantly improved PK profile versus rFVIII was confirmed in cynomolgus monkeys [mean residence time: 23.4 vs. 10.1 hours; exposure (area under the curve from time 0 to infinity): 206 vs. 48.2 IU/ml⋅h] and is in line with results from rodent studies. Finally, safety and toxicity evaluations did not indicate increased thrombogenic potential, and repeated administration of BAX 826 to monkeys and rats was well tolerated. The favorable profile and mechanism of this novel experimental therapeutic demonstrated all of the requirements for an EHL-rFVIII candidate, and thus BAX 826 was entered into clinical assessment for the treatment of hemophilia A. SIGNIFICANCE STATEMENT: Prolongation of FVIII half-life aims to reduce the burden of prophylaxis and improve treatment outcomes in patients with hemophilia. This study shows that polysialylation of PSArFVIII resulted in prolongations of rFVIII circulation time and procoagulant activity, together with a favorable nonclinical safety profile of the experimental therapeutic.

Issues complicating precision dosing for factor VIII prophylaxis.

We previously showed that personalizing prophylaxis on the basis of an individual's pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis. A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains. Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing. Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis.

The role of ultralarge multimers in recombinant human von Willebrand factor - a review of physico-and biochemical studies and findings in in vivo models and in humans with von Willebrand disease.

Ultralarge multimers (ULM) of VWF are considered to be the most active with respect to binding to platelets and to subendothelial structures and therefore are of critical importance for the function of VWF in stabilizing the primary hemostatic plug. In contrast to plasma-derived FVIII-VWF concentrates, human rVWF obtained from mammalian cell culture retains the full-spectrum of intact multimers, including ULM, as physiologically formed in the Golgi apparatus and stored in platelet α-granules and endothelial cell Weibel-Palade bodies. In the course of physico and biochemical, functional and animal studies, rVWF exhibited superiority in structure and function compared to pdVWF. These effects seemed to correlate with the multimer size and therefore might be attributed to the presence of ULM in rVWF preparations. The pharmacokinetic (PK), safety and efficacy characteristics seen in preclinical studies were further demonstrated in clinical trials.

Nonacog gamma, a novel recombinant factor IX with low factor IXa content for treatment and prophylaxis of bleeding episodes.

Nonacog gamma is a new recombinant factor IX to treat factor IX deficiency. It is indicated for control of bleeding episodes, perioperative management and routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children with hemophilia B. Nonacog gamma was first approved in the USA in June 2013 under the trade name RIXUBIS followed by market approvals in Australia and the EU in 2014, and marketing authorization decision is pending in Japan. Nonacog gamma is derived from a recombinant Chinese hamster ovary cell line using a state of the art biotechnological manufacturing process. Recombinant factor IX is produced by Baxter's protein-free fermentation technology, which was first developed for ADVATE. The product is purified and formulated in the absence of any human or animal-derived protein. Nonacog gamma was characterized both in comprehensive in vitro and in vivo non-clinical studies as well as in an extensive clinical trial program.

High shear dependent von Willebrand factor self-assembly fostered by platelet interaction and controlled by ADAMTS13.

INTRODUCTION: The paradigm of activation induced platelet aggregation has recently been refuted under blood flow conditions with shear rates exceeding 20,000s(-1). These lead to reversible rolling platelet aggregates, which were dependent on the presence of immobilized and soluble von Willebrand factor. MATERIAL AND METHODS: In vitro experiments using direct fluorescence video-microscopy were performed in wall parallel and stagnation point flow chambers with shear rates raised from 20,000 to 50,000s(-1). Washed blood cell suspension containing recombinant von Willebrand factor (rVWF) was perfused over rVWF or collagen coated surfaces. RESULTS: Here we show for the first time with the visualization of rVWF that not only colloid and polymer, i.e. platelets and VWF, form a composite, but that VWF itself is capable of entirely reversible self-assembly. On a collagen surface the platelet-VWF-conglomerates did not roll but VWF nets bound permanently to the collagen fibers and captured and immobilized platelets from the flow. Lowering the shear rate below the threshold of 20,000s(-1) no longer dissolved these deposits. Ultralarge multimer containing rVWF was most effective compared to normal sized rVWF. The presence of ADAMTS13 limited rolling aggregate and platelet-VWF-conglomerate formation to a time window of 7-8minutes. Changing wall parallel flow to stagnation point flow halved the required shear rate threshold. CONCLUSION: We conclude that flow dynamics can trigger reversible von Willebrand factor self-assembly and platelet-VWF-conglomerate accrual, which are regulated by ADAMTS13 to a time span needed by coagulation to stabilize it, e.g. in case of vessel injury.

Structural analysis of the recombinant therapeutic product rFVIII and its PEGylated variants using 2-D DIGE.

2-D DIGE is a method that circumvents the gel-to-gel variability inherent in conventional 2-DE and is particularly useful for studying proteome changes in diverse applications such as developmental biology and tissue proteomics. We developed a 2-D DIGE protocol for recombinant factor VIII (rFVIII), a therapeutic protein used for the treatment of hemophilia A. The factor VIII heterodimer is composed of heterogeneous, heavily glycosylated heavy and light chains that are held together by a divalent cation. 2-DE of rFVIII led to a separation of the various fragments whose identity could be determined by Western blot. A comparison of two rFVIII batches by 2-D DIGE revealed their identical composition, whereas an rFVIII variant lacking its central B domain was congruent with the smallest heavy and light chain fragments of rFVIII only. A simpler pattern was obtained upon removal of the terminal sialic acids of rFVIII's glycans, due to a better focusing in the first dimension. 2-D DIGE was also well suited to structurally evaluate various PEGylated rFVIII conjugates. 2-D DIGE thus proved an excellent and straightforward method for structural analysis of rFVIII. Our data suggest that the method could serve as a tool for quality control of very complex pharmaceutically active ingredients.

Preclinical testing of human recombinant von Willebrand factor: ADAMTS13 cleavage capacity in animals as criterion for species suitability.

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 ( A Disintegrin and Metalloproteinase with Thrombo Spondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested. Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects. These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.

Mutation of the surface-exposed amino acid Trp to Ala in the FVIII C2 domain results in defective secretion of the otherwise functional protein.

The C2 domain of factor VIII (FVIII) is important for FVIII-phospholipid (PL) and FVIII-von Willebrand factor (VWF) interactions. A FVIII structural model, derived by electron crystallography, suggests four hydrophobic loops at the FVIII C2 domain-PL interface. Within loop four, the solvent-exposed amino acid, Trp(2313), is believed to contribute to FVIII-PL binding. To analyse this interaction, the amino-acid exchange Trp(2313) to Ala (W2313A) was introduced into the C2 domain of B-domain-deleted FVIII (dBFVIII). Both proteins, dBFVIII and W2313A, were expressed in a mammalian expression system. Labelling experiments showed that the mutation W2313A resulted in reduced secretion but did not affect intracellular synthesis of the protein. Specific activity, kinetic parameters, binding to VWF and haemostatic potential in a murine model of haemophilia A were found to be similar for both proteins. Binding studies to synthetic 4% phosphatidyl-l-serine vesicles showed, however, a 28-fold higher K(D) for W2313A, indicating the important role of Trp(2313) in the FVIII-PL interaction. In conclusion, the C2-domain-surface-exposed residue Trp(2313), is critical for secretion of the protein. The W2313A mutation weakens binding to phosphatidyl-l-serine vesicles but the mutant protein has the same effector function as dBFVIII in vitro and in vivo.

Evaluation of the haemostatic potential of factor VIII-heparin cofactor II hybrid proteins in a mouse model.

We constructed factor VIII-heparin cofactor II (FVIII-HCII) hybrid molecules, which are more readily activated by thrombin in vitro than the respective wild-type molecules. The hybrid proteins were tested in a murine model of haemophilia A to investigate their haemostatic efficacy in vivo. Bleeding characteristics, measured using standard tail-tip cutting techniques, were total blood loss, bleeding time and survival rate. FVIII-HCII hybrids were found to be effective in preventing bleeding in FVIII knockout mice. While in vitro experiments showed that the chimaeric molecules had higher haemostatic functions than the wild-type proteins, the variables analysed in vivo were similar for both proteins.

Recombinant von Willebrand factor-insight into structure and function through infusion studies in animals with severe von Willebrand disease.

We used a canine and a murine model of von Willebrand disease (vWD) to study the in vivo effects of recombinant von Willebrand factor (vWF). Two preparations were used: (1) a fully processed mature vWF; this was achieved by coexpression of furin. (2) A preparation containing unprocessed pro-vWF, the propeptide still covalently linked to mature vWF. Both preparations induced an increase in canine and murine factor VIII:C (FVIII), which was sustained even when vWF antigen had been removed from the circulation. vWF multimers were analyzed in the plasma samples after infusion using ultra high-resolution 3% agarose gels to allow the separation of homoforms and heteroforms of the vWF polymers. Administration of pro-vWF to dogs with severe vWD resulted in the removal of the propeptide and maturation of vWF in the circulation, indicating that the propeptide cleavage from unprocessed vWF can occur extracellularly. This suggests that the vWF propeptide, besides being derived from the Weibel-Palade bodies of endothelial cells after stimulation, can also be cleaved by pro-vWF in plasma. Using a murine model of vWD, the involvement of the low-density lipoprotein receptor-related protein (LRP) in the clearance of FVIII was established. The low levels of FVIII observed in the absence of vWF are due to an enhanced clearance of FVIII by binding to LRP and removal from the circulation through endocytosis. Administration of the receptor-associated protein (RAP) as a recombinant fusion protein to vWF knockout mice significantly improved the in vivo recovery of recombinant FVIII and the survival time of otherwise rapidly cleared FVIII.