Recombinant Factor VIII Products Research Articles

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N=35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Binding to Platelets Differs Among Recombinant Factor VIII Products in Vitro

Introduction: Platelets are activated in response to injury and play a key role in blood clotting. In primary hemostasis, platelets adhere to exposed subendothelial matrix proteins (e.g., collagen) and subsequent downstream signaling pathways induce granule secretion and platelet aggregation. In secondary hemostasis, procoagulant platelets in the periphery activate clotting factors, leading to a more stable fibrin clot. Binding of factor VIII (FVIII) to procoagulant platelets ensures efficient production and localization of thrombin at the site of injury. However, it is less clear how platelet-FVIII interactions spatiotemporally control the initial thrombus formation and pave the way for successful tissue repair. In patients with hemophilia A, replacement FVIII therapy is used to treat and prevent bleeds. A number of recombinant FVIII (rFVIII) products are indicated for use in patients with hemophilia A. Simoctocog alfa (Nuwiq®) is a chemically unmodified rFVIII with no fusion protein produced in a human cell line. Damoctocog alfa pegol (Jivi®) and rurioctocog alfa pegol (Adynovate®) are pegylated rFVIII products, while efmoroctocog alfa (Eloctate®) consists of FVIII fused with a Fc protein. It is not clear whether such modifications impact the platelet binding potential of FVIII. This study aims to examine platelet binding of different rFVIII products in vitro. Methods: Platelets were isolated from healthy donors, activated using thrombin and collagen-related peptide (CRP-XL) and incubated with individual rFVIII concentrates. After 30 min, the reaction was stopped by dilution. Binding was quantified by flow cytometry using one of two approaches: rFVIII immunostaining with a fluorescent (Alexa Fluor 647; AF647) anti-FVIII antibody; or using rFVIII molecules that had been pre-labeled with AF647. For comparison among products, experiments were performed at equimolar concentrations (17.6 nM) or equal activities of rFVIII concentrates. Values were normalized to simoctocog alfa. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc testing. Results: Platelets were obtained from 10 healthy donors. At equimolar concentrations, efmoroctocog alfa and damoctocog alfa pegol bound significantly (p < 0.001) less than simoctocog alfa to stimulated platelets, as visualized via immunostaining (n = 10). Binding to platelets was significantly lower with damoctocog alfa pegol than efmoroctocog alfa (p < 0.001). Similar results were obtained from experiments with pre-labelled rFVIII (n = 6) or when using equal activities of rFVIII concentrates. In experiments with platelets from 2 healthy donors, rurioctocog alfa pegol exhibited lower platelet binding compared with simoctocog alfa, when tested using the immunostaining method and equimolar concentrations of rFVIII. Conclusions: Simoctocog alfa showed increased binding to procoagulant platelets compared with other rFVIII concentrates tested. Further studies, including additional functional platelet assays, will be performed using samples from a larger number of healthy donors and from patients with hemophilia A. The results of this study may provide new insights into the function and efficacy of different rFVIII products in patients with hemophilia A.

Immunogenicity of Current and New Therapies for Hemophilia A.

Anti-drug antibody (ADA) development is a significant complication in the treatment of several conditions. For decades, the mainstay of hemophilia A treatment was the replacement of deficient coagulation factor VIII (FVIII) to restore hemostasis, control, and prevent bleeding events. Recently, new products have emerged for hemophilia A replacement therapy, including bioengineered FVIII molecules with enhanced pharmacokinetic profiles: the extended half-life (EHL) recombinant FVIII products. However, the main complication resulting from replacement treatment in hemophilia A is the development of anti-FVIII neutralizing alloantibodies, known as inhibitors, affecting approximately 25–30% of severe hemophilia A patients. Therefore, the immunogenicity of each FVIII product and the mechanisms that could help increase the tolerance to these products have become important research topics in hemophilia A. Furthermore, patients with inhibitors continue to require effective treatment for breakthrough bleedings and procedures, despite the availability of non-replacement therapy, such as emicizumab. Herein, we discuss the currently licensed treatments available for hemophilia A and the immunogenicity of new therapies, such as EHL-rFVIII products, compared to other products available.

Cost analysis comparison of rurioctocog alfa pegol compared to turoctocog alfa pegol in the prophylaxis of Haemophilia A in Italy

Background: Hemophilia-A is an X-linked genetic disorder arising from a mutation to the X chromosome and causing either a quantitative or qualitative deficiency in blood clotting factor-VIII (FVIII). Several recombinant extended half-life FVIII products (rFVIII-EHL) are in use in Italy for the treatment of hemophilia-A. Objective: To estimate the annual average cost of the prophylactic treatment of a patient with hemophilia-A in Italy with rurioctocog alfa pegol or turoctocog alfa pegol, with particular regard to potential dose adjustments as provided in the SPCs. Methods: The comparison between the two rFVIII-EHL, expressed in terms of average annual cost per patient treated, was conducted from the NHS perspective. The average annual consumption (IU/kg) of the two rFVIII-EHL was derived from the clinical trial of reference for in label prophylactic treatment. Adjustments of doses and administration intervals allowed in the SPCs were considered on the basis of the literature. The published ex-factory price considered for both rFVIII-EHL was € 0.72. A scenario analysis focused on possible dosing adjustments was conducted. Results: With regard to the average consumption associated with the posology in label, the average annual treatment cost is almost the same between the two rFVIII-EHL. Whereas taking into account the possible dose adjustments based on once-weekly administration, rurioctocog alfa pegol shows a considerably lower average annual treatment cost compared to turoctocog alfa pegol (– € 42,719). Conclusions: Rurioctocog alfa pegol would be the more sustainable option, based on the annual average cost, for the NHS in the prophylactic treatment of hemophilia-A.

Immunogenicity, Efficacy and Safety of Rurioctocog Alfa Pegol in Previously Untreated Patients with Severe Hemophilia a: Interim Results from an Open-Label Multicenter Clinical Trial

Immunogenicity, Efficacy and Safety of Rurioctocog Alfa Pegol in Previously Untreated Patients with Severe Hemophilia a: Interim Results from an Open-Label Multicenter Clinical Trial

Monitoring of different factor VIII replacement products using a factor VIII one-stage clotting assay on cobas t 511/711 analysers.

IntroductionRecombinant coagulation factor VIII (FVIII) products are the standard of care for patients with haemophilia A. The development of modified FVIII products has provided benefit for patients but presented challenges for monitoring FVIII activity.AimThis single‐centre study evaluated the Roche FVIII one‐stage clotting assay (OSA) in measuring FVIII activity in plasma samples spiked with seven FVIII products at clinically relevant concentrations.MethodsFVIII‐deficient plasma samples were spiked with two batches of recombinant FVIII products (octocog alfa, moroctocog alfa, simoctocog alfa, efmoroctocog alfa, damoctocog alfa pegol, rurioctocog alfa pegol, lonoctocog alfa) at 1–120 IU/dL FVIII activity, according to their labelled potency. Measurement was conducted on the cobas t 511/711 analysers using the Roche FVIII OSA and the Technoclone TECHNOCHROM FVIII:C chromogenic substrate assay (CSA).ResultsUsing the OSA, FVIII activity was close to labelled potency for most analysed FVIII products including a recombinant FVIII Fc fusion protein. PEGylated FVIII product, damoctocog alfa pegol, was marginally above and single‐chain product, lonoctocog alfa, below the predefined acceptance criteria: for FVIII activity < 25 IU/dL: ± 5 IU/dL; for FVIII activity ≥ 25 IU/dL: ± 20% (relative). The different principles of OSA and CSA led to discrepancies in the estimation of all analysed FVIII products. Additionally, in vitro recovery was increased at lower levels of FVIII activity using the OSA, whereas recovery was more consistent using the CSA.ConclusionThese data allow the interpretation of FVIII activity results for different FVIII products using the Roche FVIII OSA on the cobas t 511/711 analysers.

Development of a novel fully functional coagulation factor VIII with reduced immunogenicity utilizing an in silico prediction and deimmunization approach

BackgroundUp to 30% of hemophilia A patients develop inhibitory antibodies against the infused factor VIII (FVIII). The development of a deimmunized FVIII is an unmet high medical need. Although improved recombinant FVIII (rFVIII) products evolved within the last years, the immunogenicity has not been solved. A deimmunized FVIII could reduce the probability of inhibitor development, providing safer therapy. ObjectiveTo develop a deimmunized FVIII molecule by modifying major histocompatibility complex (MHC) class II presentation, leading to a functional but less immunogenic molecule. MethodsWe performed (1) in silico prediction of potentially immunogenic T cell epitopes and their modification by amino acid substitutions in the FVIII sequence, (2) evaluation of functional and structural similarity of the modified rFVIII to unmodified FVIII and registered products, and (3) confirmation of the reduced immunogenicity by in vitro testing. ResultsA partially deimmunized fully functional FVIII molecule incorporating 19 amino acid substitutions was generated. The substitutions led to a reduction of the immunogenicity score, indicating a reduced immunogenicity based on in silico calculations. This was confirmed in an in vitro dendritic cell (DC)‐‐T cell assay. Using this assay, cells from healthy donors proved the significantly reduced immunogenicity of the modified FVIII variant by revealing less proliferation of T helper cells to this variant than to the unmodified FVIII. ConclusionIn silico predictions resulted in a partially deimmunized FVIII. This FVIII is fully functional and was demonstrated to be less immunogenic in in vitro testing. This approach may result in a reduction of the inhibitor risk for patients with hemophilia A.

Polyethylene Glycol (PEG) Exposure in Parenteral Therapies Indicated for the Pediatric Population: History and Safety

INTRODUCTIONPolyethylene glycol (PEG) is a chemically inert, amphiphilic polymer used as an excipient in many pharmaceuticals for decades, and as a conjugate with biologicals (ie, PEGylation) since 1990. To date, the US FDA has approved 14 PEGylated therapies with diverse indications. Although it is recognized that such products contain PEG, there may be a lack of awareness regarding the use of large quantities of excipient PEG in factor VIII (FVIII) and factor IX (FIX) therapies for hemophilia A and B, respectively, as well as in other parenteral therapies administered long term in the pediatric population. Due to concern about the potential toxicity of long-term exposure to PEG, particularly in children, based primarily on preclinical observations of cellular vacuolation upon supratherapeutic dosing of PEG molecules ≥ 40kDa (EMA. Safety Working Party's Response to the PDCO Regarding the Use of PEGylated Drug Products in the Pediatric Population; 2012. ), we evaluated the total PEG content of these therapies. Our objective was to estimate annual PEG exposure from each product as commonly prescribed, and to determine whether any adverse events (AEs) reported could be attributed specifically to PEG.METHODSFDA-approved parenteral therapies with pediatric indications were analyzed for PEG exposure per dose and per year. For comparative purposes, we grouped FVIII products together as primary comparators to Antihemophilic Factor (Recombinant), PEGylated (ADYNOVATE®).To quantify excipient PEG, we included free PEG of various molecular weight and excipients with chemical structures containing PEG chains (polysorbate 20 [PS20] and 80 [PS80], and poloxamer 188 [P188]). For FVIII and FIX therapies, PEG exposure was calculated based on a range of dosages recommended in the 2013 World Federation of Hemophilia Guidelines (25-40 IU/kg; Srivastava, et al. Haemophilia . 2013; 19:e1-e47). For extended half-life recombinant (r)FVIII and rFIX products and all other products, exposure was calculated based on prescribing information (PI), including a given dosage range. Three weights were used to cover the pediatric population: 10, 30, and 50 kg, with exceptions pertaining to the PI. The formula used to calculate PEG exposure per dose was:PEGylated biologic: patient weight (kg) × dose (IU or mg/kg) × (PEG MW/total MW)/specific activityExcipient PEG: patient weight (kg) × dose ((IU or mg)/kg)/(PEG concentration) × PEG ratio **Ratio: PEG/PEG=1; for PS80, PEG/PS80=0.645; for P188, PEG/P188=0.69.For this analysis, combinations of vial sizes were pragmatically chosen to minimize factor waste, leading to some variability in dosing. All PEG molecules were treated equally, whether conjugated or present as an excipient and regardless of size or structure.Safety outcomes were investigated by searching the medical literature for any report of clinical PEG-related AEs, and by attempting to identify any salient pattern of AEs listed in the PI or reported to the FDA or other reporting organization that might reflect PEG toxicity.RESULTSFor a child weighing 10 kg and receiving minimal prophylaxis dosages of FVIII, total PEG exposure ranged from <1 to 2,730 mg/year; for prophylaxis with FIX products the range was <1-168 mg/year. For other products, total PEG exposure ranged from 2 to 53,508 mg/year (Table 1). For a child weighing 50 kg and receiving maximum prophylaxis dosages of FVIII, total PEG exposure ranged from 40 to 21,840 mg/year; for prophylaxis with FIX products, the range was 13-1,342 mg/year. For other products, total PEG exposure ranged from 383 to 53,508 mg/year (Figure 1 and 2). Eloctate®, which contains PEG in the form of excipient PS20, had insufficient publicly available information to determine PEG exposure. There were no reports in the medical literature of clinical safety issues or signals specifically attributed to long-term exposure to PEG; and no recognizable patterns or trends of central nervous system, hepatic, allergic, or renal AEs.CONCLUSIONSPediatric exposure to PEG from FVIII, FIX, and other parenteral therapies has occurred for decades. Exposure to PEG as an excipient has been greater than exposure to PEG as a component of a PEGylated therapy. Notably, exposure to PEG from ADYNOVATE, including excipient and conjugated forms, is comparable to or less than that from other FVIII products. There were no discoverable reports of toxicity attributed to PEG accumulation from use of these products. [Display omitted] DisclosuresStidl:Shire: Employment. Schwartz:Shire: Employment. Denne:Shire: Employment. Goldstine:Shire: Employment. Kadish:PAREXEL International: Employment. Korakas:PAREXEL International: Employment. Turecek:Shire: Employment.

Individualized Prophylaxis with BAY 81-8973: Clinical Experience in an Italian Patient with Severe Hemophilia A

Introduction: Prophylaxis with a factor VIII (FVIII) product is the standard of care for patients with severe hemophilia A to prevent bleeding episodes and development of target joints and hemophilic arthropathy. In patients with established joint disease, prophylaxis can slow progression of joint damage, which can improve health-related quality of life. Adherence to prophylaxis is critical to its success. Missing or skipping a prophylaxis infusion can cause FVIII levels to fall below the desired target level, which can increase the risk of breakthrough bleeding episodes. To promote adherence to prophylaxis, healthcare providers and patients must work together to ensure that the prophylaxis regimen is manageable for each patient. In this case report, we describe the strategy we used to improve adherence, including changes to the prophylaxis dosing regimen, in a patient with severe hemophilia A.Demographics and medical history: A 54-year-old patient was diagnosed with severe hemophilia A in early childhood and treated on demand with a plasma-derived FVIII product, despite meeting criteria for use of prophylaxis. In 2000, he began tertiary prophylaxis with a standard-acting recombinant FVIII (rFVIII) product given twice weekly. At that time, he had target joints in both knees and both elbows and a Hemophilia Joint Health Score (HJHS) of 16. In 2007, he underwent surgical replacement of the left knee. In 2010, prophylaxis with a rFVIII was started at 3000 IU (32 IU/kg) 3 times per week. The patient's job as a vendor in outdoor markets affected his ability to adhere to this prophylaxis regimen. To facilitate adherence, prophylaxis dosing was decreased to twice weekly. The patient's comorbidities included obesity and hepatitis C infection, which did not respond to combined treatment with interferon and ribavirin.Current prophylaxis treatment: In 2015, because of weight gain (to 110 kg), joint bleeding episodes (2-3 per year), repeated occurrence of left ankle pain, and further joint deterioration (HJHS score, 25), prophylaxis dosing was increased to 4000 IU (40 IU/kg) 3 times per week. Adherence to this regimen was again difficult for the patient. In March 2017, based on pharmacokinetic results obtained in this patient with BAY 81-8973 (Kovaltry®, Bayer, Berkeley, CA; Table 1), he was transitioned to BAY 81-8973 5000 IU (45 IU/kg) twice weekly (every 84 hours, on Monday morning and Wednesday evening). This prophylaxis regimen accommodated his work schedule, and no bleeding episodes occurred after the patient started BAY 81-8973 prophylaxis.Conclusion: This case illustrates that the prophylaxis dose and dosing interval can be individually tailored, keeping in mind the clinical goal of treatment and the patient's bleeding phenotype and daily activities. In this patient, tailored prophylaxis dosing with BAY 81-8973 was key to improved adherence, outcomes, and health-related quality of life. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Ancestral Sequence Reconstruction of Von Willebrand Factor Reveals Highly Conserved Structure/Function

Von Willebrand factor (VWF) is a large multimeric glycoprotein of critical importance for both primary and secondary hemostasis due to its role in platelet activation and stabilization of coagulation factor VIII (FVIII), respectively. Mutations affecting VWF synthesis, secretion, or function result in one of 6 distinct subtypes of von Willebrand disease (VWD) with mutations inhibiting FVIII binding classified as type 2N. Due to low circulating levels of plasma FVIII, type 2N patients can be misdiagnosed with hemophilia A. Because the clearance of FVIII is dominated by its association with VWF, and given recent evidence demonstrating an immuno-protective role of VWF for FVIII, understanding the molecular interaction between FVIII and VWF remains critically important for both hemophilia A and type 2N VWD therapeutic development. However, limited structural information remains a major limitation for rational design studies. Recently, ancestral sequence reconstruction (ASR) was performed to infer the sequences of FVIII predicted to have existed throughout mammalian evolution. ASR provides a platform for high-resolution mapping of sequential differences within a phylogeny while maintaining a high probability of retaining function. Sequential changes in the amino acid sequence of FVIII appears to have resulted in altered biochemical properties including biosynthesis and stability. In this study, we demonstrate that a previously characterized ancestral (An) FVIII molecule with 95% identity to human FVIII, termed An53-FVIII, exhibits a 2.5 to 5-fold increase in binding to human VWF over commercial recombinant FVIII products, suggesting a dynamic association throughout evolution. We sought to further explore the FVIII-VWF interaction through ASR of VWF and biochemical analysis.A phylogenetic tree was constructed for VWF and the ancestral amino acid sequences predicted through Bayesian inference. A compiled list of 59 extant sequences allowed accurate prediction of the sequences extending beyond the class Mammalia. Of the resulting 50, 5 An-VWF sequences representing significant branch points in primate, rodent, and ungulate evolution were selected for synthesis de novo and subsequent biochemical characterization. All sequences, including an additional human (h) VWF cDNA, were human codon optimized (co). These sequences are predicted to span 105 million years of evolution and contain 81 to 364 amino acid substitutions (Table 1). Heterologous expression in HEK293 cells demonstrated that all An-VWF sequences resulted in secreted antigen as determined by ELISA using anti-human VWF polyclonal antibodies. Furthermore, immunohistochemistry of HEK293 cells expressing An-VWF revealed the formation of pseudo Weibel-Palade bodies by all VWF proteins. Stably transected, clonal producer cell lines were generated and utilized for recombinant VWF production and purification using heparin-sepharose affinity chromatography. Consistent with our IHC studies, multimer analysis of the purified proteins revealed the presence of high molecular weight multimers equivalent to human plasma VWF. To test the functionality of An-VWF, the VWF:GPIbM assay was performed. Compared to an observed specific activity of 45 IU/mg VWF for cohVWF, An-VWF molecules displayed specific activities ranging from 6 to 94 IU/mg. These data suggest that VWF and GPIb may have co-evolved to regulate platelet adhesion and activation. Hydrodynamic injection of An63-, An88-, and An101-VWF plasmid DNA into VWF deficient mice revealed plasma VWF levels of 1.44 ± 0.48, 0.72 ± 0.16, and 2.9 μg/mL at 24 hours after administration, respectively (n = 4, 3, and 1). Additionally, endogenous mouse (m) FVIII levels in these mice were elevated to 61 ± 9, 75 ± 16, and 67% of normal levels from a basal 12.9 ± 2.4%. This data demonstrates that liver synthesized An-VWF retains FVIII association and stabilization. Molar ratios of An88-VWF:mFVIII are 6:1 compared to 14:1 and 25:1 for An63- and An101-VWF, respectively, supporting the coevolution hypothesis for VWF and FVIII. Cumulatively, this study further validates the utility of ASR for the generation of functional sequences for complex proteins with a high propensity for inactivating mutations. Further analysis of ancestral sequences may provide greater resolution of amino acid sequences responsible for biochemical properties including clearance and FVIII association. [Display omitted] DisclosuresGaucher:General Genomics: Equity Ownership.

Nonhuman glycans can regulate anti–factor VIII antibody formation in mice

AbstractRecombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal–knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.

Personalised Prophylaxis in a Child with Haemophilia A and Type 1 Diabetes.

Poor management of either type 1 diabetes or haemophilia A can lead to complications such as organ dysfunction and haemarthropathy. Here, we describe the case of an 8-year-old boy diagnosed with severe haemophilia A shortly after birth. At 2 years old, he was also diagnosed with type 1 diabetes. After six years, the haemophilia treatment was changed from a plasma-derived factor VIII (FVIII) concentrate (octanate®, Octapharma, Lachen, Switzerland) to Nuwiq® (simocotocog alfa, Octapharma, Lachen, Switzerland), a recombinant FVIII (rFVIII) product from a human cell line, which allowed for a personalised treatment schedule that supported good adherence. The dosing regimen could be reduced to two weekly rFVIII infusions. The patient has experienced no spontaneous bleeds since switching to rFVIII and shows no signs of joint damage after over seven years of FVIII prophylaxis. rFVIII was well tolerated, with no treatment-related adverse events observed. This case illustrates the importance of treatment personalisation for young patients and their families managing concomitant diseases.

Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products

BackgroundTherapeutic products with coagulation factor VIII (FVIII) have a wide range of specific activities, implying presence of protein with altered structure. Previous studies showed that recombinant FVIII products (rFVIII) contain a fraction (FVIIIFT) unable to bind von Willebrand factor (VWF) and reported to lack activity. Because of loss of function(s), FVIIIFT can be defined as a product‐related impurity, whose properties and levels in rFVIII products should be investigated. ObjectiveTo isolate and characterize the FVIIIFT fraction in rFVIII products. MethodsProtein fractions unable (FVIIIFT) and able (FVIIIEL) to bind VWF were isolated from rFVIII products using immobilized VWF affinity chromatography (IVAC) and characterized by gel electrophoresis, immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results and ConclusionsA robust IVAC methodology was developed and applied for analysis of 10 rFVIII products marketed in the United States. FVIIIFT was found at various contents (0.4%‐21.5%) in all products. Compared with FVIIIEL, FVIIIFT had similar patterns of polypeptide bands by gel electrophoresis, but lower functional activity. In several representative products, FVIIIFT was found to have reduced sulfation at Tyr1680, important for VWF binding, decreased interaction with a low‐density lipoprotein receptor‐related protein 1 fragment, and faster plasma clearance in mice. These findings provide basic characterization of FVIIIFT and demonstrate a potential for IVAC to control this impurity in rFVIII products to improve their efficacy in therapy of hemophilia A.

Factor VIII products: key aspects of development, clinical research and use (part 2)

Continuous replacement therapy with clotting factor products can lead to serious complications in haemophilia A patients. One of potential reasons of such complications is an undesirable immune response to a blood clotting factor VIII (FVIII) product, which undermines the treatment effectiveness. The aim of the study was to systematise and summarise data on undesirable immunogenicity of plasma-derived and recombinant FVIII products, formation of immunological tolerance, and modern approaches to the development of clinical trial programmes for such products. The analysis was based on scientific literature, as well as Russian and international guidelines, including the updated document of the European Medicines Agency. The paper presents clinical trial data on pharmacokinetics, efficacy, and safety of FVIII products, including data on manifestations of unwanted immunogenicity. It highlights molecular mechanisms of interaction between inhibitors and FVIII, and analyses the main factors (genetic characteristics, immune status of patients, dosage regimen, etc.) affecting the frequency and intensity of the immune response to the product. The authors summarised approaches to the clinical trial design, including selection of patients and studied parameters. They substantiate the need for post-authorisation studies to collect additional clinical data on both efficacy and safety of the routine use of the product, including additional assessment of immunogenicity and other adverse reactions. It is concluded that the successful use of high-quality FVIII products ensures by harmonisation of requirements of Russian and international regulatory documents.

Recombinant FVIII Products (Turoctocog Alfa and Turoctocog Alfa Pegol) Stable Up to 40°C.

PurposeThe stability under high-temperature conditions of factor VIII (FVIII) concentrates for replacement therapy is of critical importance to patients, particularly those who reside in, or travel to, regions with high ambient temperatures. Concerns about product stability may limit or prevent access to treatment for patients and may limit their ability to live a close-to-normal life. This study evaluated the effect of hot and humid storage conditions on the long-term stability of the recombinant FVIII products, turoctocog alfa and turoctocog alfa pegol.MethodsTuroctocog alfa samples were assessed for stability at 30°C for 9 months or 40°C for 3 months following storage at 5°C for 21 or 27 months, respectively, while turoctocog alfa pegol samples were assessed at 30°C for 12 months or 40°C for 3 months following storage at 5°C for 18 or 27 months, respectively. In addition, turoctocog alfa and turoctocog alfa pegol dry powders were evaluated for stability at 5°C/ambient humidity (AH) for 30 months, 30°C/75% relative humidity (RH) for 12 months and 40°C/75% RH for 6 months. Both studies utilized a range of product strengths. Key stability assessments included oxidized forms, potency, water content and high molecular weight protein (HMWP).ResultsBoth turoctocog alfa and turoctocog alfa pegol remained stable following storage at 40°C/75% RH for 3 months, and at single temperatures (5°C/AH, 30 and 40°C/75% RH), without any major increase in HMWP or any impairment of potency or water content.ConclusionTuroctocog alfa and turoctocog alfa pegol offer stability at 40°C for up to 3 months without jeopardizing the quality of each product. These stability characteristics may offer patients flexibility with product storage and daily use.

Characterization of Protein Unable to Bind Von Willebrand Factor in Recombinant Factor VIII Products: Can We Reduce Their Immunogenicity?

Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

Direct comparison of two extended half-life PEGylated recombinant FVIII products: a randomized, crossover pharmacokinetic study in patients with severe hemophilia A

An open-label, crossover randomized study was performed to compare the pharmacokinetics (PK) of damoctocog alfa pegol and rurioctocog alfa pegol, two recombinant factor VIII (FVIII) products indicated in patients with hemophilia A, both conjugated to polyethylene glycol to reduce clearance and extend time in circulation. Adult patients (N = 18) with severe hemophilia A (FVIII < 1 IU/dL), previously treated with any FVIII product for ≥ 150 exposure days, were randomized to receive a single 50 IU/kg infusion of damoctocog alfa pegol followed by rurioctocog alfa pegol, or vice versa, with ≥ 7-day washout between doses. FVIII activity was measured using the one-stage clotting assay. PK parameters, including area under the curve from time 0 to the last data point (AUC0–tlast, primary parameter), dose-normalized AUC (AUCnorm), and time to threshold, were calculated based on 11 time points between 0.25 and 120 h post-dose and evaluated using a noncompartmental model. Due to differences in batch-specific vial content used for the study, actual administered median doses were 54.3 IU/kg for damoctocog alfa pegol and 61.4 IU/kg for rurioctocog alfa pegol. Based on actual dosing, a significantly higher geometric mean (coefficient of variation [%CV]) AUCnorm was observed for damoctocog alfa pegol (43.8 h kg/dL [44.0]) versus rurioctocog alfa pegol (36.0 h kg/dL [40.1, P < 0.001]). Based on population PK modeling, median time to reach 1 IU/dL was 16 h longer for damoctocog alfa pegol compared with rurioctocog alfa pegol. No adverse events or any immunogenicity signals were observed. Overall, damoctocog alfa pegol had a superior PK profile versus rurioctocog alfa pegol. Trial registration number: NCT04015492 (ClinicalTrials.gov identifier). Date of registration: July 9, 2019

Biological mechanisms underlying inter-individual variation in factor VIII clearance in haemophilia.

Previous studies have highlighted marked inter‐individual variations in factor VIII (FVIII) clearance between patients with haemophilia (PWH). The half‐life of infused FVIII has been reported to vary from as little as 5.3 hours in some adult PWH, up to as long as 28.8 hours in other individuals. These differences in clearance kinetics have been consistently observed using a number of different plasma‐derived and recombinant FVIII products. Furthermore, recent studies have demonstrated that half‐life for extended half‐life (EHL‐) FVIII products also demonstrates significant inter‐patient variation. Since time spent with FVIII trough levels <1% has been shown to be associated with increased bleeding risk in PWH on prophylaxis therapy, this variability in FVIII clearance clearly has major clinical significance. Recent studies have provided significant novel insights into the cellular basis underlying FVIII clearance pathways. In addition, accumulating data have shown that endogenous plasma VWF levels, ABO blood group and age, all play important roles in regulating FVIII half‐life in PWH. Indeed, multiple regression analysis suggests that together these factors account for approximately 34% of the total inter‐individual variation in FVIII clearance observed between subjects with severe haemophilia A. In this review, we consider these and other putative modulators of FVIII half‐life, and discuss the biological mechanisms through which these factors impact upon FVIII clearance in vivo.

Critical Review of the Pivotal Studies of Four rFVIII Products for the Treatment of Hemophilia A Patients: The Role of Octocog Alfa

INTRODUCTION: Hemophilia A is a rare congenital bleeding disorder caused by a deficiency of clotting factor VIII (FVIII). The severe form of the disease is characterized by spontaneous bleeds, especially into the joints. Prophylaxis, based on regularly intravenous administration of the missing factor to avoid hemorrhages, represents the gold standard of treatment. In recent years, new products that significantly improve the treatment management options for patients with hemophilia have become available in the market.OBJECTIVE: To critically evaluate the pivotal studies of recombinant FVIII (rFVIII) products, approved in Europe within the first half of 2018 having obtained the indication for a prophylaxis dosing regimen based also on a twice weekly infusion frequency or even less, highlighting their limitations or strengths.METHODS: A systematic literature search was conducted, and several databases (PubMed and Embase) were consulted.RESULTS: Nine clinical trials involving patients with severe hemophilia A without inhibitor were included in this analysis. Four rFVIII products (Elocta®, Biogen, Cambridge, MA, USA; Kovaltry®, Bayer HealthCare Pharmaceuticals, Germany; Afstyla®, CSL Behring GmbH, Germany; Adynovi®, Baxalta Innovation GmbH, Austria) with different pharmacokinetic profiles were evaluated. The trials included in this analysis had different designs and heterogeneous methods were utilized to assess the study outcomes. The baseline characteristics of the patients enrolled in the studies were also often different and sometimes not adequately described. LEOPOLD II, a trial to compare prophylaxis to on-demand therapy with an unmodified rFVIII product (Kovaltry®, octocog alfa), was the only completely randomized trial that enrolled a more critical patient population in terms of compromised joint condition than the other studies. Based on these side-by-side comparison, Octocog alfa reported similar efficacy, in terms of annualized bleeding rate, to the other rFVIII products, including extended half-life.CONCLUSIONS: Even without structural modifications, octocog alfa may be considered a useful treatment option for two times a week prophylaxis in a selected population of haemophilia patients.