rFVIII Products Research Articles

Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies.

Standard treatment of congenital haemophilia A is based on replacement therapy with coagulation factor VIII (FVIII) products. A major complication of FVIII therapy is the occurrence of IgG alloantibodies (inhibitors) that neutralize FVIII activity. The aim of the analysis was estimating the risk of high-titre inhibitor associated with the second-generation full-length product compared to third-generation full-length product and other recombinant FVIII (rFVIII). We conducted a combined analysis of individual patient data from three large studies in previously untreated patients (PUPs) with severe haemophilia A. A total of 1109 PUPs were treated from 1993 to 2013 including 787 PUPs treated from 2004 onwards (primary analysis cohort). A total of 322 patients (29.0%) developed an inhibitor, of which 192 (17.3%) a high-titre inhibitor. In the primary analysis set, 29.9% of patients developed an inhibitor and 17.2% a high-titre inhibitor. The combined analysis indicated a lower risk of high-titre inhibitor development for the third-generation rFVIII product compared to the second-generation rFVIII product (primary analysis: adjusted hazard ratio (HR)=0.72, 95% CI: 0.49 to 1.06). Adjusted HR for all inhibitor development was significantly lower for the third-generation product compared to the second-generation product. The trend of an increased risk of inhibitor development in PUPs for one recombinant product illustrates that extrapolation from one recombinant factor VIII product to other products might not be justified.

RO4 COST-EFFECTIVENESS ANALYSIS OF RFVIIIFC, PEGYLATED RFVIII, AND EMICIZUMAB FOR THE PROPHYLACTIC TREATMENT OF SEVERE HEMOPHILIA A PATIENTS WITHOUT INHIBITORS IN THE UNITED STATES

Recombinant factor VIII Fc fusion protein (rFVIIIFc) was the first extended half-life (EHL) rFVIII product approved for hemophilia A in the US. Recently, another EHL (PEGylated rFVIII) and monoclonal antibody emicizumab have also been approved. This study aimed to evaluate the cost-effectiveness of rFVIIIFc, PEGylated rFVIII, and emicizumab for the prophylactic treatment of severe hemophilia A patients without inhibitors in the US. A Markov model was constructed to assess the lifetime costs and outcomes associated with rFVIIIFc, PEGylated rFVIII, and emicizumab from a third-party US payer perspective. Efficacy data were sourced from published pivotal trial documentation. Dosing information were identified via product labels and literature searches. The impact of treatment on joint health was assessed via the rates of target joint development and resolution, as well as joint function outcomes via the modified Hemophilia Joint Health Score (mHJHS). Health-related quality of life (HRQoL) data were sourced from published literature. Costs and outcomes were discounted at 3% per annum. Based on the literature review, rFVIIIFc was associated with improved joint health over time measured by mHJHS; no data regarding improved mHJHS were identified for PEGylated rFVIII or emicizumab, hence no effect was assumed in the base-case. The model included the impact of improved mHJHS on HRQoL and showed that compared with PEGylated rFVIII, rFVIIIFc provided more quality-adjusted life-years (QALYs) (26.15 vs. 25.82) at a reduced cost ($15.64m vs. $17.07m) over a lifetime horizon. Compared with emicizumab, rFVIIIFc provided more QALYs (26.15 vs. 25.83) at a reduced cost ($15.64m vs. $16.10m). Sensitivity analyses showed consistent results. rFVIIIFc is the only EHL rFVIII treatment with published evidence demonstrating improved mHJHS. This cost-effectiveness analysis, which includes the impact of treatment on joint health, indicates that rFVIIIFc is associated with lower costs and more QALYs compared to PEGylated rFVIII and emicizumab.

Comparative analysis of marketed factor VIII products: reply

Development of factor VIII (FVIII) inhibitors following replacement therapy with FVIII is one of the major challenges faced when treating patients with hemophilia A. Inhibitors develop in 20–32% of previously untreated patients with severe and in 3–13% with moderate or mild hemophilia A 1, 2. The cause of the immunogenicity is not well understood. There is evidence that both genetic and non-genetic factors influence patients’ susceptibility to develop these antibodies 3, 4. Novel hypothesis and research approaches are required to obtain more clarity on the molecular basis of FVIII immunogenicity and how FVIII triggers unwanted immune responses in some patients but not in others. The aim of our paper ‘Comparative analysis of marketed factor VIII products: recombinant products are not alike vis-a-vis soluble protein aggregates and subvisible particles’ 5 was to advance science and help to generate new hypotheses for future research on the molecular basis of FVIII immunogenicity. Our aim was to provide scientific transparency of our rationale and to stay on the level of a scientific debate rather than discussing brands or products. We believe that when it comes to treatment decisions clinical evidence on a robust basis is required. Disclosure of brand names as recently suggested by M Makris and A Farrugia in a letter to the editor of JTH 6 could influence treatment decisions by hypotheses rather than robust clinical evidence. Data from other protein products suggest that critical quality variables such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics 7. We analyzed SPAs and SVPs concentrations in commercially available recombinant FVIII (rFVIII) products to understand if there are differences between these products after reconstitution. Moreover, we wanted to know if and how levels of SPAs and SVPs change upon exposure of rFVIII products to relevant stress conditions such as agitation and sheer stress. Pre-existing SPAs and SVPs may act as seeds that nucleate further protein aggregation upon exposure to stress 8-10. In addition to the baseline levels of SPAs and SVPs in each rFVIII product, our data indicate that product mishandling after reconstitution can increase the concentration of SPAs and SVPs. Similar findings for FVIII products were recently published by Tsutomo et al. 15. Thus, it is important to educate end-users about proper product handling to avoid an amplification of potential adverse effects due to increases in SPAs and SVPs induced by mishandling. 15. In conclusion, we believe the research community needs to pay more attention to the presence of SPAs and SVPs in FVIII products and how these variables influence the immunogenicity of the products in patients. Our current understanding does not yet allow specific conclusions on how levels of SPAs and SVPs in FVIII products translate into product immunogenicity in patients but we believe that thorough assessment of these variables is important. B. M. Reipert wrote the manuscript. J. Anzengruber and F. Scheiflinger revised the manuscript and all authors approved the final version. We thank E. Langdon-Neuner for editing the English language of the manuscript. This work was funded by Shire. All authors are employees of Shire.

The Atlanta Protocol: Immune Tolerance Induction in Pediatric Patients with Hemophilia a and Inhibitors on Emicizumab

Introduction: The formation of neutralizing anti-factor VIII (fVIII) antibodies, called inhibitors, is a challenging complication in hemophilia A care. While novel non-factor therapies significantly reduce bleeding symptoms in patients with hemophilia A and inhibitors, the absence of fVIII tolerance remains unchanged. Additionally, there are concerns regarding the hemostatic efficacy and safety of bypassing agents necessary for the management of breakthrough bleeds in patients with inhibitors on these novel therapies. Immune tolerance induction (ITI) remains the primary method for eradicating inhibitors and restoring the hemostatic response to fVIII. Here we report on the response to ITI in pediatric patients following initiation of the bispecific humanized monoclonal antibody Emicizumab.Methods: Patients between the ages of 1 - 11 years old with hemophilia A and an active inhibitor (≥0.6 Bethesda Units (BU)/mL) treated at the Emory Pediatric Hemophilia Treatment Center were included in this analysis. In general, ITI was initiated after four weekly loading doses of Emicizumab with standard or extended half-life recombinant (rfVIII) or plasma-derived (pdfVIII) fVIII at 100 units/kg three times per week. Factor choice was determined by the fVIII product used for the ITI attempt prior to initiation of Emicizumab or a standard half-life third generation rfVIII product if no prior ITI attempts. FVIII pharmacokinetics during ITI including the estimated fVIII half-life, fVIII incremental recovery of expected, and inhibitor titers were measured with chromogenic-based assays using bovine reagents. Bleeding symptoms and treatment regimens for bleeds/procedures were also monitored.Results: Eight patients are currently receiving ITI according to this Atlanta Protocol. Details regarding this cohort are outlined in Table 1. Three of the 8 patients required central venous access for ITI fVIII infusions. These eight patients have historical peak inhibitor titers, defined as the highest inhibitor titer prior to this ITI regimen, ranging from 2.0 - 198 BU/mL. Four patients have just recently started ITI. The other 4 patients (patients 1, 2, 4, and 5) have been treated with Emicizumab and ITI for a median duration of 15 weeks (range 13-18 weeks). Two of the 4 patients are on ITI with a standard half-life third generation rfVIII product, 1 is on a standard half-life third generation B domain deleted (BDD) rfVIII product, and 1 is on an extended half-life (EHL) rfVIII-Fc fusion product. Their last measured inhibitor titers after initiation of ITI range from 0.3 - 3.7 chromogenic BU (CBU). All inhibitor titers declined with the start of ITI, and none of the 4 patients experienced an amnestic response (Figure 1). The fVIII incremental recovery improved in 3 of the 4 patients from a median of 30% of expected (range 17-69%) prior to Emicizumab with ITI to 85% of expected (range 67-85%) at the last clinical evaluation. The 1 patient remaining has an fVIII incremental recovery <10% of expected at the last clinical evaluation. Only one patient has required treatment with a single dose of recombinant activated factor VII (rfVIIa) for a right knee hemarthrosis during ITI without complication. There were 3 additional mild bleeding symptoms in 2 patients that did not require intervention including 1 trauma-induced forehead hematoma that self-resolved and 2 episodes self-resolving epistaxis lasting <5 minutes. Two of the 4 patients reported no bleeding symptoms. There were no episodes of thrombosis or thrombotic microangiopathy.Conclusions: This is the first report detailing the Atlanta Protocol for ITI in hemophilia A patients with inhibitors receiving Emicizumab prophylaxis. Although early, these results suggest that ITI can be safely administered in these patients and is able to achieve continued improvement in clinical indicators of tolerance. DisclosuresBatsuli:Bayer: Other: Advisory Board; Genentech: Other: Advisory Board; Octapharma: Other: Advisory Board. Zimowski:National Hemophilia Association/Shire: Other: Funding for clinical fellowship in Hemostasis/Thrombosis. Meeks:Bioverativ: Other: Advisory Board; Bayer: Other: Advisory Board; CSL Behring: Other: Advisory Board; Catalyst Biosciences: Other: Advisory Board; Genentech: Other: Advisory Board; HEMA Biologics: Other: Advisory Board; Shire: Other: Advisory Board; Pfizer: Research Funding. Sidonio:Genentech: Other: Advisory Board, Research Funding; Bioverativ: Other: Advisory Board, Research Funding; Biomarin: Other: Advisory Board; Novo Nordisk: Other: Advisory Board; Octapharma: Other: Advisory Board; Grifols: Other: Advisory Board, Research Funding; Uniqure: Other: Advisory Board; CSL Behring: Other: Advisory Board; Shire: Other: Advisory Board, Research Funding; Kedrion: Research Funding.

Improved Pharmacokinetic Profile for BAY 81-8973 Due to Increased Branching and Sialylation of N-Linked Glycans of Recombinant Factor VIII

The circulatory half-life of recombinant factor VIII (rFVIII) products is affected by glycosylation of the FVIII protein, including N-linked glycan branching and terminal sialic acid occupancy, primarily through receptor-mediated hepatic clearance (eg, asialoglycoprotein receptor [ASGPR] and lipoprotein receptor-related protein [LRP]). BAY 81-8973 (Kovaltry®, Bayer, Berkeley, CA) is an unmodified full-length rFVIII for treatment of hemophilia A. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns and pharmacokinetic (PK) characteristics of BAY 81-8973 and 2 other rFVIII products (sucrose-formulated rFVIII [rFVIII-FS; Kogenate® FS, Bayer] and antihemophilic factor (recombinant) plasma/albumin-free method [rAHF-PFM; Advate®, Shire, Westlake Village, CA]).N-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81-8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from 2 separate phase 1 crossover studies in which the PK profile of BAY 81-8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50-IU/kg dose of each product.BAY 81-8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81-8973 versus 11.5% for rFVIII-FS and 4.8% to 5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% versus 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81-8973, 94% for rFVIII-FS, and 78% to 81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81-8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM.Increases in the percentage of sialylated tri-antennary and tetra-antennary N-glycans correlated well with longer half-life of rFVIII in humans (adjusted R2=0.978 and 0.892 for tri-antennary and tetra-antennary N-glycans, respectively). Higher percentages of sialylation (ie, sialic acid capping) correlated with a longer half-life (adjusted R2=0.697), but the relationship was not as strong as that between glycan branching and half-life. Improved PK for BAY 81-8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which may prolong the time BAY 81-8973 remains in the circulation. DisclosuresTeare:Bayer: Employment. Kates:Bayer: Employment. Shah:Bayer: Employment. Garger:Bayer: Employment.

Bay 94-9027 Can be Accurately Measured across Regions with Appropriate One-Stage Assay Reagent Use

Introduction: Differences in coagulation assay reagents may lead to variable results for some extended-half-life (EHL) recombinant factor VIII (rFVIII) products, with the potential to adversely affect patient care. Laboratories assessing EHL-rFVIII activity can choose from many available one-stage assay and chromogenic assay reagents. A field study was conducted to evaluate the ability of clinical laboratories to accurately measure FVIII activity in plasma samples spiked with BAY 94-9027, an EHL rFVIII, when using diverse assay reagents and when guided in the choice of reagent. In a regional subanalysis, BAY 94-9027 field study results from clinical laboratories in the United States and Canada were compared with those from Europe and Israel.Methods: In this 2-part study, a broad range of laboratories in the United States, Canada, Europe (Austria, Germany, Italy, Romania, Spain, Switzerland, United Kingdom), and Israel were provided samples containing defined concentrations of BAY 94-9027 or an unmodified rFVIII (antihemophilic factor [recombinant] plasma/albumin-free method [rAHF-PFM]) as a control. In part 1, each laboratory measured FVIII activity using their routine methods (one-stage assay, chromogenic assay, or both); laboratories that had ≥2 routine one-stage assays were asked to perform sample testing with one of the less commonly used assays in the laboratory to ensure that part 1 captured both the prevalence and heterogeneity of one-stage assays used in the geographic regions studied. In part 2, laboratories used one-stage assays with SynthASil and Pathromtin SL reagents provided by Bayer; SynthASil and Pathromtin SL have previously been shown to accurately measure BAY 94-9027 and full-length rFVIII products. The field study data were analyzed to identify the most commonly used reagents in the 2 regions (United States/Canada and Europe/Israel) and to assess if regional differences resulted in differences in accuracy of FVIII activity measurement.Results: 52 laboratories (US/Canada, n=25; Europe/Israel, n=27) participated in the field study. The one-stage reagent SynthASil was commonly used in all countries (n=15) in part 1, but differences were seen in the frequency of use of other reagents. PTT-A (n=6) and Actin FSL (n=5) were frequently used in the US/Canada and Actin FS (n=6) and Pathromtin SL (n=6) in Europe/Israel. Regional differences in the choice of assay, with US/Canada more likely to use assays that did not accurately measure BAY 94-9027, affected the ability of laboratories to accurately measure FVIII activity in the spiked samples (Figure). In part 1 of the study, regional differences in median recovery of BAY 94-9027 were seen among laboratories when using their own in-house one-stage assays. These differences were not seen in part 2 of the study when laboratories used provided reagents (SynthASil and Pathromtin SL), despite using the same instrumentation as used in part 1. Regional differences in part 1 might have been exacerbated by use of less common, and potentially inappropriate, one-stage assay reagents, affecting the accuracy of BAY 94-9027 measurement. Chromogenic assay use was more common in Europe/Israel (n=11) than US/Canada (n=5). No regional differences in chromogenic assay results were observed.Conclusions: Most clinical laboratories were able to accurately measure BAY 94-9027 activity using their in-house assays. However, regional differences in the accurate measurement of BAY 94-9027 activity were influenced by regional choice of one-stage reagents used in part 1 of the study; the activity and accuracy differences were eliminated when all laboratories used the same provided one-stage reagents with their in-house instruments (part 2 of study). Therefore, standardization of laboratory procedures and use of newer, more accurate one-stage assay reagents should improve measurement of FVIII activity with EHL products. [Display omitted] DisclosuresCastellone:Bayer: Membership on an entity's Board of Directors or advisory committees; Wilmer-Hale: Consultancy. Church:Bayer: Employment. Leong:Bayer: Employment. Kitchen:Bayer: Consultancy, Other: travel reimbursement, Speakers Bureau.

The Irish Personalized Approach to the Treatment of Haemophilia (iPATH) - Determinants of Inter-Individual Variation in FVIII Pharmacokinetics

Introduction:Standard factor VIII (FVIII) prophylaxis aims to minimise bleeding episodes in people with haemophilia A (PWHA) using a weight based dosing strategy. However, it is well recognised that FVIII half-life (FVIII t1/2) varies markedly between individual PWHA. Consequently, some PWHA continue to experience repeated bleeding episodes even after commencing standard dose prophylaxis therapy. In essence, these patients ‘bleed their way’ to eventually reaching their required optimal FVIII prophylactic regimen. Given that even a small number of joint bleeds have been shown to represent a risk factor for hemophilic arthropathy, there is a clear unmet need for the development of personalised treatment approaches for PWHA that include consideration of individual FVIII pharmacokinetics (PK) to guide prophylaxis dosing. With the introduction of limited plasma FVIII sampling and Bayesian analysis, evaluation of FVIII pharmacokinetics (PK) are now possible in routine clinical practice. However, real life clinical experience to date has been limited to small cohorts of PWHA. Critically, the influence of PK in modulating clinical phenotype has not been systematically studied. As part of the Irish Personalised Approach to the Treatment of Haemophilia (iPATH) study, we integrated extensive clinical data and individual PK profiles to investigate modulators of FVIII PK, the role of limited versus extended sampling FVIII PK and the impact of PK on FVIII prophylaxis and clinical phenotype.Methods:Written informed consent was obtained prior to recruitment to the iPATH study. All PK samples were obtained following administration of a single rFVIII product (antihemophilic factor [recombinant]; ADVATE®, Shire, Lexington, MA, USA) and using myPKFiT™ software (Shire, Lexington, MA, USA). Utilising limited sampling (2 timed FVIII:C levels, taken at 3‒6 and 24‒32 hours post rFVIII administration) and Bayesian analysis, PK curves were constructed using myPKFiT™. For each individual the FVIII t1/2, Von Willebrand Factor antigen (VWF:Ag), blood group, weight, age at PK and Haemophilia Joint Health Score (HJHS) was recorded. rFVIII prescribed over the preceding 5 years was analysed; all patients used antihemophilic factor (recombinant) throughout this time period. Mann-Whitney analyses were used for non-parametric comparisons and correlations were performed using the Pearson test on Prism 7.0c for Mac OSX.Results:Complete PK profiles were available for 53 patients with FVIII deficiency recruited to the iPATH study (3 moderate, 50 severe FVIII deficiency). FVIII t1/2 varied considerably, ranging from 7.7‒20.1 hours (median=11.4 hours). Patients with blood group O had a significantly shorter FVIII t1/2 (n=31, median=10.9 hours) than non-O patients (n=22, median=12.2 hours, p=0.014). Both increased age (r2=0.2932, p<0.0001) and higher plasma VWF:Ag levels (r2=0.3, p<0.0001) at time of PK were associated with a significantly longer FVIII t1/2. HJHS significantly increased with age (r2=0.494, p<0.0001) but no significant correlation between FVIII t1/2 and HJHS was observed, likely reflecting the multifactorial nature of arthropathy in PWHA.In a subset of 10 PWHA we compared the FVIII t1/2 results generated from extended (10 point) to those from limited sampling (2 point) PK. Each limited sampling study included a 24 hour time point as well as either a 3, 4 or 6 hour sample. The 10 point results significantly correlated with limited sampling results using either the 3/24 hour (r2=0.9801, p<0.0001), 4/24 hour (r2=0.9762, p<0.0001) or 6/24 hour sampling combination (r2=0.9693, p<0.0001), underscoring the clinical utility of limited PK sampling.Prior to the introduction of routine PK-guided dosing in 2018, prescribed rFVIII dose was altered according to bleed rate at clinical review. Analysing the prescribed rFVIII dose over the past 5 years, a clear relationship to rFVIII t1/2 was identified (r2=0.3517, p<0.0001), suggesting that bleeding events in PWHA guided them towards their PK dose.Conclusions:This study highlights the accuracy of simplified vs extended sampling for generation of PK profiles on antihemophilic factor (recombinant) using myPKFiT™. Bleed rate adjusted prophylaxis was found to correlate with FVIII t1/2 in an Irish population of PWHA, suggesting that early evaluation of PK and implementation of PK guided dosing of prophylaxis may be of benefit in minimising bleeds in PWHA. DisclosuresLavin:Shire: Honoraria, Research Funding, Speakers Bureau. Reipert:Shire: Employment, Equity Ownership. Pipe:Shire: Consultancy, Research Funding; HEMA Biologics: Consultancy; Pfizer: Consultancy; Nove Nordisk: Consultancy; Bioverativ: Consultancy; CSL Behring: Consultancy; Genentech: Consultancy; Ainylam: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy; Apcintex: Consultancy; Catalyst Biosciences: Consultancy; Spark Therapeutics: Consultancy; Freeline: Consultancy; Bayer: Consultancy; uniQure: Consultancy; Biomarin: Consultancy; R2 Diagnostics: Research Funding; Siemens: Research Funding. Turecek:Shire: Employment, Equity Ownership. O'Donnell:Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Leo Pharma: Speakers Bureau; Octapharma: Speakers Bureau; CSL Behring: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Research Funding; Baxter: Research Funding, Speakers Bureau; Shire: Research Funding, Speakers Bureau.

In‐depth comparison of N‐glycosylation of human plasma‐derived factor VIII and different recombinant products: from structure to clinical implications

Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, LewisX/Y and Galα1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.

Patent mining and landscaping of emerging recombinant factor VIII through network analysis.

A landscape analysis of the recombinant factor VIII patent sector using network analysis to map the cooperation network among assignees shows emerging technologies that focus on improved methods for rFVIII production and bioactivity performance.

BAY 81-8973, a full-length recombinant factor VIII for the treatment of hemophilia A: product review

BAY 81-8973 (Kovaltry®) is an unmodified, full-length recombinant factor VIII (rFVIII) approved for the prevention and treatment of bleeding episodes in patients with hemophilia A. The amino acid sequence for BAY 81-8973 is identical to that of sucrose-formulated rFVIII (rFVIII-FS; Kogenate® FS/KOGENATE®, Bayer), but the two products differ in their manufacturing approaches. The manufacture of BAY 81-8973 includes several modifications and enhancements, such as the introduction of the gene for human heat shock protein 70, a molecular chaperone protein that facilitates folding of proteins; no addition of human- or animal-derived proteins in the cell culture, purification process, or final formulation; and use of a 20-nm filter to remove any potential aggregates and pathogens. BAY 81-8973 was extensively studied in the LEOPOLD clinical development program, which enrolled participants of all age groups (children, adolescents, and adults) with severe hemophilia A. The pharmacokinetic profile of BAY 81-8973 was shown to be noninferior to, and for some variables more favorable than, rFVIII-FS and another commercial full-length rFVIII product. BAY 81-8973 was shown to be efficacious when used for prophylaxis, on-demand treatment, and perioperative hemostasis. The efficacious prophylaxis dose of BAY 81-8973 was approximately 20-40 IU/kg given two or three times per week, which achieved low annualized bleeding rates. Either the one-stage or the chromogenic assay provides accurate measurements for postinfusion monitoring of BAY 81-8973 levels, with no product-specific calibration standard needed. The incidence of treatment-related adverse events was ⩽7% across all LEOPOLD studies, and no previously treated patient developed anti-BAY 81-8973 inhibitors in the completed primary studies.

Comparative analysis of marketed factor VIII products: recombinant products are not alike vis‐a‐vis soluble protein aggregates and subvisible particles

Background Recombinant protein technologies have facilitated the development of novel factor VIII (FVIII) therapeutics with improved production efficiency, potency and half-live, and a low risk of viral transmission. The increasing number of recombinant FVIII (rFVIII) products and information on their efficacy, safety and cost allow patients and healthcare professionals to adjust treatment to individual needs. Nonetheless, 20-32% of previously untreated patients with severe hemophilia A develop inhibitory antibodies to rFVIII following treatment. The root cause of the immunogenicity of rFVIII products is not well understood. Data for human interferon and human insulin products suggest that critical quality parameters such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics. Therefore, we analyzed SPA and SVP concentrations in commercially available rFVIII products and determined how these parameters change upon exposure of rFVIII products to relevant stress conditions. Objectives Compare critical quality parameters such as SPA and SVP concentrations in rFVIII products under intended use and use-relevant stress conditions. Methods Nine rFVIII products (≥ 3 lots each) were analyzed by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and flow cytometry-based particle analysis. Results/conclusions SPAs and SVPs were present at different concentrations in all freshly reconstituted rFVIII products: SPA concentrations ranged from 0.2% to 11.6%; SVPs were 0.7 × 106 to 114.0 × 106 / 1000 IU. Under use-relevant stress conditions (agitation and shear stress) the products formed additional SPAs and SVPs to different degrees. The collected data indicate that product quality determines its propensity to form SVPs and SPAs, and highlights differences between marketed rFVIII products.

Defining extended half-life rFVIII-A critical review of the evidence.

Recent haemophilia treatment advances include new recombinant FVIII (rFVIII) products with improved pharmacokinetic (PK) properties that aim to reduce the burden of prophylaxis. These treatments are commonly referred to as extended half-life rFVIII products (EHL rFVIII). There is no uniform definition of what constitutes an EHL rFVIII. Such a definition would help physicians, patients and funders understand the properties of standard and EHL rFVIIIs and thus provide clarity when selecting an EHL in clinical settings. To critically assess the published evidence on new and emerging rFVIII products in order to propose a definition to classify EHL rFVIIIs. We systematically searched PubMed, EMBASE and regulatory authorities (FDA/EMA/Health Canada) websites for publications and regulatory submissions describing prospective crossover PK studies evaluating rFVIIIs that demonstrate improved PK parameters in adults and adolescents with severe haemophilia A. Following critical analyses of the published data, we developed a holistic approach to defining rFVIIIs as EHLs, which requires all of the following: (i) using technology designed to extend rFVIII half-life; (ii) lacking bioequivalence with a standard rFVIII comparator-above the FDA/EMA cut-off of 125% for the 90% confidence intervals for area under the curve ratio; and (iii) having an extended half-life ratio measured in a PK comparator crossover study. In this systematic review, a pragmatic definition of EHL rFVIII has been proposed that should provide better clarity in clinical discussions surrounding the appropriate use of rFVIII products. At present, only products using PEGylation or Fc fusion half-life extension technology meet the proposed criteria for definition of EHL rFVIII.

Recombinant factor VIII: past, present and future of treatment of hemophilia A.

The development of recombinant factor VIII (rFVIII) was initially driven by the necessity to treat hemophilia A (HA) patients with FVIII concentrates without the risk of transmitting infectious agents. Over the last three decades the safety of rFVIII has been further improved by completely removing animal or human proteins from the manufacturing process, so that patients would not be exposed to known or emerging pathogens. Recent efforts have concentrated on improving the expression of rFVIII, reducing its immunogenicity and enhancing its pharmacokinetic (PK) behavior. These new goals have been possible thanks to the develop-ment of biotechnology and a better knowledge of the function and structure of FVIII. Several approaches such as deletion of the B-domain, expression of FVIII by human cell lines, sequence modification, structural modification, co-expression with other proteins, fusion with the Fc fragment of immunoglobulins and PEGylation have been utilized. As a result of these efforts, different rFVIII products have been validated in terms of efficacy, immunogenicity and PK profile. Other technologies are currently being explored to improve the PK of FVIII and allow its subcutaneous administration. Although nonreplacement therapies and HA gene therapy appear to be promising alternatives for HA, rFVIII will very likely remain as a critical component for the treatment of HA because of its physiological activity and mode of action, as well as its unique ability to induce or restore tolerance to exogenous FVIII. This review summarizes the principal features of past, current and emerging rFVIII products for HA.

Cost-utility analysis of life-long prophylaxis with recombinant factor VIIIFc vs recombinant factor VIII for the management of severe hemophilia A in Sweden

Aims: Prophylaxis with recombinant factor VIII (rFVIII) is the standard of care for severe hemophilia A in Sweden. The need for frequent injections with existing rFVIII products may, however, result in poor adherence to prophylaxis, leading to increased bleeding and long-term joint damage. Recombinant FVIIIFc (rFVIIIFc) is an extended half-life fusion protein which can offer prolonged protection and reduced dosing frequency. The objective of this study was to evaluate the cost-utility of prophylaxis with rFVIIIFc in severe hemophilia A from the perspective of the Swedish health system.Methods: A Markov model was built to estimate lifetime costs and benefits of prophylaxis with rFVIIIFc vs rFVIII products. Clinical outcomes were represented by annualized bleeding rate (ABR) and quality of life via disutility applied to bleeding events and injection frequency. Costs included the cost of FVIII for routine prophylaxis and bleed resolution. The pooled comparator was costed by weighting the cost of individual products by their market share.Results: In the base case, rFVIIIFc was dominant vs the pooled comparator. Savings of SEK 9.0 million per patient resulted from lower factor consumption for prophylaxis and bleed resolution. Fewer bleeds and reduced injection frequency yielded an estimated 0.59 quality-adjusted life years (QALYs). Results were sensitive to drug dosage and robust to variation in other parameters. Probabilistic sensitivity analysis suggested a greater than 85% probability of rFVIIIFc being cost-effective at a willingness-to-pay threshold of 500,000 SEK/QALY.Limitations: Due to unavailibilty of patient-level data, treatment benefit was based on a non-adjusted indirect comparison. Dosing and treatment outcomes were assumed to persist over the model duration in the absence of long-term outcome data.Conclusion: The results suggest that rFVIIIFc may be a cost-effective option for hemophilia A prophylaxis, generating greater quality of life and reduced costs for the Swedish payer compared to more frequently administered rFVIII alternatives.

Meta-analysis on incidence of inhibitors in patients with haemophilia A treated with recombinant factor VIII products.

: Recent cohort studies showed differences in inhibitor incidence in previously untreated patients (PUPs) with haemophilia A treated with recombinant factor VIII (rFVIII) concentrates. We carried out a systematic literature search and meta-analysis for all randomized clinical trials and observational studies published from 1 January 1988 to 31 August 2015, to assess the incidence of inhibitor development and the relationship with rFVIII product used in PUPs and minimally treated patients (MTPs, ≤5 previous exposure days), with severe haemophilia. The primary outcome measure was development of all inhibitors and high-titre inhibitors. We computed pooled meta-analytic estimates according to the rFVIII product used with the inverse-variance method, assuming a fixed, or a random-effects model if significant between-studies heterogeneity was present. Out of 781 articles, 16 published between 1998 and 2015 were included in the meta-analysis, involving a total of 2094 haemophilia A PUPs or MTPs. The pooled estimate of all inhibitors was 0.27 (95% confidence interval 0.23-0.31). No significant difference in pooled inhibitor incidence across products was found (P = 0.72). Meta-analysis of studies reporting inhibitor hazard ratios with different rFVIII products, adjusted to different risk factors, showed that PUPs/MTPs treated with Advate had a pooled inhibitor hazard ratio estimate of 0.63 (95% confidence interval 0.48-0.83) as compared with Kogenate FS. The overall inhibitor incidence in PUPs/MTPs was 27%. Differences between products were found considering hazard ratios in which potential confounders were taken into account.

Long-term efficacy and safety of prophylaxis with recombinant factor VIII in Chinese pediatric patients with hemophilia A: a multi-center, retrospective, non-interventional, phase IV (ReCARE) study

Background: The first recombinant factor VIII (rFVIII) product was launched in China in 2007. However, until now, no study has been conducted to describe the efficacy and safety of prophylaxis with rFVIII in Chinese pediatric patients with hemophilia A (HA).Objective: To summarize the efficacy and safety data on prophylaxis with rFVIII in Chinese pediatric patients with HA.Methods: ReCARE (Retrospective study in Chinese pediatric hemophilia A patients with rFVIII contained regular prophylaxis) was a retrospective study conducted in 12 hemophilia treatment centers (HTCs) across China. The primary endpoints included reduction in annualized bleeding rate (ABR); the secondary endpoints included evaluation of joint function (number and sites of target joints) using Gilbert score and Hemophilia Joint Health Score (HJHS), quality of life (QoL) and factors affecting treatment choices. Safety assessment of rFVIII was also conducted.Results: We analyzed a total of 183 male pediatric patients (mean age, 7.1 ± 4.23 years) who received prophylaxis between 1 November 2007 and 31 May 2013. Compared with baseline, prophylaxis with rFVIII significantly reduced overall annualized joint bleed rate (AJBR) (p < .001) and ABR (p < .001). Inhibitor formation was reported in 5 (2.7%) patients and hemarthrosis was reported in 1 patient. The mean number of target joints was positively related to age (p < .001) and weight (p = .003) at baseline. Responses from survey questionnaires reported that effective bleeding control, joint protection, improvement in quality of life, favorable medical insurance policies, and economic capability were reasons for choosing prophylaxis.Conclusion: Prophylaxis with rFVIII reduced bleeding and number of target joints, even with a low-dose regimen, in Chinese pediatric patients with HA. Other than the efficacy and safety, factors such as poor disease control, improved economic stability and stable financial support made prophylaxis as an attractive treatment option.ClinicalTrials.gov ID: NCT02263066.

Efficacy and safety of a recombinant factor VIII produced from a human cell line (simoctocog alfa)

ABSTRACTIntroduction: The development of inhibitors against infused factor VIII (FVIII) has a detrimental impact on health and quality of life of patients with hemophilia A. Several observational studies and a recently published randomized trial indicate that the inhibitor risk in previously untreated patients (PUPs) is higher following the use of recombinant FVIII (rFVIII) products compared with plasma-derived FVIII concentrates. There is currently a great interest towards newer rFVIII products that adopt various technological solutions to reduce the inhibitor risk.Areas covered: This review describes the efficacy and safety of simoctocog alfa (Nuwiq), a human cell-line derived rFVIII developed for the prevention and treatment of bleeding in hemophilia A patients. Particular relevance will be given to the safety aspects of this product, with special emphasis on the rate of inhibitor development, presenting the results of the phase II and III clinical trials on previously treated patients (PTPs) and the interim data from the phase III trial on PUPs.Expert opinion: The final results from PTP studies and the preliminary data from the PUP study document the high efficacy and safety profile of this rFVIII product, which has the potential to reduce the inhibitor risk in PUPs with severe hemophilia A.

BAY 81‐8973, a full‐length recombinant factor VIII: manufacturing processes and product characteristics

BAY 81-8973 (Kovaltry® , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20-nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human- or animal-derived proteins are added to the cell culture process, purification or final formulation. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81-8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A.

Differential Glycosylation Between Recombinant Factor VIII Produced in Baby Hamster Kidney and Chinese Hamster Ovary Cells Confers Differences in Immunogenicity in a Humanized Hemophilia Α Mouse Model

IntroductionThe formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations.MethodsFactor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS.Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT.FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay.ResultsLectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p<0.01), and greater levels of sialic acid capping (p<0.01) and fucosylated glycans (p<0.01). Mass spectra confirmed higher levels of sialic acid and identified two additional N-linked sites bearing high mannose glycans on CHO-rFVIII. In this mouse model we observed that BHK-rFVIII had a circulating half-life of 6.06 hours compared to the 10.01 hour half-life of CHO-rFVIII (p<0.0001).The immunogenicity of the BHK- and CHO-rFVIII products was next evaluated in vivo. In mice primed with a single 6 IU dose of BHK-rFVIII, we identified a higher proportion of FVIII-specific IFN-γ secreting splenocytes after seven days. Furthermore, long-term studies showed that 100% of mice subcutaneously exposed to BHK-rFVIII developed anti-FVIII IgG compared to the 47% that received CHO-rFVIII (p<0.01). Coincidently, when FVIII inhibitors were measured, we observed an incidence of 100% vs 37% (p<0.01), respectively. While the titres of FVIII-specific IgG were higher in mice exposed to BHK-rFVIII (p<0.01), there were no significant differences in the inhibitor concentrations. Similarly, we observed increased titres of FVIII-specific IgG in mice exposed intravenously (1 ug LPS with the first infusion) to BHK-rFVIII compared to CHO-rFVIII. However, there were no differences in the incidence of FVIII-specific IgG, nor in the incidence and concentration of inhibitors between the intravenously-infused mice.ConclusionsOur results demonstrate that BHK-rFVIII exhibits altered pharmacokinetic and immunogenic properties compared to CHO-rFVIII in humanized hemophilia A mice. The observed early increase in the proportion FVIII-specific IFN-γ producing cells in the spleen suggests an intrinsic immunogenic element of BHK-rFVIII. Similarly, the substantially increased immunogenicity of BHK-rFVIII in mice when treated subcutaneously complements the previously-reported clinical evidence. These differences may be attributed to the significant disparities in N-linked glycosylation, most notably high mannose and sialic acid containing glycans. Additional studies are underway to directly address the role of the these specific glycans and their potential impact on immunogenicity of rFVIII. DisclosuresLillicrap:Octapharma: Research Funding; Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding.

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin

Inhibitor Development in Previously Untreated Patients with Severe Hemophilia a Treated with Nuwiq®, a New Generation Recombinant FVIII of Human Origin