Lack of evidence for increased inhibitor incidence in patients switched from plasma-derived to recombinant factor VIII

De novo inhibitor development is a rare event in PTPs switched from pdFVIII to rFVIII. Based on previously published data of clinical studies a change in FVIII product is unlikely to provoke inhibitor formation.     

Product‐dependent anti‐factor VIII antibodies

The development of anti‐factor (F)VIII antibodies in haemophilia A (HA) subjects undergoing replacement therapy has been well documented. The correlation between antibody development and the FVIII product used for replacement therapy remains a subject of discussion. The aim of this study was to evaluate the presence of anti‐FVIII antibodies towards three commercial rFVIII products in 34 HA subjects’ plasmas. Antibodies were quantitated by a Multiplex Fluorescence Immunoassay. All plasmas contained anti‐FVIII antibodies at variable concentrations ranging from 50 nm to 570 μm . Eleven of the 20 HA subjects treated with one (r)FVIII product contained inhibitory anti‐FVIII antibodies (0.8‐3584 BU). The inhibitory antibody titre and the molar concentrations of total antibody were mildly correlated (r² = 0.6). Pronounced differences in antibody recognition with the three rFVIII products were observed. For the group treated with Product ‘A’, the titre towards this product was 2.4‐fold higher than that observed with another full‐length rFVIII‐containing product (Product ‘B’) and almost four‐fold higher than that measured with a B domain‐less rFVIII product (Product ‘C’). For the group of 14 HA subjects treated with FVIII other than Product ‘A’, only one showed higher antibody titre when measured with this product. Our data suggest that the development of anti‐FVIII antibodies is biased towards the product used for treatment and that a significant fraction of antibodies bind to the B domain of FVIII.     

Clinical observation on safety and efficacy of a plasma‐ and albumin‐free recombinant factor VIII for on‐demand treatment of Chinese patients with haemophilia A

Summary. Recombinant FVIII (rFVIII) has become the best choice for treating bleeding of haemophilia A patients. A plasma‐ and albumin‐free recombinant FVIII (rAHF‐PFM, ADVATE^®), as the third generation rFVIII, virtually eliminates the risk of blood‐borne disease transmission by excluding all human blood derived additives throughout cell culture, purification and formulation. In this multicentre prospective clinical study we evaluated the efficacy, safety and immunogenicity of ADVATE^® in Chinese patients with haemophilia A. Fifty‐eight patients enrolled and received ADVATE^® treatment. Of the patients enrolled, eight (13.79%) had severe haemophilia, 45 (77.59%) had moderate haemophilia and five (8.62%) had mild haemophilia. Fifty‐four patients completed 6 months of observation. A total of 781 bleeds occurred in these 58 subjects, all evaluable per‐protocol. A total of 984 infusions were administered with a mean of 17.0 ± 11.1 infusions per patient. On average, each patient received a mean of 15030.2 ± 7972.7 IU ADVATE^® (median 13 625 IU, range 9500–19 750 IU) during 6 months. The majority of bleeding episodes (95.9%) were successfully treated with one or two infusions of ADVATE^®. Overall, response to the first ADVATE^® treatment was rated as either ‘excellent’ (82.8%) or ‘improved’ (17.2%) in all subjects. All patients tolerated ADVATE^® infusions well. One patient (1/58, 1.7%) developed an inhibitor of 4 Betheseda units at day 180 visit. The results of this clinical observational study support that ADVATE^® is efficacious, safe and well tolerated in the treatment of Chinese patients with haemophilia 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.     

The incidence of factor VIII inhibitors in severe haemophilia A following a major switch from full‐length to B‐domain‐deleted factor VIII: a prospective cohort comparison

Although it has been suggested that switching of factor VIII (FVIII) products may increase inhibitor formation this is disputed. Half of UK patients changed rFVIII brands because of national contracting in 2010, presenting an opportunity to compare inhibitor incidence of switchers with non‐switchers. Centres were requested to test all the patients for inhibitors prior to the switching date and 6‐monthly thereafter. Positive and negative inhibitor test data were also collected to analyse for testing bias. A total of 1198 patients with severe haemophilia A and treated with Advate, Kogenate/Helixate or Refacto AF preswitch were included in the analysis, of whom 516 switched to Refacto‐AF and 682 did not switch products. Five new inhibitors were reported amongst previously treated patients (>50 exposure days) with a median titre at the time of detection of 1.25 BU mL^?1 (IQR 0.7–23.05). One inhibitor occurred in a non‐switcher using Kogenate, an incidence of 1.5 per 1000 treatment‐years (95% CI 0.2–10.5). Four inhibitors arose in patients who had switched from Kogenate (two) or Advate (two) to ReFacto‐AF, an incidence of 7.8 per 1000 treatment‐years (95% CI 2.9–20.8). These incidence rates did not differ significantly from one another (incidence rate ratio 5.3 (95% CI 0.5–260.3) or from the historical rate of 6.05 inhibitors/1000 treatment‐years (95% CI 5.18–7.06). Only one inhibitor (non‐switcher) persisted. Non‐switchers were significantly older (P = 0.03), and used significantly less FVIII per year (P = 0.005) prior to switching. Following switching, factor usage increased similarly (P = 0.53) in both groups. Switching from FLRFVIII to Refacto‐AF (BDDRFVIII) was not associated with an increased inhibitor development.     

Potency estimation of recombinant factor VIII: effect of assay method and standard

Large potency discrepancies between the chromogenic and one-stage clotting methods have been reported for patients' plasma samples following the infusion of recombinant factor VIII (rFVIII) concentrates. We have investigated the potency estimation of two different full-length rFVIII concentrates using both assay methods relative to both plasma and concentrate standards. Potencies by the chromogenic method were significantly higher (53% and 45%) than potencies by the one-stage clotting method when a plasma standard was used. In contrast, there was no significant potency difference between methods when a concentrate standard was used. Time-course studies into thrombin and activated factor X (FXa) generation, in modified clotting and chromogenic methods, respectively, revealed that the two rFVIII concentrates behaved very similarly to the concentrate standard, whereas the plasma standard showed slightly more rapid thrombin generation and markedly slower FXa generation. The different behaviour of rFVIII and plasma FVIII in the chromogenic method is proposed as the main cause of the methods-based potency discrepancy. The results support the use of a concentrate standard to measure rFVIII in post-infusion plasma.     

Profiling of factor VIII mutations in Korean haemophilia A

Summary.  Haemophilia A (HA) is an X chromosome-linked inherited bleeding disorder caused by heterogeneous mutations of coagulation factor VIII (FVIII). Although more than 900 mutations of FVIII gene are reported in the HAMSTeRS database, the mutation data regarding the FVIII gene in the Korean population is currently insufficient. The aim of this study was to profile the mutations of FVIII in Korean HA, 38 unrelated Korean HA male patients were examined. Peripheral blood samples were obtained from the patients. Long distance-PCR was performed for the identification of inversions in intron 22 and intron 1. Then gross exon deletion was examined to the inversion-negative patients by multiplex-PCR. Finally, direct sequencing was performed on exons 1–26, 5'- and 3'-UTR. We identified 33 mutations from the 38 patients. These included 15 inversions in intron 22 (39.5%), one inversion in intron 1 (2.6%), one gross exon deletion (2.6%), five deletions (13.2%), two insertions (5.3%), six missense (15.8%) and three nonsense mutations (7.9%). Mutation types for five patients (13.2%) were not identified in this study. We determined that the most common defect in FVIII in this study was an inversion mutation in intron 22; this is consistent with the findings of other studies. For the first time in Korean HA, a patient with intron 1 inversion was found. In addition, we report eight novel mutation types which never been reported in HAMSTeRS database. The mutation data in this study should prove useful as a reference for the diagnosis of HA and the detection of carriers in the Korean population.     

Therapeutic factor VIII does not trigger TLR1.2 and TLR2.6 signalling in vitro

The administration of therapeutic factor VIII (FVIII) to patients with haemophilia A induces the development of inhibitory anti‐FVIII IgG in a substantial number of patients. For an antigen‐specific immune response to develop, antigen‐presenting cells (APCs) need to mature and procure appropriate co‐stimulatory signals to T cells at the time of presentation of the endocytosed antigen. The nature of the danger signals that induce APC maturation, thus initiating the anti‐FVIII immune response, are yet ill‐characterized. Contradictory reports on a direct effect of therapeutic FVIII on APC maturation have been released. Here, we investigated whether FVIII directly triggers Toll‐like receptor 2 (TLR2) signalling. The capacity of human recombinant FVIII to promote the maturation of a mouse bone marrow macrophage cell line (BMA) was investigated by flow cytometry. In parallel, the triggering of TLR1.2 or TLR2.6‐expressing HEK293 cells by FVIII was analysed following transfection of the cells with a reporter construct for NFκB activity. In contrast, to zymosan, a known TLR2 agonist, human recombinant FVIII did not induce the maturation of mouse BMA macrophages, as analysed by the levels of expression of CD80, CD86, CD40 and I‐Ab at the cell surface. Furthermore, incubation of FVIII with cells expressing TLR2 paired with TLR1 or TLR6, failed to activate NFκB, whereas NKκB activity was triggered in the presence of zymosan. Our results confirm that FVIII alone is insufficient to trigger the maturation of APCs that is required to initiate an immune response.     

Pharmacokinetics and safety of OBI-1, a recombinant B domain-deleted porcine factor VIII, in subjects with haemophilia A

Summary. OBI‐1 is a recombinant B‐domain deleted porcine factor VIII (FVIII). FVIII treatment in those with haemophilia A may be complicated by the development of anti‐FVIII antibodies (inhibitors) leading to a failure to respond to treatment with human FVIII. To compare the pharmacokinetics and safety of a single dose of OBI‐1 with Hyate:C in subjects with haemophilia A and inhibitors, subjects were randomized to receive either Hyate:C followed by placebo or placebo followed by OBI‐1 in a double‐blind fashion. FVIII levels were assayed using both a one‐stage coagulation assay (OSCA) and chromogenic assay. Pharmacokinetic parameters for FVIII were calculated for 6/9 subjects randomized; in three subjects baseline anti‐porcine FVIII inhibitors led to a lack of measurable FVIII activity. Mean C_max appeared higher for OBI‐1 (OSCA: 176.00 U dL^?1, standard deviation ± 88.00; chromogenic: 151.00 ± 31.51 U dL^?1) than Hyate:C (OSCA: 82.3 ± 19.22 U dL^?1; chromogenic: 52.67 ± 13.8 U dL^?1). Mean AUC also appeared higher for OBI‐1 (OSCA: 2082.87 ± 1323.43 U h^?1dL^?1; chromogenic: 1817.28 ± 625.14 U h^?1dL^?1) than Hyate:C (OSCA: 1177.8 ± 469.49 U h^?1dL^?1; chromogenic: 707.61 ± 420.05 U h^?1dL^?1). Two infusion‐related events occurred: one with Hyate:C, one with placebo. Four of five subjects without anti‐porcine FVIII inhibitors at baseline remained porcine FVIII inhibitor negative 29 days after infusion. A single dose of OBI‐1 appears to have higher bioavailability than Hyate:C in subjects with haemophilia A without measurable anti‐porcine FVIII inhibitors, and is well tolerated. These results should be confirmed in a larger phase 2/3 study.     

Practical and cost‐effective measurement of B‐domain deleted and full‐length recombinant FVIII in the routine haemostasis laboratory

The assessment of recombinant FVIII (rFVIII) activity (FVIII:C) in plasma of patients is dependent on the assay. Notably, a calibration with a product‐specific laboratory standard is recommended when measuring Refacto‐AF^R activity in plasma with a one‐stage assay. The objective of this study was to facilitate the measurement of rFVIII, taking into account the recent demonstration that a calibration curve does not have to be included in each run. FVIII:C was measured in patients' samples after infusion of different types of rFVIII with a one‐stage and a chromogenic assay calibrated either with pooled normal plasma or a product‐specific laboratory standard. Results obtained with the one‐stage coagulation assay were compared with these provided by a chromogenic assay. We confirmed that a calibration curve can be used for a prolonged period of time without loss of precision and accuracy. In such conditions, a stable relation between the calibration curves generated with a product‐specific laboratory standard and plasma can be established. In patients' plasma, Refacto‐AF levels measured with a one‐stage FVIII assay calibrated with plasma or a product‐specific laboratory standard diverged from ?58% to ?17% and from ?25% to +18%, respectively, from the activity determined with a chromogenic substrate assay. By comparison, FVIII:C levels of full‐length rFVIII measured with the one‐stage assay calibrated with plasma were 6–49% lower than with the chromogenic assay. In a monocentric setting, the long‐term stability of the calibration curves allows the implementation of a practical and cost‐effective approach to determine rFVIII:C levels.