Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products.

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one-stage clotting assays.

Essentials Performance of the one-stage clotting (OSC) assay varies with the clotting activator used. Recombinant FIX-albumin fusion protein (rIX-FP) was reliably monitored with most OSC reagents. rIX-FP shows comparable reagent-dependent variability to other rFIX products in the OSC assay. Actin® FS and kaolin-based reagents underestimated rIX-FP activity by around 50% in the OSC assay. SUMMARY: Background Measuring factor IX activity (FIX:C) with one-stage clotting (OSC) assays, based on the activated partial thromboplastin time (APTT), is the current mainstay of diagnostic techniques for hemophilia B. Assessing the performance of new recombinant FIX (rFIX) products in OSC assays is essential, as APTT reagents from different manufacturers yield different potency estimates for rFIX. Objectives To evaluate the extent to which choice of reagent composition influences rFIX potency measurements of recombinant FIX-albumin fusion protein (rIX-FP, IDELVION) activity in OSC assays. Methods rIX-FP was added to FIX-deficient plasma, and FIX:C was assessed centrally and locally in a multicenter international field study with a variety of commercial OSC APTT reagents. Paired sample analysis of clinical samples was performed to compare values of FIX:C from local and central laboratories. In-house bioanalytical investigations with spiked samples were conducted to compare the APTT-reagent dependent variability of rIX-FP with unmodified rFIX and rFIX Fc fusion protein (rFIXFc). Results Central and local assessments of FIX:C from 10 countries and 21 participating centers showed comparable results to those from the central laboratory across the majority of 18 different APTT reagents from both clinical and spiked samples. There was a consistent underestimation of rIX-FP activity of ≈ 50% with OSC assays using Actin FS or kaolin-based APTT reagents. In the bioanalytical study, rIX-FP showed comparable variability in OSC assays to unmodified rFIX and rFIXFc. Conclusions rIX-FP activity can be accurately measured by the use of OSC assays with the majority of commercial reagents. Actin FS or kaolin-based reagents will probably lead to a 50% underestimation of activity.

Calibration of the 7th British Working Standard for factors II, IX and X, concentrate.

Seven laboratories from 5 different countries participated in the calibration of the 7th British Working Standard (BWS) for blood coagulation factors II, IX and X. The candidate, 15/182, was assayed for Factors II and X potencies against the 4th International Standard (IS) for Factors II and X, Concentrate (11/126) and for Factor IX potency against the 5th IS for Factor IX, Concentrate (14/148). Intra-laboratory GCVs for all 3 factors were less than 10%, with the majority less than 5%. Inter-laboratory GCVs were 3.4%, 3.2% and 2.3% for FII, IX and X respectively. All participants agreed with the value assigned and preparation 15/182 was established by NIBSC in October 2017 as the 7th BWS for FII, IX, X Concentrate with potencies of 6.0 IU/ampoule, 6.7 IU/ampoule and 4.9 IU/ampoule for FII, IX and X respectively.

Current laboratory practices in the diagnosis and management of haemophilia: a global assessment.

Haemophilia management is complicated by the extreme variability in laboratory practices. Lack of consistency or comparability in testing makes it difficult to establish diagnostic criteria or disease severity, and complicates response assessment. A global survey was conducted to document current practices. A 35-min survey was completed by 30 laboratory scientists in each of seven countries (France, Germany, Italy, Japan, Spain, UK, USA; 210 in total); results were weighted by average country testing volume in haemophilia. Eighty-three per cent of participants reported participation in a Quality Assurance scheme. Ninety per cent reported using clotting tests in haemophilia A and 88% in haemophilia B (55% and 53% frequent use respectively). Sixty-eight per cent reported chromogenic assays were used in haemophilia A, with only 23% reporting frequent use, compared to only 11% reporting any use in haemophilia B. Twenty-nine separate activated partial thromboplastin time (aPTT) reagents were reported for haemophilia A and 27 aPTT reagents were reported for haemophilia B, with one-quarter or less obtaining reagents or kits from any single manufacturer. Fifty-four per cent run a calibration curve with every factor VIII (FVIII) assay. The mean number of plasma dilutions varied from 2 to 4 for FVIII assays and from 1 to 3 for FIX assays. Results indicate very low consistency in materials and practices used to test for factor activity in haemophilia. A number of responses suggest that some laboratory scientists' understanding of best practices or guidelines in haemophilia could be improved. More education and broader understanding is recommended regarding assay types, assay components, test material and instrument features and capabilities.

Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators.

A workshop organized by the European Medicines Agency and the European Directorate for the Quality of Medicines and HealthCare was held in London, UK on November 28-29, 2013, to provide an overview of the current knowledge of the characterization of new factor VIII (FVIII) and factor IX (FIX) concentrates with respect to potency assays and testing of postinfusion material. The objective was to set the basis for regulatory authorities' discussion on the most appropriate potency assay for the individual products, and European Pharmacopoeia (Ph. Eur.) discussion on whether to propose revision of the Ph. Eur. monographs with respect to potency assays in the light of information on new FVIII and FIX concentrates. The workshop showed that for all products valid assays vs. the international concentrate standards were obtained and potency could be expressed in International Units. The Ph. Eur. chromogenic potency assay gave valid assay results which correlate with in vivo functionality of rFVIII products. For some modified rFVIII products and all modified rFIX products, one-stage clotting assay methods result in different potencies depending on the activated partial thromboplastin time reagent. As a consequence, monitoring of patients' postinfusion levels is challenging but it was pointed out that manufacturers are responsible for providing the users with appropriate information for use and laboratory testing of their product. Strategies to avoid misleading determination of patents' plasma levels, e.g. information on suitable assays, laboratory standards or correction factors were discussed.

Virus removal from factor IX by filtration: validation of the integrity test and effect of manufacturing process conditions.

Virus removal from a high purity factor IX, Replenine-VF, by filtration using a Planova 15N filter has been investigated. A wide range of relevant and model enveloped and non-enveloped viruses, of various sizes, were effectively removed by this procedure. Virus removal was confirmed to be effective when different batches of filter were challenged with poliovirus-1. It was confirmed that intentionally modified filters that failed the leakage test had completely lost the ability to remove virus, thus confirming that this test demonstrates gross filter failure. In the case of the more sensitive integrity test based on gold particle removal, it was found that a pre-wash step was not essential. Planova filters that had been modified by sodium hydroxide treatment to make them more permeable, and filters manufactured with varying pore-sizes over the range of 15-35 nm, were tested. The integrity test value that resulted in the removal of >4 log(10) of poliovirus-1 from the product correlated with that recommended by the filter manufacturer. Virus removal from the product was not influenced by filter load mass, flow-rate or pressure. These studies confirm the robustness of this filtration procedure and allow suitable process limits to be set for this manufacturing step.

Effect of manufacturing process parameters on virus inactivation by solvent-detergent treatment in a high-purity factor IX concentrate.

BACKGROUND AND OBJECTIVES: Treatment with solvent-detergent is widely used for ensuring the virus safety of plasma products. Laboratory studies have shown this procedure to be effective for inactivating enveloped viruses under manufacturing conditions. In the present study, the effect of different manufacturing process parameters on virus inactivation by treatment with polysorbate 80 and tri-n-butyl phosphate were investigated for a high-purity factor IX concentrate in order to evaluate the robustness of this step. MATERIALS AND METHODS: Samples of factor IX intermediate were obtained, virus was added and the kinetics of virus inactivation followed during incubation. The effect of altering the conditions on virus inactivation was tested. RESULTS: Solvent-detergent treatment was confirmed to effectively inactivate, i.e. by > or = 5 log, a wide range of representative enveloped viruses under standard conditions. Virus inactivation was consistently effective in a number of different manufacturing batches. Of the parameters tested, only solvent-detergent concentration and temperature significantly effected virus inactivation. CONCLUSIONS: The robustness of the solvent-detergent step for virus inactivation has been confirmed. Using the data generated, appropriate limits can be set for this manufacturing process step.

Hepatitis G virus in clotting factor concentrates.

Blood-borne hepatitis is a well-known complication in patients with bleeding disorders. A recently discovered parentally transmitted virus, hepatitis G [GB virus C (GBV-C)] has an increased prevalence in patients with haemophilia. Clotting factor concentrates derived from pools of human plasma currently undergo viral inactivation techniques known to be effective against hepatitis B, C and HIV; however, the effectiveness of current purification and viral inactivation techniques against newly discovered viruses such as GBV-C is unknown. A total of 37 vials of clotting factor concentrates manufactured in the USA from 1981 to 1995 were tested for the presence of GBV-C virus. All samples that did not undergo a specific viral inactivation step were positive for GBV-C. Viral inactivation techniques that did not uniformly remove GBV-C included vapour heat treatment and dry heat treatments for less than 144 h. All samples treated by pasteurization, solvent detergent or dry heat for 144 h, were negative for the presence of GBV-C.

[Technology optimization and properties study of factor IV complex concentrates].

OBJECTIVE: To produce high quality factor IX complex concentrates with domestic ion exchanger. A human solvent-detergent (S/D) treated factor IX complex concentrates has been produced from fresh-frozen plasma using domestic DEAE-Sepharose fast flow ion exchange chromatagraphy and Ca3(PO4)2 adsorption. The properties of this new concentrates were compared with those of the concentrates obtained by foreign DEAE-Sapharose CL-6B. RESULTS: The recovery of activity was 93.67 +/- 2.56% (n = 6) and 88.71 +/- 2 (n = 6), respectively. The specific activity of the concentrates was 7.36 +/- 0.96 U Factor IX: C/mg protein (n = 6). This new concentrates retained all the useful components with higher purity and lower thrombogenicity. Conclusion Domestic ion exchanger could replace foreign ion exchanger in producing high quality factor IX complex concentrates.

Concentrate safety and efficacy.

Safety from transmission of infections through plasma-derived clotting factor concentrates is assured by improved donor screening, serological testing of individual donations and direct viral testing of small plasma pools. Modern viral-inactivation techniques are highly effective. Recombinant concentrates stabilized in human albumin are being superaeded by those with other stabilizers. Recently reported discrepancies between estimates of concentrate potency from in vitro assays versus in vivo recovery, depending upon type of assay and reference standard used, are not fully resolved.

Recombinant factor IX recovery and inhibitor safety: a Canadian post-licensure surveillance study.

As part of the Canadian post-licensure surveillance on the safety of recombinant factor IX (rFIX. BeneFIX), factor IX recovery and inhibitor development were studied. The recovery following rFIX infusion in 126 patients (mean = 0.77, median 0.72, range 0.36-1.85, 95% CI of mean 0.74-0.81, expressed as FIX activity increase in U/dL per IU FIX concentrate/kg body weight infused) was significantly lower than that following the last plasma-derived factor IX (pdFIX) infusion in 74 patients (mean 1.05, median 1.00, range 0.37-2.29, 95% CI of mean 0.99-0.97). The recovery for rFIX for patients aged < or =15 years (n = 41, mean recovery 0.64) and that for patients aged >15 years (n = 85, mean 0.84) was each significantly lower than that for pdFIX (aged < or =15 years: n = 21, mean recovery 0.91; aged >15 years: n = 53, mean recovery 1.10). For both rFIX and pdFIX concentrates, the recovery was lower in patients < or =15 years of age compared to those > 15 years of age. Similar data and conclusions were obtained on 66 patients with paired recovery data from rFIX and pdFIX. Overall, our data are similar to those obtained in formal clinical trials. Two of 244 patients treated with rFIX for up to 5 years have developed de novo inhibitors associated with anaphylaxis, an incidence that is similar to that reported for pdFIX. No other serious adverse events, including thrombotic episodes, were reported. To the best of our knowledge, this is the first formal report of recovery and inhibitor formation on rFIX in a peer-reviewed manuscript form.

Recombinant factor IX (BeneFix) by adjusted continuous infusion: a study of stability, sterility and clinical experience.

The safety and efficacy of adjusted continuous infusion (CI) of recombinant factor IX (FIX; BeneFix) was assessed in vitro and in a clinical study. BeneFix was reconstituted at 100 IU mL-1 with or without unfractionated heparin (4 U mL-1) and stored at either 4 degrees C or room temperature. Reconstituted BeneFix retained at least 90% activity over 14 days if stored at 4 degrees C but stability was reduced at room temperature. BeneFix reconstituted in a sterile pharmacy was free of bacterial contamination. Six patients with haemophilia B received seven CIs of BeneFix to cover routine surgery and severe bleeding episodes. The CIs lasted between 3 and 10 days. In all cases, haemostasis was excellent and the desired therapeutic FIX level was easily maintained. No thrombotic episodes or inhibitor development occurred but two patients developed thrombophlebitis at the infusion site when heparin was not added to the infusion. BeneFix is not currently licensed for CI and we suggest that studies to enable licensing should be established as soon as possible.

Inactivation and clearance of viruses during the manufacture of high purity factor IX.

Haemophilia is a bleeding disorder characterised by a deficiency in Factor IX. Replacement therapy in the form of a Factor IX concentrate is a widely accepted practice. In this paper we describe a double virus inactivated chromatographic process for producing a high purity Factor IX product, MonoFIX((R))-VF. The process involves separation of the prothrombin complex by cryoprecipitation, fraction I precipitation and DEAE-cellulose adsorption, further ion-exchange chromatography of crude Factor IX, followed by solvent/detergent treatment. Heparin affinity chromatography is then used to further purify Factor IX. Final nanofiltration is sequential through 35 nm then 15 nm membrane filters. The principal virus inactivation/removal steps are solvent/detergent treatment and nanofiltration and the partitioning of relevant and model viruses provides further reduction in virus load through the production process.Solvent/detergent treatment was shown to achieve log reduction factors of 4.5 for HIV-1, 5.1 for Sindbis virus, 6.1 for vesicular stomatitis virus (VSV), 5.1 for bovine viral diarrhoea virus (BVDV) and 5.3 for pseudorabies virus (PRV). BVDV is a model for hepatitis C virus (HCV), and pseudorabies virus (PRV), like hepatitis B virus (HBV) is an enveloped DNA virus. Using scaled down models of the production process, we have also demonstrated the neutralization/partitioning of at least 6 logs of hepatitis A virus (HAV) during cryoprecipitation, Fraction I precipitation, and the DEAE adsorption and elution step, and a further 1.6 log reduction in HAV load as a result of heparin affinity chromatography. The log reduction factors for HAV as a result of the second ion-exchange chromatography step and as a result of enhanced neutralisation associated with solvent/detergent treatment were not significant. Nanofiltration was shown to contribute a further log reduction factor of 6.7 for HAV and 5.8 for BVDV indicating that log reduction factors of this order would be obtained with other viruses of a similar or larger size, such as HIV, HBV and HCV.Overall, these studies indicate that MonoFIX-VF is a product with an extremely high level of viral safety.

Virus safety of prothrombin complex concentrates and factor IX concentrates.

Substantial progress in virus safety has been achieved during the past 15 years. Therefore only a few virus transmissions with plasma-derived products have been observed since 1985. Specific steps to eliminate, remove, or inactivate viruses were developed. Although virus safety is of decisive importance, chemical or physical treatment during the manufacturing process need not activate labile coagulation factors that cause the risk of thrombogenicity nor need not create neoantigens that mediate the risk of inhibitor formation. Thus, any new virus elimination procedure has to be evaluated and validated for all safety aspects. A comprehensive framework of regulations and efforts has been set up involving plasma donors, donation centres, manufacturers, regulatory authorities, politicians responsible for legislation, physicians, and patients. Blood and plasma donation centres and pharmaceutic industry follow "Good Manufacturing Practice" and are subject to regular audits and official inspections. Every single donation as well as plasma pools are tested for virus markers. The final products need both a marketing authorization and official batch release; in Germany, supervised by the Paul-Ehrlich-Institute. European integration is the purpose of the European Medicines Evaluation Agency. An alert pharmacovigilance system enables scientifically adequate reactions in any case of a safety problem. The ultimate evidence of product safety is provided by clinical surveillance. By participating in clinical studies, patients themselves are able to contribute significantly to the safety of plasma-derived products. The currently achieved high level of safety should encourage us to take further steps to stabilize this success and to look for further progress, wherever possible.

The manufacturing process for recombinant factor IX.

Advances in recombinant DNA manufacturing technology have now made possible the production of a highly purified and active recombinant factor IX (rFIX) product. Recombinant factor IX was developed by (1) stable insertion of the genes for both factor IX and PACE-SOL (a truncated, soluble serine protease needed to enhance the capacity of cells to remove the amino-terminal propeptide from rFIX) into Chinese hamster ovary cells; (2) selection of a cell line that was capable of expressing high amounts of active rFIX while growing in bioreactors containing a completely defined culture medium that does not contain blood or plasma products; and (3) inclusion of four independent chromatography steps, none of which require monoclonal antibodies. Furthermore, rFIX has been extensively tested to demonstrate similarity to plasma-derived factor IX and has been shown to be a consistent, high-purity product. For example, a high-specific-activity product (276+/-23 IU/mg) has been consistently produced throughout 65 consecutive batches from five consecutive manufacturing campaigns. Thus, rFIX offers a consistent and high-purity source of factor IX treatment for patients with hemophilia B.