Potency Labeling of Novel Factor VIII and Factor IX Concentrates: Past Experience and Current Strategy Anthony R. Hubbard, PhD1
Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, United Kingdom Semin Thromb Hemost
Abstract
Keywords
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hemophilia factor VIII factor IX coagulation
Address for correspondence Anthony R. Hubbard, PhD, Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom (e-mail: [email protected]).
Various strategies to produce “longer-lasting” factor VIII and factor IX concentrates through chemical and genetic modifications are currently under evaluation. It is now clear that these new molecules are amenable to testing using conventional methods for biological activity (one-stage clotting and chromogenic) and there is a preference to maintain labeling in International Units (IU) traceable to the WHO International Standard Concentrates. This is an achievable goal; however, many of the new molecules are associated with potency discrepancies both between methods and also within methods, for instance, when different activated partial thromboplastin time reagents are used. In the interests of global harmonization, it is important for licensing authorities to reach agreement on the choice of the potency labeling method. This choice should be supported by a thorough characterization of product potency, both in vitro and in vivo, to anticipate future issues and with a view to maintaining equivalence of the IU compared with existing licensed products. In cases where the product potency is defined using specific reagents, the robustness of the manufacturer’s product standard will be crucial for product consistency. The sensitivity of measured potency to different methodologies will require manufacturers to provide guidance to clinical laboratories on suitable postinfusion testing approaches.
Replacement therapy for hemophilia is entering a new era, which will see the introduction of bioengineered factor VIII (FVIII) and factor IX (FIX) products with improved properties over the natural molecules. The objective of most new developments is to reduce the frequency of infusion through a prolonged plasma half-life as a result of various strategies, such as pegylation, protein fusion, or increased stability (single chain FVIII).1–3 Results from preclinical and clinical studies are encouraging, particularly for the modified FIX molecules, where half-life extensions up to fivefold have been reported.4 Despite the advanced technologies applied to the development of these new products, they are still classified as biological medicines, which means their activity is measured
Issue Theme Current Issues in Hemophilia: Recognizing Clinical Heterogeneity, Replacement Therapy and Outcome Assessment; Guest Editor: Alok Srivastava, MD, FRACP, FRCPA, FRCP.
relative to a reference standard. Discrepancies in potency measurements, when different methods or assay reagents are used, have been reported to various degrees for most of the new products. This poses a significant challenge to the current system of potency labeling, where all FVIII and FIX concentrates are labeled in International Units (IU) traceable to single World Health Organization International Standard (WHO IS) Concentrates.5–7 The diversity of the new products means that a “one solution fits all” approach to labeling will not be possible and each product should be considered on a case-by-case basis. However, in the interests of global harmonization for product consistency and effective treatment it is imperative to have agreement on the strategy for potency
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DOI http://dx.doi.org/ 10.1055/s-0034-1395353. ISSN 0094-6176.
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1 Haemostasis Section, Biotherapeutics Group, National Institute for
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labeling at the international level. Strategies for addressing discrepant potency issues in the new products may be derived from an appreciation of how the current potency labeling system has survived for over 40 years and our past experience of similar discrepancy issues.
Product Labeling in International Units Maintenance of the WHO IS/IU system has relied on two main components: (1) well-defined methods for potency labeling and (2) comparison of similar standard and test materials (“like vs. like”).
Well-Defined Methods for Factor VIII and Factor IX Potency Potency labeling of the final therapeutic product has evolved into an extremely precise and robust exercise to satisfy the requirement for product consistency, accurate labeling, and hence predictable efficacy in the patient. Two methods are currently used to label FVIII concentrates, the one-stage clotting method and the chromogenic method. The former method has been applied to the labeling of most concentrates licensed in the United States whereas the latter method is recommended for product labeling in Europe and was adopted by the European Pharmacopoeia (EP) in 1995.8 The one-stage clotting method has a long history, being initially developed in the 1950s and refined in the 1960s, whereas the chromogenic method for FVIII was developed and optimized in the 1970s and 1980s.9–12 Although both methods allow the quantification of FVIII clotting activity, they differ in some respects; for instance, the one-stage clotting method is sensitive to the activation status of FVIII in the test samples whereas the chromogenic method is not.13 This may be relevant to the potency estimation of modified FVIII molecules if there is a difference in the profile of FVIII activation during the assay, between the test and standard samples, and may help to explain the variability of potency estimates seen with the one-stage clotting method.5,14–16 In contrast, the chromogenic method for FVIII is designed to achieve full activation of FVIII in the test and standard materials early in the first stage of the procedure and may, therefore, be less affected by differences in the FVIII activation profile. The onestage method is also performed using an extremely diverse range of activated partial thromboplastin time (APTT) reagents and this could also contribute to increased potency variability.17,18 Despite these differences, the application of these methods to product potency estimation follows a similar protocol based on the relative comparison of dose– response relationships of test sample with the reference standard through the assay of multiple dilutions.19 Critical steps and reagents for the chromogenic method as well as instructions on data analysis are described in significant detail in the EP.8 Up to now, FIX concentrates have only been labeled using the one-stage clotting method and this procedure is subject to the same wide range of APTT reagents seen with one-stage FVIII clotting assays. A detailed methodology is described in the EP and this approach has been applied to the potency Seminars in Thrombosis & Hemostasis
estimation of new products under development.20–23 The clotting/chromogenic discrepancy issue has, therefore, been avoided for FIX concentrates up to now; however, this will certainly be a consideration for the future. There is already information that the recently introduced chromogenic methods for FIX may return different potency values for recombinant FIX (rFIX) compared with the one-stage clotting method.24
Comparison of “Like versus Like” Standard and Test Materials The principle of assaying “like versus like” is probably the most powerful tool for the potency testing of biological therapeutics where potency estimation relies on comparison relative to a standard. Over 40 years ago, it was observed that the interlaboratory variability for estimates of FVIII in concentrates was reduced when assayed relative to a concentrate standard rather than a plasma standard.25 This principle is well established in most areas of coagulation factor testing and there are both plasma and concentrate WHO ISs for diagnostic and product testing, respectively, for many factors.7 The testing of “like” samples (test vs. standard) has two advantages, reducing the variability of results between laboratories and minimizing potency discrepancies between methods, such as the one-stage clotting and chromogenic methods for FVIII.26 The ability to obtain valid potency estimates for all FVIII and FIX concentrates relative to single WHO Concentrate ISs is an indication that sufficient similarity has existed between the therapeutic concentrates and the WHO IS to support the single IU. This probably reflects the fact that most FVIII and FIX therapeutics comprised full-length molecules similar to the composition of the relevant WHO IS and that valid comparisons of plasma-derived and recombinant molecules were possible.24,27 We are now faced with extending this comparison to include the new modified therapeutics for IU labeling, and it is worthwhile considering previous challenges to the WHO IS/IU system, associated with large interlaboratory variability or discrepancies between the one-stage clotting and chromogenic methods.
Past Challenges to the World Health Organization International Standard/ International Units Labeling System Recombinant FVIII The introduction of the first generation of recombinant FVIII (rFVIII) concentrates in the 1990s was accompanied by reports of anomalous postinfusion recoveries and methodsbased potency discrepancies and it became clear that rFVIII products were behaving differently to conventional plasmaderived products.28,29 The importance of the choice of assay method and standard was reproduced in vitro where the potency of rFVIII by one-stage and chromogenic methods were not significantly different, relative to a concentrate standard, but differed by more than 40% relative to a plasma standard.30 Equally worrying were the results from the first collaborative studies where rFVIII was tested relative to the WHO IS Concentrate. Estimates for rFVIII were associated
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with very high interlaboratory variability for all methods (geometric coefficient of variation [GCV] 1-stage 39.4%, 2-stage 136.8%, and chromogenic 53.7%), indicating that the rFVIII and the conventional FVIII standards were not behaving as “like” materials.31 These results raised concerns that the WHO IS Concentrate may not be suitable for the potency estimation of rFVIII products and that a separate standard might be required.32 However, laboratory investigations identified methodological details which were able to reduce assay variability; for instance, inclusion of 1% (w/v) albumin in dilution buffers to improve the stability of dilutions during the assay and the predilution of test and standard concentrates in hemophilic plasma or FVIII deficient plasma with normal levels of von Willebrand factor (VWF). These steps markedly reduced the interlaboratory variability of potency estimation for recombinant and very high purity FVIII concentrates to a level where comparisons against the WHO IS Concentrate were acceptable and a separate standard for rFVIII was not required. These methodological modifications were recommended by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC/ISTH) in 1993 and remain relevant to the testing of the modified FVIII products today.33 More recent studies, such as the value assignment of the WHO 8th IS FVIII Concentrate, have confirmed the validity of assaying rFVIII relative to a plasma-derived standard in terms of parallelism and linearity of dose–response relationships.27 However, these comparisons were associated with wider interlaboratory variability (GCV, 14.5–19.7%) than the comparison of plasma-derived versus plasma-derived products (GCVs < 5%). This might reflect a greater sensitivity of rFVIII to local assay conditions such as the need for predilution in FVIIIdeficient plasma with normal levels of VWF as suggested in “SSC field trials” of rFVIII potency.34
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stage incubation time (factor X activation stage) in the chromogenic method.14,16 The potential for potency variability in the chromogenic method was addressed by a revision of the EP monograph for the FVIII assay, which introduced a tighter specification for the first stage incubation time for factor X activation.8 To support postinfusion testing in clinical laboratories, primarily using the one-stage clotting method, a product-specific reference material (ReFacto Laboratory standard, Pfizer Inc., New York, NY) was made available which reinstated the “like versus like” comparison of test and standard samples. This initiative overcame the methods-based potency discrepancy as well as allowed clinical laboratories to continue using their local one-stage clotting methods.17,29 The ability of product-specific standards to overcome large one-stage clotting/chromogenic discrepancies has also been demonstrated for full-length rFVIII.35 Experience with B-DD FVIII emphasized the need for a rigorous evaluation of potency characteristics in vitro prior to licensing to anticipate problems and also the importance of agreement between licensing authorities in the approach to potency labeling when products display large methods-based discrepancies.36 Unfortunately, the latter requirement was not followed when the latest generation of (albumin-free) B-DD FVIII was licensed and labeled using different methodologies in the United States (Xyntha, Pfizer Inc., New York, NY) and Europe (ReFacto AF). We are now in a difficult situation where 1 IU of the product licensed in the United States is equivalent to 1.38 IU of the same material licensed in Europe. Apparently, the large methods discrepancy associated with ReFacto does not apply to a second licensed B-DD product (NovoEight, NovoNordisk A/S, Bagsvaerd, Denmark), and this indicates that subtle differences between molecules with similar modifications can have an effect on assay behavior.37
Recombinant Factor IX B-Domain-Deleted Factor VIII Past experience with the first licensed bioengineered molecule, B-domain-deleted (B-DD) FVIII (ReFacto, Pfizer), is probably the most relevant to the current challenges presented by the new modified products. Following licensing in 1999, there was confusion over postinfusion recovery values, even compared with the full-length rFVIII products, with reports of 50% of expected levels.29 Such anomalous results were the outcome of using different methods and standards for product labeling (chromogenic/concentrate standard) and postinfusion testing (one-stage clotting/plasma standard). The situation was further aggravated by the inherent 20 to 30% potency discrepancy between chromogenic and clotting methods when B-DD FVIII was measured relative to concentrate standards.14,26 Fortunately, with respect to product consistency in the international market, the first version of this product was labeled in IU using the same method (chromogenic) in both Europe and the United States. In addition to potency discrepancies between methods, it was also apparent that different potency estimates could be obtained within the same method when different reagents or procedures were followed. For instance, the relative potency was sensitive to the composition of the APTT reagent in the one-stage clotting method and to the first
The first hints of methods-based potency discrepancies with FIX products are associated with the first rFIX concentrate for hemophilia B therapy (Benefix, Pfizer Inc., New York, NY). Since licensing in 1998, there have been reports of a 30% or greater reduction for in vivo recovery compared with plasmaderived products and it has been suggested that this may be linked to differences in the posttranslational modification of the recombinant molecule leading to increased clearance.38–40 However, the possible contribution of in vitro testing effects, caused by different properties of rFIX and plasma-derived FIX (pdFIX), has also been proposed. For instance, it has been suggested that the potency estimation of rFIX relative to conventional pdFIX standards could be influenced by the different activation rates of rFIX and pdFIX or by the higher content of activated FIX in rFIX products.41,42 The different behavior of rFIX and pdFIX in vitro is supported by the finding that rFIX has a lower potency by the newly developed chromogenic methods compared with the onestage clotting method, when measured relative to a pdFIX reference, whereas no such discrepancy is found with pdFIX.24 Although this type of discrepancy can be reproduced in vitro by spiking with purified activated FIX (FIXa), the amounts required to produce a significant discrepancy are far Seminars in Thrombosis & Hemostasis
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greater than those measured in rFIX products.43 It, therefore, appears unlikely that FIXa content is the main reason for the methods discrepancy or the anomalous postinfusion recovery results. It has also been shown that use of a rFIX reference can overcome the clotting/chromogenic discrepancy and this has been confirmed in a multicenter study where two rFIX products were assayed relative to a rFIX standard, leading to good agreement between methods and reduced interlaboratory variability.44 This is another example where restoration of a “like versus like” comparison can overcome method discrepancies and can be used to support the development of a rFIX reference standard.
Potency Labeling for New Factor VIII and Factor IX Therapeutics Previous experience dealing with products with novel or discrepant potency issues has highlighted some key points pertinent to the potency labeling of the new modified therapeutics, namely, 1. The need to anticipate issues of potency discrepancy on the final product, prior to licensing, with agreement on a single method for potency labeling. To avoid confusion at the point of administration, it is important that potency issues are resolved upstream by the manufacturer and licensing authorities. 2. Recognition that product-specific standards can overcome methods-based potency discrepancies and allow different methods to be used for potency estimation. 3. Provision of guidance to clinical laboratories on the best approach to postinfusion testing. The requirement for a transparent and harmonized approach to the potency labeling of new products, which encompasses and facilitates the above issues, was addressed by an initiative through the SSC/ISTH, and the “Recommendations on the potency labeling of factor VIII and factor IX concentrates” were published in 2013.45 These recommendations address the key issues in the potency estimation of new products, covering potency labeling of the purified product, the manufacturer’s product standard, and postinfusion testing. The first section of the recommendations describes the necessary product characterization on which the decision for potency labeling should be based. To anticipate problems of methods-based potency discrepancies, the product should be evaluated using different methodologies (clotting and chromogenic) and different reagents (APTT) relative to the WHO IS Concentrate. Labeling the new products in IU is only possible if assays relative to the WHO IS Concentrate are valid in terms of parallelism of the dose–response relationships. Where there is little or no potency discrepancy when different methods and reagents are used, the choice of potency labeling method should be fairly straightforward; however, where significant discrepancies exist between methods, then it is important that there is agreement between manufacturers and all licensing authorities on a single method for labeling if we are to avoid a situation similar to that seen with Xyntha/ ReFacto. Where it is not possible to obtain valid tests, relative Seminars in Thrombosis & Hemostasis
to the WHO IS concentrate, the recommendations indicate that it may be necessary to label using product-specific units. Since publication in 2013, the interest in the recommendations, from both regulators and manufacturers, has been very encouraging. This was obvious during a workshop on the “Characterisation of new clotting factor concentrates (FVIII, FIX) with respect to potency assays used for labelling and testing of post infusion samples,” held at the European Medicines Agency (EMA) (London, UK) in November 2013 when almost every manufacturer of new FVIII and FIX products reported their approach to potency labeling by reference to the SSC recommendations.46 Manufacturers reported that valid assays, relative to the WHO IS Concentrates, were possible for all the new FVIII and FIX products under development and there was a clear preference to retain product labeling in IU. It was confirmed that the new FVIII products could be tested using both the one-stage clotting and chromogenic methods with some displaying discrepancies between methods (e.g., some pegylated products), whereas others did not (e.g., B-DD FVIII).5,37 These data confirmed that a single approach to potency labeling was not possible for all the products; however, some manufacturers expressed a preference for labeling new FVIII products by the chromogenic method when the potency by the onestage clotting method was highly dependent on the source of APTT reagent. At present, the chromogenic method is not considered a viable option for the potency labeling of the new FIX products, and all manufacturers have used the one-stage clotting method despite the issue of potency discrepancies when different APTT reagents are used.23 Valuable information on the assay of full-length recombinant and new “longerlasting” FIX products has recently become available through a multicenter collaborative study organized by the National Institute for Biological Standards and Control.44 In summary, this study confirmed that valid potency estimates could be obtained, relative to the WHO IS Concentrate, by both onestage clotting and chromogenic FIX methods. However, all products displayed some degree of methods-based potency discrepancies and there was particularly large interlaboratory variability for the new longer-lasting products. The overriding common finding with many of the new FVIII and FIX modified products is that several valid, but sometimes widely different, potencies can be obtained relative to the WHO IS Concentrates and this complicates the choice of method for potency labeling. The SSC/ISTH recommendations already indicate that results from in vivo efficacy studies should be included in the decision. However, another important consideration is maintaining the equivalence of the IU between existing licensed products and the new product under development. Ideally, these products would be administered using a similar IU/kg dosage, albeit with different infusion frequency, since large differences in the recommended IU/kg dosage between established products and the new products could lead to confusion and undermine the credibility of the IU. The choice of potency labeling method should, therefore, take into account potency comparisons with existing products in vitro, using the same methods and reagents, as well as information on comparative
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hemostatic efficacy from in vivo studies.4,47 In 2014, the first “longer-lasting” concentrate for hemophilia B therapy was licensed (recombinant FIX-Fc fusion, Alprolix, Biogen Idec Inc., Cambridge, MA); this product is labeled in IU using the one-stage clotting method relative to the WHO IS Concentrate. It is encouraging to see that the expected increase in plasma FIX levels following infusion (1 IU/kg giving a rise of 1%) is similar to that for other licensed products.48 Once the potency labeling method for the new product has been decided, this will be used to calibrate the manufacturer’s in-house standard. For some products, where the potency is sensitive to particular reagents, the definition of potency labeling in IU, relative to the WHO IS Concentrate, may need to specify not only the method used (e.g., clotting or chromogenic) but also the specific reagents (e.g., APTT reagent). This may mean that the link of the product to the WHO IS Concentrate could be affected by the reliability and consistency of reagents beyond the control of the product manufacturer. In these situations, the consistency of product potency labeling will rely more heavily on the manufacturer’s in-house product standard and greater emphasis on its stability and replacement strategy will be necessary. The recommendation that manufacturers should provide guidance to clinical laboratories on postinfusion testing remains valid. This information can be obtained through formal pharmacokinetic studies and also through in vitro studies designed to evaluate the use of many different methodologies. For example, field studies on a B-DD FVIII product and a FVIII-Fc fusion product have allowed the manufacturers to conclude that a product-specific standard is not required by clinical laboratories to assess postinfusion levels.15,49 Similarly, a study comparing different APTT reagents on the measurement of pegylated factor VIII spiked into hemophilic plasma was able to recommend the use of ellagic acid-based reagents for postinfusion monitoring.5 In some cases, where the postinfusion recovery cannot be reliably monitored using local clotting methods, it might be necessary for clinical laboratories to adopt specific laboratory test procedures; for instance, the use of recommended reagents or product-specific standards.17 One of the suggestions from the EMA workshop was to elaborate a decision tree for postinfusion testing as a guide through all of the possibilities (e.g., choice of method, reagents, local or product-specific standard, correction factors, etc.) because a single solution will not be applicable to all products.
cases, offer an array of possible valid potency values. The choice of potency method is, therefore, not straightforward and it is important that the discrepant potency issues are resolved between the manufacturer and licensing authority rather than passed on to the end user. One major consideration is maintenance of the credibility of the IU and its equivalence with existing products by both in vitro and in vivo comparisons, which will ideally result in similar dosage requirements in terms of IU/kg. This may require licensing authorities to be more flexible in the choice of potency labeling method in the interests of global harmonization. For new products with large potency discrepancies, relative to the WHO IS Concentrate, the consistency of product labeling will become extremely reliant on the stability of the manufacturer’s product standard. Past experience has shown that the postinfusion recovery testing of products with known methods discrepancies can be extremely problematic when clinical laboratories use local methods and reference standards.28,29 For the new products, it will be necessary for manufacturers to provide guidance and education in this area. It is unlikely that there will be a single approach applicable to all products and various strategies, such as the use of specific APTT reagents or product-specific standards, may be considered. In conclusion, potency labeling the new FVIII and FIX products in IU, relative to the WHO IS Concentrates, is an achievable goal; however, this will depend on a detailed appreciation of the product potency characteristics and a harmonized approach to the choice of labeling method at the global level.
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replacement therapy. J Thromb Haemost 2013;11(Suppl 1):84–98 2 Shapiro AD. Long-lasting recombinant factor VIII proteins for
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Conclusion It is now clear that the potency of most new modified FVIII and FIX therapeutics can be measured using the conventional one-stage clotting and chromogenic methods and that valid estimates can be obtained relative to the WHO IS Concentrates.24,46 This is an essential requirement to support the preference for all manufacturers to label the new products in IU. However, the chemical and genetic manipulations used to produce the new generation of modified FVIII and FIX therapeutics have resulted in varying degrees of methods-based and reagent-based potency discrepancies, which, in some
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hemophilia A. Hematology (Am Soc Hematol Educ Program) 2013;2013:37–43 Schaub RG. Recent advances in the development of coagulation factors and procoagulants for the treatment of hemophilia. Biochem Pharmacol 2011;82(2):91–98 Oldenburg J, Albert T. Novel products for haemostasis - current status. Haemophilia 2014;20(Suppl 4):23–28 Gu JM, Ramsey P, Evans V, et al. Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A. Haemophilia 2014;20(4):593–600 Barrowcliffe TW, Hubbard AR, Kitchen S. Standards and monitoring treatment. Haemophilia 2012;18(Suppl 4):61–65 Hubbard AR. International biological standards for coagulation factors and inhibitors. Semin Thromb Hemost 2007;33(3): 283–289 Assay of human coagulation factor VIII (2.7.4). In: European Pharmacopoeia. 8th ed. Strasbourg, France: Council of Europe; 2014;8.0:236 Langdell RD, Wagner RH, Brinkhous KM. Effect of antihemophilic factor on one-stage clotting tests; a presumptive test for hemophilia and a simple one-stage antihemophilic factor assy procedure. J Lab Clin Med 1953;41(4):637–647 Proctor RR, Rapaport SI. The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Am J Clin Pathol 1961;36:212–219 Seminars in Thrombosis & Hemostasis
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factor VIII: C potency. Med Lab Sci 1978;35(4):347–354 Rosén S. Assay of factor VIII: C with a chromogenic substrate. Scand J Haematol Suppl 1984;40:139–145 Barrowcliffe TW. Standardization of FVIII & FIX assays. Haemophilia 2003;9(4):397–402 Hubbard AR, Weller LJ, Bevan SA. Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies. Br J Haematol 2002;117(4):957–960 Viuff D, Barrowcliffe T, Saugstrup T, Ezban M, Lillicrap D. International comparative field study of N8 evaluating factor VIII assay performance. Haemophilia 2011;17(4):695–702 Mikaelsson M, Oswaldsson U, Sandberg H. Influence of phospholipids on the assessment of factor VIII activity. Haemophilia 1998; 4(4):646–650 Ingerslev J, Jankowski MA, Weston SB, Charles LA; ReFacto Field Study Participants. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII: C activity in hemophilic plasma using one-stage clotting assays. J Thromb Haemost 2004;2(4):623–628 Barrowcliffe TW, Mertens K, Preston FE, Ingerslev J. Laboratory aspects of haemophilia therapy. Haemophilia 2002;8(3):244–249 Finney DJ. Statistical method in biological assay. 3rd ed. London: Charles Griffin & Company Ltd.; 1978 Assay of human coagulation factor IX (2.7.11). In: European Pharmacopoeia 8th ed. Strasbourg, France: Council of Europe; 2014;8.0:248 Shapiro AD, Ragni MV, Valentino LA, et al. Recombinant factor IXFc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients. Blood 2012;119(3):666–672 Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S. Genetic fusion to albumin improves the pharmacokinetic properties of factor IX. Thromb Haemost 2009;102(4):634–644 Østergaard H, Bjelke JR, Hansen L, et al. Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide. Blood 2011;118(8): 2333–2341 Gray E, Pickering W, Hockley J, et al. Collaborative study for the establishment of replacement batches for human coagulation factor IX concentrate reference standards. Pharmeur Bio 2008; 2008(1):19–30 Bangham DR, Brozović M. Factor VIII international units and reference materials. Thromb Diath Haemorrh 1974;31(1):3–11 Hubbard AR, Weller LJ, Bevan SA. A survey of one-stage and chromogenic potencies in therapeutic factor VIII concentrates. Br J Haematol 2002;117(1):247–248 Raut S, Daniels S, Heath AB; SSC Sub-Committee on Factor. Value assignment of the WHO 8th International Standard for factor VIII, concentrate (07/350). J Thromb Haemost 2012;10(6):1175–1176 Lusher JM, Hillman-Wiseman C, Hurst D. In vivo recovery with products of very high purity—assay discrepancies. Haemophilia 1998;4(4):641–645 Mikaelsson M, Oswaldsson U. Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products. Semin Thromb Hemost 2002;28(3):257–264 Hubbard AR, Bevan SA, Weller LJ. Potency estimation of recombinant factor VIII: effect of assay method and standard. Br J Haematol 2001;113(2):533–536 Barrowcliffe TW. Clotting factor concentrates in clinical practice. Standardization and assay. Semin Thromb Hemost 1993;19(1): 73–79
assessment of recombinant FVIII concentrates. Haemophilia 1998; 4(4):634–640 Barrowcliffe TW. Factor VIII and factor IX Sub-Committee. Recommendations for the assay of high-purity factor VIII concentrates. Thromb Haemost 1993;70(5):876–877 Raut S, Sands D, Heath AB, Barrowcliffe TW. Variability in factor VIII concentrate measurement: results from SSC field collaborative studies. J Thromb Haemost 2003;1(9):1927–1934 Lee CA, Owens D, Bray G, et al. Pharmacokinetics of recombinant factor VIII (recombinate) using one-stage clotting and chromogenic factor VIII assay. Thromb Haemost 1999;82(6):1644–1647 Farrugia A. Potency assessment of the new generation of coagulation factor concentrates—time for a new paradigm? Thromb Haemost 2003;90(6):968–970 Christiansen ML, Balling KW, Persson E, et al. Functional characteristics of N8, a new recombinant FVIII. Haemophilia 2010;16(6): 878–887 White G, Shapiro A, Ragni M, et al. Clinical evaluation of recombinant factor IX. Semin Hematol 1998;35(2, Suppl 2):33–38 Ewenstein BM, Joist JH, Shapiro AD, et al; Mononine Comparison Study Group. Pharmacokinetic analysis of plasma-derived and recombinant F IX concentrates in previously treated patients with moderate or severe hemophilia B. Transfusion 2002;42(2): 190–197 Chevreux G, Faid V, Andre MH, Tellier Z, Bihoreau N. Differential investigations from plasma-derived and recombinant Factor IX revealed major differences in post-translational modifications of activation peptides. Vox Sang 2013;104(2):171–174 Barrowcliffe TW. Insights from factor IX activation studies with chromogenic assays: implications of disparate product results. Haemophilia 2010;16(Suppl 6):9–12 Yu Y, Millar CM. Measurement of factor IX activity in plasmaderived and recombinant concentrates: insights from thrombin generation and activation-based assays. J Thromb Haemost 2014; 12(1):62–70 Wilmot HV, Hogwood J, Gray E. Recombinant factor IX: discrepancies between one-stage clotting and chromogenic assays (epub ahead of print). Haemophilia 2014 Kitchen S, Gray E, Mertens K. Monitoring of modified factor VIII and IX products. Haemophilia 2014;20(Suppl 4):36–42 Hubbard AR, Dodt J, Lee T, et al; Factor VIII and Factor IX Subcommittee of The Scientific and Standardisation Committee of The International Society on Thrombosis and Haemostasis. Recommendations on the potency labelling of factor VIII and factor IX concentrates. J Thromb Haemost 2013;11(5):988–989 European Medicines Agency. Report of Workshop on Characterisation of New Clotting Factor Concentrates (FVIII, FIX) with Respect to Potency Assays Used for Labelling and Testing of Post Infusion Samples. 28-29 November 2013. Doc Ref. EMA/135928/ 2014. London, 26 June 2014. Available at: http://www.ema. europa.eu/docs/en_GB/document_library/Report/2014/07/ WC500169760.pdf Powell JS, Josephson NC, Quon D, et al. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients. Blood 2012;119(13):3031–3037 Product information for Alprolix (Coagulation factor IX, recombinant, Fc fusion protein). Cambridge, MA: Biogen Idec Inc. Sommer JM, Moore N, McGuffie-Valentine B, et al. Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories. Haemophilia 2014;20(2):294–300
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Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, United Kingdom Semin Thromb Hemost
Abstract
Keywords
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hemophilia factor VIII factor IX coagulation
Address for correspondence Anthony R. Hubbard, PhD, Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom (e-mail: [email protected]).
Various strategies to produce “longer-lasting” factor VIII and factor IX concentrates through chemical and genetic modifications are currently under evaluation. It is now clear that these new molecules are amenable to testing using conventional methods for biological activity (one-stage clotting and chromogenic) and there is a preference to maintain labeling in International Units (IU) traceable to the WHO International Standard Concentrates. This is an achievable goal; however, many of the new molecules are associated with potency discrepancies both between methods and also within methods, for instance, when different activated partial thromboplastin time reagents are used. In the interests of global harmonization, it is important for licensing authorities to reach agreement on the choice of the potency labeling method. This choice should be supported by a thorough characterization of product potency, both in vitro and in vivo, to anticipate future issues and with a view to maintaining equivalence of the IU compared with existing licensed products. In cases where the product potency is defined using specific reagents, the robustness of the manufacturer’s product standard will be crucial for product consistency. The sensitivity of measured potency to different methodologies will require manufacturers to provide guidance to clinical laboratories on suitable postinfusion testing approaches.
Replacement therapy for hemophilia is entering a new era, which will see the introduction of bioengineered factor VIII (FVIII) and factor IX (FIX) products with improved properties over the natural molecules. The objective of most new developments is to reduce the frequency of infusion through a prolonged plasma half-life as a result of various strategies, such as pegylation, protein fusion, or increased stability (single chain FVIII).1–3 Results from preclinical and clinical studies are encouraging, particularly for the modified FIX molecules, where half-life extensions up to fivefold have been reported.4 Despite the advanced technologies applied to the development of these new products, they are still classified as biological medicines, which means their activity is measured
Issue Theme Current Issues in Hemophilia: Recognizing Clinical Heterogeneity, Replacement Therapy and Outcome Assessment; Guest Editor: Alok Srivastava, MD, FRACP, FRCPA, FRCP.
relative to a reference standard. Discrepancies in potency measurements, when different methods or assay reagents are used, have been reported to various degrees for most of the new products. This poses a significant challenge to the current system of potency labeling, where all FVIII and FIX concentrates are labeled in International Units (IU) traceable to single World Health Organization International Standard (WHO IS) Concentrates.5–7 The diversity of the new products means that a “one solution fits all” approach to labeling will not be possible and each product should be considered on a case-by-case basis. However, in the interests of global harmonization for product consistency and effective treatment it is imperative to have agreement on the strategy for potency
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DOI http://dx.doi.org/ 10.1055/s-0034-1395353. ISSN 0094-6176.
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1 Haemostasis Section, Biotherapeutics Group, National Institute for
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labeling at the international level. Strategies for addressing discrepant potency issues in the new products may be derived from an appreciation of how the current potency labeling system has survived for over 40 years and our past experience of similar discrepancy issues.
Product Labeling in International Units Maintenance of the WHO IS/IU system has relied on two main components: (1) well-defined methods for potency labeling and (2) comparison of similar standard and test materials (“like vs. like”).
Well-Defined Methods for Factor VIII and Factor IX Potency Potency labeling of the final therapeutic product has evolved into an extremely precise and robust exercise to satisfy the requirement for product consistency, accurate labeling, and hence predictable efficacy in the patient. Two methods are currently used to label FVIII concentrates, the one-stage clotting method and the chromogenic method. The former method has been applied to the labeling of most concentrates licensed in the United States whereas the latter method is recommended for product labeling in Europe and was adopted by the European Pharmacopoeia (EP) in 1995.8 The one-stage clotting method has a long history, being initially developed in the 1950s and refined in the 1960s, whereas the chromogenic method for FVIII was developed and optimized in the 1970s and 1980s.9–12 Although both methods allow the quantification of FVIII clotting activity, they differ in some respects; for instance, the one-stage clotting method is sensitive to the activation status of FVIII in the test samples whereas the chromogenic method is not.13 This may be relevant to the potency estimation of modified FVIII molecules if there is a difference in the profile of FVIII activation during the assay, between the test and standard samples, and may help to explain the variability of potency estimates seen with the one-stage clotting method.5,14–16 In contrast, the chromogenic method for FVIII is designed to achieve full activation of FVIII in the test and standard materials early in the first stage of the procedure and may, therefore, be less affected by differences in the FVIII activation profile. The onestage method is also performed using an extremely diverse range of activated partial thromboplastin time (APTT) reagents and this could also contribute to increased potency variability.17,18 Despite these differences, the application of these methods to product potency estimation follows a similar protocol based on the relative comparison of dose– response relationships of test sample with the reference standard through the assay of multiple dilutions.19 Critical steps and reagents for the chromogenic method as well as instructions on data analysis are described in significant detail in the EP.8 Up to now, FIX concentrates have only been labeled using the one-stage clotting method and this procedure is subject to the same wide range of APTT reagents seen with one-stage FVIII clotting assays. A detailed methodology is described in the EP and this approach has been applied to the potency Seminars in Thrombosis & Hemostasis
estimation of new products under development.20–23 The clotting/chromogenic discrepancy issue has, therefore, been avoided for FIX concentrates up to now; however, this will certainly be a consideration for the future. There is already information that the recently introduced chromogenic methods for FIX may return different potency values for recombinant FIX (rFIX) compared with the one-stage clotting method.24
Comparison of “Like versus Like” Standard and Test Materials The principle of assaying “like versus like” is probably the most powerful tool for the potency testing of biological therapeutics where potency estimation relies on comparison relative to a standard. Over 40 years ago, it was observed that the interlaboratory variability for estimates of FVIII in concentrates was reduced when assayed relative to a concentrate standard rather than a plasma standard.25 This principle is well established in most areas of coagulation factor testing and there are both plasma and concentrate WHO ISs for diagnostic and product testing, respectively, for many factors.7 The testing of “like” samples (test vs. standard) has two advantages, reducing the variability of results between laboratories and minimizing potency discrepancies between methods, such as the one-stage clotting and chromogenic methods for FVIII.26 The ability to obtain valid potency estimates for all FVIII and FIX concentrates relative to single WHO Concentrate ISs is an indication that sufficient similarity has existed between the therapeutic concentrates and the WHO IS to support the single IU. This probably reflects the fact that most FVIII and FIX therapeutics comprised full-length molecules similar to the composition of the relevant WHO IS and that valid comparisons of plasma-derived and recombinant molecules were possible.24,27 We are now faced with extending this comparison to include the new modified therapeutics for IU labeling, and it is worthwhile considering previous challenges to the WHO IS/IU system, associated with large interlaboratory variability or discrepancies between the one-stage clotting and chromogenic methods.
Past Challenges to the World Health Organization International Standard/ International Units Labeling System Recombinant FVIII The introduction of the first generation of recombinant FVIII (rFVIII) concentrates in the 1990s was accompanied by reports of anomalous postinfusion recoveries and methodsbased potency discrepancies and it became clear that rFVIII products were behaving differently to conventional plasmaderived products.28,29 The importance of the choice of assay method and standard was reproduced in vitro where the potency of rFVIII by one-stage and chromogenic methods were not significantly different, relative to a concentrate standard, but differed by more than 40% relative to a plasma standard.30 Equally worrying were the results from the first collaborative studies where rFVIII was tested relative to the WHO IS Concentrate. Estimates for rFVIII were associated
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with very high interlaboratory variability for all methods (geometric coefficient of variation [GCV] 1-stage 39.4%, 2-stage 136.8%, and chromogenic 53.7%), indicating that the rFVIII and the conventional FVIII standards were not behaving as “like” materials.31 These results raised concerns that the WHO IS Concentrate may not be suitable for the potency estimation of rFVIII products and that a separate standard might be required.32 However, laboratory investigations identified methodological details which were able to reduce assay variability; for instance, inclusion of 1% (w/v) albumin in dilution buffers to improve the stability of dilutions during the assay and the predilution of test and standard concentrates in hemophilic plasma or FVIII deficient plasma with normal levels of von Willebrand factor (VWF). These steps markedly reduced the interlaboratory variability of potency estimation for recombinant and very high purity FVIII concentrates to a level where comparisons against the WHO IS Concentrate were acceptable and a separate standard for rFVIII was not required. These methodological modifications were recommended by the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC/ISTH) in 1993 and remain relevant to the testing of the modified FVIII products today.33 More recent studies, such as the value assignment of the WHO 8th IS FVIII Concentrate, have confirmed the validity of assaying rFVIII relative to a plasma-derived standard in terms of parallelism and linearity of dose–response relationships.27 However, these comparisons were associated with wider interlaboratory variability (GCV, 14.5–19.7%) than the comparison of plasma-derived versus plasma-derived products (GCVs < 5%). This might reflect a greater sensitivity of rFVIII to local assay conditions such as the need for predilution in FVIIIdeficient plasma with normal levels of VWF as suggested in “SSC field trials” of rFVIII potency.34
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stage incubation time (factor X activation stage) in the chromogenic method.14,16 The potential for potency variability in the chromogenic method was addressed by a revision of the EP monograph for the FVIII assay, which introduced a tighter specification for the first stage incubation time for factor X activation.8 To support postinfusion testing in clinical laboratories, primarily using the one-stage clotting method, a product-specific reference material (ReFacto Laboratory standard, Pfizer Inc., New York, NY) was made available which reinstated the “like versus like” comparison of test and standard samples. This initiative overcame the methods-based potency discrepancy as well as allowed clinical laboratories to continue using their local one-stage clotting methods.17,29 The ability of product-specific standards to overcome large one-stage clotting/chromogenic discrepancies has also been demonstrated for full-length rFVIII.35 Experience with B-DD FVIII emphasized the need for a rigorous evaluation of potency characteristics in vitro prior to licensing to anticipate problems and also the importance of agreement between licensing authorities in the approach to potency labeling when products display large methods-based discrepancies.36 Unfortunately, the latter requirement was not followed when the latest generation of (albumin-free) B-DD FVIII was licensed and labeled using different methodologies in the United States (Xyntha, Pfizer Inc., New York, NY) and Europe (ReFacto AF). We are now in a difficult situation where 1 IU of the product licensed in the United States is equivalent to 1.38 IU of the same material licensed in Europe. Apparently, the large methods discrepancy associated with ReFacto does not apply to a second licensed B-DD product (NovoEight, NovoNordisk A/S, Bagsvaerd, Denmark), and this indicates that subtle differences between molecules with similar modifications can have an effect on assay behavior.37
Recombinant Factor IX B-Domain-Deleted Factor VIII Past experience with the first licensed bioengineered molecule, B-domain-deleted (B-DD) FVIII (ReFacto, Pfizer), is probably the most relevant to the current challenges presented by the new modified products. Following licensing in 1999, there was confusion over postinfusion recovery values, even compared with the full-length rFVIII products, with reports of 50% of expected levels.29 Such anomalous results were the outcome of using different methods and standards for product labeling (chromogenic/concentrate standard) and postinfusion testing (one-stage clotting/plasma standard). The situation was further aggravated by the inherent 20 to 30% potency discrepancy between chromogenic and clotting methods when B-DD FVIII was measured relative to concentrate standards.14,26 Fortunately, with respect to product consistency in the international market, the first version of this product was labeled in IU using the same method (chromogenic) in both Europe and the United States. In addition to potency discrepancies between methods, it was also apparent that different potency estimates could be obtained within the same method when different reagents or procedures were followed. For instance, the relative potency was sensitive to the composition of the APTT reagent in the one-stage clotting method and to the first
The first hints of methods-based potency discrepancies with FIX products are associated with the first rFIX concentrate for hemophilia B therapy (Benefix, Pfizer Inc., New York, NY). Since licensing in 1998, there have been reports of a 30% or greater reduction for in vivo recovery compared with plasmaderived products and it has been suggested that this may be linked to differences in the posttranslational modification of the recombinant molecule leading to increased clearance.38–40 However, the possible contribution of in vitro testing effects, caused by different properties of rFIX and plasma-derived FIX (pdFIX), has also been proposed. For instance, it has been suggested that the potency estimation of rFIX relative to conventional pdFIX standards could be influenced by the different activation rates of rFIX and pdFIX or by the higher content of activated FIX in rFIX products.41,42 The different behavior of rFIX and pdFIX in vitro is supported by the finding that rFIX has a lower potency by the newly developed chromogenic methods compared with the onestage clotting method, when measured relative to a pdFIX reference, whereas no such discrepancy is found with pdFIX.24 Although this type of discrepancy can be reproduced in vitro by spiking with purified activated FIX (FIXa), the amounts required to produce a significant discrepancy are far Seminars in Thrombosis & Hemostasis
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greater than those measured in rFIX products.43 It, therefore, appears unlikely that FIXa content is the main reason for the methods discrepancy or the anomalous postinfusion recovery results. It has also been shown that use of a rFIX reference can overcome the clotting/chromogenic discrepancy and this has been confirmed in a multicenter study where two rFIX products were assayed relative to a rFIX standard, leading to good agreement between methods and reduced interlaboratory variability.44 This is another example where restoration of a “like versus like” comparison can overcome method discrepancies and can be used to support the development of a rFIX reference standard.
Potency Labeling for New Factor VIII and Factor IX Therapeutics Previous experience dealing with products with novel or discrepant potency issues has highlighted some key points pertinent to the potency labeling of the new modified therapeutics, namely, 1. The need to anticipate issues of potency discrepancy on the final product, prior to licensing, with agreement on a single method for potency labeling. To avoid confusion at the point of administration, it is important that potency issues are resolved upstream by the manufacturer and licensing authorities. 2. Recognition that product-specific standards can overcome methods-based potency discrepancies and allow different methods to be used for potency estimation. 3. Provision of guidance to clinical laboratories on the best approach to postinfusion testing. The requirement for a transparent and harmonized approach to the potency labeling of new products, which encompasses and facilitates the above issues, was addressed by an initiative through the SSC/ISTH, and the “Recommendations on the potency labeling of factor VIII and factor IX concentrates” were published in 2013.45 These recommendations address the key issues in the potency estimation of new products, covering potency labeling of the purified product, the manufacturer’s product standard, and postinfusion testing. The first section of the recommendations describes the necessary product characterization on which the decision for potency labeling should be based. To anticipate problems of methods-based potency discrepancies, the product should be evaluated using different methodologies (clotting and chromogenic) and different reagents (APTT) relative to the WHO IS Concentrate. Labeling the new products in IU is only possible if assays relative to the WHO IS Concentrate are valid in terms of parallelism of the dose–response relationships. Where there is little or no potency discrepancy when different methods and reagents are used, the choice of potency labeling method should be fairly straightforward; however, where significant discrepancies exist between methods, then it is important that there is agreement between manufacturers and all licensing authorities on a single method for labeling if we are to avoid a situation similar to that seen with Xyntha/ ReFacto. Where it is not possible to obtain valid tests, relative Seminars in Thrombosis & Hemostasis
to the WHO IS concentrate, the recommendations indicate that it may be necessary to label using product-specific units. Since publication in 2013, the interest in the recommendations, from both regulators and manufacturers, has been very encouraging. This was obvious during a workshop on the “Characterisation of new clotting factor concentrates (FVIII, FIX) with respect to potency assays used for labelling and testing of post infusion samples,” held at the European Medicines Agency (EMA) (London, UK) in November 2013 when almost every manufacturer of new FVIII and FIX products reported their approach to potency labeling by reference to the SSC recommendations.46 Manufacturers reported that valid assays, relative to the WHO IS Concentrates, were possible for all the new FVIII and FIX products under development and there was a clear preference to retain product labeling in IU. It was confirmed that the new FVIII products could be tested using both the one-stage clotting and chromogenic methods with some displaying discrepancies between methods (e.g., some pegylated products), whereas others did not (e.g., B-DD FVIII).5,37 These data confirmed that a single approach to potency labeling was not possible for all the products; however, some manufacturers expressed a preference for labeling new FVIII products by the chromogenic method when the potency by the onestage clotting method was highly dependent on the source of APTT reagent. At present, the chromogenic method is not considered a viable option for the potency labeling of the new FIX products, and all manufacturers have used the one-stage clotting method despite the issue of potency discrepancies when different APTT reagents are used.23 Valuable information on the assay of full-length recombinant and new “longerlasting” FIX products has recently become available through a multicenter collaborative study organized by the National Institute for Biological Standards and Control.44 In summary, this study confirmed that valid potency estimates could be obtained, relative to the WHO IS Concentrate, by both onestage clotting and chromogenic FIX methods. However, all products displayed some degree of methods-based potency discrepancies and there was particularly large interlaboratory variability for the new longer-lasting products. The overriding common finding with many of the new FVIII and FIX modified products is that several valid, but sometimes widely different, potencies can be obtained relative to the WHO IS Concentrates and this complicates the choice of method for potency labeling. The SSC/ISTH recommendations already indicate that results from in vivo efficacy studies should be included in the decision. However, another important consideration is maintaining the equivalence of the IU between existing licensed products and the new product under development. Ideally, these products would be administered using a similar IU/kg dosage, albeit with different infusion frequency, since large differences in the recommended IU/kg dosage between established products and the new products could lead to confusion and undermine the credibility of the IU. The choice of potency labeling method should, therefore, take into account potency comparisons with existing products in vitro, using the same methods and reagents, as well as information on comparative
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hemostatic efficacy from in vivo studies.4,47 In 2014, the first “longer-lasting” concentrate for hemophilia B therapy was licensed (recombinant FIX-Fc fusion, Alprolix, Biogen Idec Inc., Cambridge, MA); this product is labeled in IU using the one-stage clotting method relative to the WHO IS Concentrate. It is encouraging to see that the expected increase in plasma FIX levels following infusion (1 IU/kg giving a rise of 1%) is similar to that for other licensed products.48 Once the potency labeling method for the new product has been decided, this will be used to calibrate the manufacturer’s in-house standard. For some products, where the potency is sensitive to particular reagents, the definition of potency labeling in IU, relative to the WHO IS Concentrate, may need to specify not only the method used (e.g., clotting or chromogenic) but also the specific reagents (e.g., APTT reagent). This may mean that the link of the product to the WHO IS Concentrate could be affected by the reliability and consistency of reagents beyond the control of the product manufacturer. In these situations, the consistency of product potency labeling will rely more heavily on the manufacturer’s in-house product standard and greater emphasis on its stability and replacement strategy will be necessary. The recommendation that manufacturers should provide guidance to clinical laboratories on postinfusion testing remains valid. This information can be obtained through formal pharmacokinetic studies and also through in vitro studies designed to evaluate the use of many different methodologies. For example, field studies on a B-DD FVIII product and a FVIII-Fc fusion product have allowed the manufacturers to conclude that a product-specific standard is not required by clinical laboratories to assess postinfusion levels.15,49 Similarly, a study comparing different APTT reagents on the measurement of pegylated factor VIII spiked into hemophilic plasma was able to recommend the use of ellagic acid-based reagents for postinfusion monitoring.5 In some cases, where the postinfusion recovery cannot be reliably monitored using local clotting methods, it might be necessary for clinical laboratories to adopt specific laboratory test procedures; for instance, the use of recommended reagents or product-specific standards.17 One of the suggestions from the EMA workshop was to elaborate a decision tree for postinfusion testing as a guide through all of the possibilities (e.g., choice of method, reagents, local or product-specific standard, correction factors, etc.) because a single solution will not be applicable to all products.
cases, offer an array of possible valid potency values. The choice of potency method is, therefore, not straightforward and it is important that the discrepant potency issues are resolved between the manufacturer and licensing authority rather than passed on to the end user. One major consideration is maintenance of the credibility of the IU and its equivalence with existing products by both in vitro and in vivo comparisons, which will ideally result in similar dosage requirements in terms of IU/kg. This may require licensing authorities to be more flexible in the choice of potency labeling method in the interests of global harmonization. For new products with large potency discrepancies, relative to the WHO IS Concentrate, the consistency of product labeling will become extremely reliant on the stability of the manufacturer’s product standard. Past experience has shown that the postinfusion recovery testing of products with known methods discrepancies can be extremely problematic when clinical laboratories use local methods and reference standards.28,29 For the new products, it will be necessary for manufacturers to provide guidance and education in this area. It is unlikely that there will be a single approach applicable to all products and various strategies, such as the use of specific APTT reagents or product-specific standards, may be considered. In conclusion, potency labeling the new FVIII and FIX products in IU, relative to the WHO IS Concentrates, is an achievable goal; however, this will depend on a detailed appreciation of the product potency characteristics and a harmonized approach to the choice of labeling method at the global level.
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replacement therapy. J Thromb Haemost 2013;11(Suppl 1):84–98 2 Shapiro AD. Long-lasting recombinant factor VIII proteins for
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Conclusion It is now clear that the potency of most new modified FVIII and FIX therapeutics can be measured using the conventional one-stage clotting and chromogenic methods and that valid estimates can be obtained relative to the WHO IS Concentrates.24,46 This is an essential requirement to support the preference for all manufacturers to label the new products in IU. However, the chemical and genetic manipulations used to produce the new generation of modified FVIII and FIX therapeutics have resulted in varying degrees of methods-based and reagent-based potency discrepancies, which, in some
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factor VIII: C potency. Med Lab Sci 1978;35(4):347–354 Rosén S. Assay of factor VIII: C with a chromogenic substrate. Scand J Haematol Suppl 1984;40:139–145 Barrowcliffe TW. Standardization of FVIII & FIX assays. Haemophilia 2003;9(4):397–402 Hubbard AR, Weller LJ, Bevan SA. Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies. Br J Haematol 2002;117(4):957–960 Viuff D, Barrowcliffe T, Saugstrup T, Ezban M, Lillicrap D. International comparative field study of N8 evaluating factor VIII assay performance. Haemophilia 2011;17(4):695–702 Mikaelsson M, Oswaldsson U, Sandberg H. Influence of phospholipids on the assessment of factor VIII activity. Haemophilia 1998; 4(4):646–650 Ingerslev J, Jankowski MA, Weston SB, Charles LA; ReFacto Field Study Participants. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr Factor VIII: C activity in hemophilic plasma using one-stage clotting assays. J Thromb Haemost 2004;2(4):623–628 Barrowcliffe TW, Mertens K, Preston FE, Ingerslev J. Laboratory aspects of haemophilia therapy. Haemophilia 2002;8(3):244–249 Finney DJ. Statistical method in biological assay. 3rd ed. London: Charles Griffin & Company Ltd.; 1978 Assay of human coagulation factor IX (2.7.11). In: European Pharmacopoeia 8th ed. Strasbourg, France: Council of Europe; 2014;8.0:248 Shapiro AD, Ragni MV, Valentino LA, et al. Recombinant factor IXFc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients. Blood 2012;119(3):666–672 Metzner HJ, Weimer T, Kronthaler U, Lang W, Schulte S. Genetic fusion to albumin improves the pharmacokinetic properties of factor IX. Thromb Haemost 2009;102(4):634–644 Østergaard H, Bjelke JR, Hansen L, et al. Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide. Blood 2011;118(8): 2333–2341 Gray E, Pickering W, Hockley J, et al. Collaborative study for the establishment of replacement batches for human coagulation factor IX concentrate reference standards. Pharmeur Bio 2008; 2008(1):19–30 Bangham DR, Brozović M. Factor VIII international units and reference materials. Thromb Diath Haemorrh 1974;31(1):3–11 Hubbard AR, Weller LJ, Bevan SA. A survey of one-stage and chromogenic potencies in therapeutic factor VIII concentrates. Br J Haematol 2002;117(1):247–248 Raut S, Daniels S, Heath AB; SSC Sub-Committee on Factor. Value assignment of the WHO 8th International Standard for factor VIII, concentrate (07/350). J Thromb Haemost 2012;10(6):1175–1176 Lusher JM, Hillman-Wiseman C, Hurst D. In vivo recovery with products of very high purity—assay discrepancies. Haemophilia 1998;4(4):641–645 Mikaelsson M, Oswaldsson U. Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products. Semin Thromb Hemost 2002;28(3):257–264 Hubbard AR, Bevan SA, Weller LJ. Potency estimation of recombinant factor VIII: effect of assay method and standard. Br J Haematol 2001;113(2):533–536 Barrowcliffe TW. Clotting factor concentrates in clinical practice. Standardization and assay. Semin Thromb Hemost 1993;19(1): 73–79
assessment of recombinant FVIII concentrates. Haemophilia 1998; 4(4):634–640 Barrowcliffe TW. Factor VIII and factor IX Sub-Committee. Recommendations for the assay of high-purity factor VIII concentrates. Thromb Haemost 1993;70(5):876–877 Raut S, Sands D, Heath AB, Barrowcliffe TW. Variability in factor VIII concentrate measurement: results from SSC field collaborative studies. J Thromb Haemost 2003;1(9):1927–1934 Lee CA, Owens D, Bray G, et al. Pharmacokinetics of recombinant factor VIII (recombinate) using one-stage clotting and chromogenic factor VIII assay. Thromb Haemost 1999;82(6):1644–1647 Farrugia A. Potency assessment of the new generation of coagulation factor concentrates—time for a new paradigm? Thromb Haemost 2003;90(6):968–970 Christiansen ML, Balling KW, Persson E, et al. Functional characteristics of N8, a new recombinant FVIII. Haemophilia 2010;16(6): 878–887 White G, Shapiro A, Ragni M, et al. Clinical evaluation of recombinant factor IX. Semin Hematol 1998;35(2, Suppl 2):33–38 Ewenstein BM, Joist JH, Shapiro AD, et al; Mononine Comparison Study Group. Pharmacokinetic analysis of plasma-derived and recombinant F IX concentrates in previously treated patients with moderate or severe hemophilia B. Transfusion 2002;42(2): 190–197 Chevreux G, Faid V, Andre MH, Tellier Z, Bihoreau N. Differential investigations from plasma-derived and recombinant Factor IX revealed major differences in post-translational modifications of activation peptides. Vox Sang 2013;104(2):171–174 Barrowcliffe TW. Insights from factor IX activation studies with chromogenic assays: implications of disparate product results. Haemophilia 2010;16(Suppl 6):9–12 Yu Y, Millar CM. Measurement of factor IX activity in plasmaderived and recombinant concentrates: insights from thrombin generation and activation-based assays. J Thromb Haemost 2014; 12(1):62–70 Wilmot HV, Hogwood J, Gray E. Recombinant factor IX: discrepancies between one-stage clotting and chromogenic assays (epub ahead of print). Haemophilia 2014 Kitchen S, Gray E, Mertens K. Monitoring of modified factor VIII and IX products. Haemophilia 2014;20(Suppl 4):36–42 Hubbard AR, Dodt J, Lee T, et al; Factor VIII and Factor IX Subcommittee of The Scientific and Standardisation Committee of The International Society on Thrombosis and Haemostasis. Recommendations on the potency labelling of factor VIII and factor IX concentrates. J Thromb Haemost 2013;11(5):988–989 European Medicines Agency. Report of Workshop on Characterisation of New Clotting Factor Concentrates (FVIII, FIX) with Respect to Potency Assays Used for Labelling and Testing of Post Infusion Samples. 28-29 November 2013. Doc Ref. EMA/135928/ 2014. London, 26 June 2014. Available at: http://www.ema. europa.eu/docs/en_GB/document_library/Report/2014/07/ WC500169760.pdf Powell JS, Josephson NC, Quon D, et al. Safety and prolonged activity of recombinant factor VIII Fc fusion protein in hemophilia A patients. Blood 2012;119(13):3031–3037 Product information for Alprolix (Coagulation factor IX, recombinant, Fc fusion protein). Cambridge, MA: Biogen Idec Inc. Sommer JM, Moore N, McGuffie-Valentine B, et al. Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories. Haemophilia 2014;20(2):294–300
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