Special Issue 2019

New developments in treatment

Clinical and Laboratory Aspects

New developments in haemophilia treatment

Special Issue 9 Editorial 2019 The ninth Special issue has the theme “New developments in haemophilia treatment; clinical and laboratory aspects”. These new developments and their impact on laboratory management raise many ECAT Foundation P.O. Box 107 questions regarding laboratory diagnostics and that is why this topic deserves our attention. 2250 AC Voorschoten The first section focuses on the clinical aspects. The first contribution gives an overviewofnew The Netherlands Website: developments in the treatment of haemophilia (J. Tarrant, D. Lillicrap). The updated management approaches www.ECAT.nl () and therapies which have been recently licensed or are currently in development are E-mail: [email protected] outlined. Next there follows an article focusing on the role of gene therapy in the future treatment of Phone: haemophilia patients (W. Miesbach). The third contribution describes personalising therapy in haemophilia; +31.(0)71.3030910 Fax: pharmacokinetically guided dosing of factor VIII and factor IX concentrates (T. Goedhart, M. Cnossen). +31.(0)71.3030919 The second part of this special issue focuses on laboratory aspects and starts with a contribution about new developments in the treatment of haemophilia and the implications for laboratory testing. Are changes Editor in Chief: necessary in the near future when the new drugs, not based on the infusion of factors, will be P. ter Hark - van Velp widely used? (A. Tripodi). An article about potency labelling of extended half-life FVIII and FIX products gives Editorial Board: information about the importance of ensuring the consistency of production and the efficacy of a product P. ter Hark - van Velp P. Meijer (E. Gray), followed by a contribution about laboratory measurement of extended half-life FVIII (S. Tiefen- Advisory Committee: bacher) and extended half-life FIX products (A. Bowyer, S. Kitchen). In the last contribution the quality E. van Cott assurance of replacement therapy and future perspectives are discussed (P. Meijer, M. de Maat). K. Devreese D. Peetz The editorial board and advisory committee want to thank all the authors for their useful contributions. A. Stroobants Special thanks are due to Prof. Dr. M. de Maat for her significant contribution as guest editor of this issue. Guest editor: M. de Maat We wish you interesting reading. Petra ter Hark—van Velp

Content

New Developments in The Treatment of Haemophilia Pages 2—7

Role of gene therapy in the future treatment of haemophilia patients Pages 8—10

Personalizing therapy in hemophilia Pharmacokinetic-guided dosing of factor VIII and factor IX concentrates Pages 11—14

New developments in the Treatment of Haemophilia: Implications for Laboratory Testing Pages 15—18

Potency labelling of extended half-life FVIII and FIX products Pages 19—20

Laboratory measurement of extended half-life FVIII products Pages 21—27

Laboratory measurement of extended half-life FIX products Pages 28—31

Quality Assurance of replacement therapy and future perspectives Pages 32—35

Page 1 New developments in haemophilia treatment

New Developments in The Treatment of Haemophilia

Julie L. Tarrant, MBChB, MRCP(UK) FRCPath(Haem)1, David P bleeding risk – for example planned sports [17]. It is desira- Lillicrap PhD, MD, FRCPC1 ble to assess an individual’s pharmacokinetic (PK) profile for FVIII in order to provide economical personalised prophylax- 1Department of Pathology & Molecular Medicine, Queen’s is, although frequent sampling over multiple days, limits the University, Kingston, Ontario, Canada feasibility of this approach. Traditionally, treatment success is simply assessed by the presence of breakthrough bleeds in Haemophilia A and B are X-linked recessive bleeding dis- conjunction with the measurement of trough factor activity orders characterised by missing or defective coagulation immediately before a planned (often delayed) infusion at a Factor VIII (FVIII) or Factor IX (FIX) respectively. The resulting routine clinic visit. Population data-based clinical tools to phenotype is dependent on the circulating factor level with determine an individual’s PK profile only require a few well- severe disease defined as less than 0.01 IU/mL (or less than timed plasma samples and no washout period. These tools 1% of normal) [1]. Untreated severe disease is associated have now been extensively used through programs such as with spontaneous joint and soft tissue bleeds and prolonged the WAPPS-Hemo and the myPKFit services [18,19]. Addi- bleeding following trauma thus resulting in debilitating ar- tionally, a population PK model has been created specifically thropathy and life-threatening haemorrhage [2]. for use perioperatively, which takes into account carious Factor replacement therapy has been the gold standard FVIII concentrates [20]. of treatment since the 1970s [3,4]. Plasma-derived products were used initially, which carried the risk of transmitting viral Extended Half-Life Products infections, before safer manufacturing processes and recom- Extended half-life (EHL) products are desirable to either binant factor concentrates were made available following reduce frequency of administration or to elevate trough lev- cloning of the F8 and F9 genes [5,6,7]. However, the incon- els and reduce the occurrence of bleeds [21]. EHL products venience of regular intravenous administration of these might also make subcutaneous dosing possible where intra- products along with the ongoing issue of inhibitory drug anti- venous access is a challenge [22,23]. Achieving EHL FVIII body (inhibitor) development (which render treatment in- products (~1.4-fold increase in half-life) has been more chal- effective in approximately 30% of severe haemophilia A pa- lenging that FIX (2.4-4.8 fold increase) as FVIII circulates with tients) has driven the need for alternative treatment options its chaperone protein von Willebrand Factor (VWF), domi- [8]. Additionally, the role of plasma-derived products has nating the FVIII clearance process [24]. been reconsidered since they have recently been document- One method to achieve EHL is by pegylation i.e. covalent ed to be associated with a lower risk of developing inhibitors attachment of polyethylene glycol (PEG) to the protein. This [9]. non-immunogenic polymer is considered to be non-toxic Here we outline the updated management approaches [25]. It increases the molecule’s size and molecular weight, (pharmacokinetics) and therapies which have been recently increasing retention time in the circulation and possibly de- licensed or are currently in development. The latter can be creasing immunogenicity. Adynovate® (Shire/Takeda) has a divided into: Extended half-life products, non-factor coagu- half-life of 14 hours due to random pegylation with 20kDa lation products, rebalancing agents, gene therapy and oral- PEG mostly bound to the B domain [26]. (Table 1) Esperoct® ly administered therapeutics. Within these groups, thera- (Novo Nordisk) is a recombinant glyco-pegylated FVIII which pies are now available with extended half-lives or reduced increases the half-life by a factor of 1.6 in adults and 1.9 in immunogenicity. Alternative bypassing agents can be used to children [27]. Although initially investigated as a potential for treat patients with inhibitors [10,11]. Additionally, long-term daily subcutaneous dosing to maintain a trough level of be- cure for haemophilia with a single peripheral intravenous tween 5 and 10%, Esperoct® has been licensed by the FDA administration of gene therapy is currently recruiting for this year for intravenous use every 4 days (twice per week in phase III trials [12,13]. children) and can also be used for perioperative manage- ment [28]. Jivi® (Bayer) is a recombinant pegylated FVIII de- Pharmacokinetics rived from a baby hamster kidney (BHK) cell line which has a The half-lives of wild-type FVIII and FIX molecules are 8- half-life of 17-18 hours. It was approved in 2018 for patients 12 and 18-34 hours, respectively [14,15]. To achieve ade- over 12 years of age and the PROTECT VIII KIDS trial is due to quate prophylaxis to maintain trough levels and prevent be completed in November 2019 [29]. Rebinyn/Refixia® spontaneous bleeds, infusion 3-4 times per week is neces- (Novo Nordisk) is a glycol-pegylated FIX molecule with a half- sary for severe haemophilia A and twice a week for severe life of 111 hours [28]. haemophilia B. Patients receive prophylaxis when their level Another method to achieve EHL is the genetic fusion of is below 0.01 IU/mL [16]. Infusions can also be timed to pro- human recombinant albumin to the C-terminus of FIX via a vide additional protection during episodes of increased cleavable linker in Idelvion® (CSL Behring) which results in a

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New developments in haemophilia treatment

Table 1. Therapeutics recently licensed or in development

Haemophilia A Prophylactic Therapies Mechanism Trade Generic name Research Company Cell Mean T1/2 Route Stage of name name line or dose develop- ment Random pegylation Adynovi/ Rurioctocog alfa pegol BAX 855 Shire/Takeda CHO 14.7 hrs IV Licenced Adynovate® Site-specific pegylation Jivi® BAY 94-9027 Bayer BHK 18.7 hrs IV Licenced Site-specific pegylation Esperoct® pegol N8-GP Novo Nordisk CHO 19 hrs IV (SC) Licenced Glycosylation Nuwiq® Simoctocog alfa OCTA101 Octapharma HEK 17.1 hrs IV (SC) Licenced Glycosylation & Eloctate/ BIIB-031 Bioverativ/ HEK 19.7 hrs IV Licenced Fc fusion Elocta® Sanofi Sialylation NA NA SHP826 Shire/Takeda CHO TBC IV Animal (BAX826) models HSP 70/ enhanced Kovaltry® Octocog alfa BAY 81-8973 Bayer BHK 14.3 hrs IV Licenced protein folding Single Chain Afstyla® Ionoctocog alfa CSL627 CSL Behring CHO 14.2 hrs IV Licenced rFVIII-Fc-VWF-XTEN NA NA BIVV001 Bioverativ/ HEK 37 hrs IV Clinical Sanofi/Amunix Trial Nanobody NA NA KB013bv Inserm, France NS x2 IV Animal models Haemophilia B Prophylactic Therapies Fc fusion Alprolix® Eftrenonacog alfa BIIB-029 Bioverativ/ HEK 86.5 hrs IV Licenced Sanofi Albumin fusion Idelvion® Albutrepenonacog alfa CSL654 CSL Behring CHO 104 hrs IV Licenced Glyco-pegylated Rebinyn/ Nonacog beta pegol N9-GP Novo Nordisk CHO 111 hrs IV Licenced Refixia® Rational protein design DalcA® Dalcinonacog alfa CB 2679d/ Catalyst NS Daily dose SC Clinical ISU304 Trial rFIXFc-XTEN NA NA BIVV002 Bioverativ/ NS Weekly SC Clinical Amunix dose Trial Sanofi Haemophilia A or B Prophylactic Therapy FVIIa Substitutions x4 MarzAA® Marzeptacog alfa CB 813 Catalyst NS 3.5 hrs SC Clinical activated Daily dose Trial Non-Factor Coagulation Products FVIIIa bispecific mAB HemLibra® Emicizumab ACE 910 Roche NA Weekly SC Licenced Si-EMI load, 2-4 weekly dose FVIIIa bispecific mAB NA NA BS-027125 Bioverativ NA NS NS Early develop- ment FVIIIa bispecific mAB NA NA NIBX-2101 Shire NA NS NS Early develop- ment

2019 Page 3 New developments in haemophilia treatment

Rebalancing Agents AT3 siRNA Fitusiran® NA ALN-AT3 Alnylam NA Monthly SC Phase II SAR439774 dose trials TFPI mAB NA NA BAY- Bayer NA Weekly SC Phase II 1093884 dose trials TFPI mAB NA Concizumab mAb 2021 Novo Nordisk NA Daily load, SC Phase II 4 day dose trials TFPI mAB NA Marstacimab PF- Pfizer NA Weekly SC Phase III 06741086 dose trials TFPI mAB NA NA MG-1113A GC Pharma NA Weekly SC Pre clini- dose cal study APC inhibition NA NA KRK α­1AT/ University of NA 5-7 days SC Animal SerpinPC Cambridge, UK dose models TFPI mAB NA NA HAPC1573 Bayer NA NS SC Animal models Protein S siRNA NA NA s72206 Life NA NS SC Animal Technologies models

mAB = monoclonal antibody; Fc = fragment crystalisable region of immunoglobulin; HSP70 = Heat Shock Protein 70; rFVIII = recombinant Factor VIII; XTEN = unstructured polypeptide linker; FVIIa = Activated FVII; AT3 = Antithrombin III; siRNA = small interfering Ribonucleic Acid; TFPI = Tissue Factor Pathway Inhibitor; APC = activated protein C; hrs = hours; IV = intravenous; SC = subcutaneous; CHO = Chinese Hamster Ovary; BHK = baby hamster kidney; HEK = human embryonic kidney; NA = not applicable; NS = not stated; Licensed = Licensed in adults in US.

half-life of 104 hours. Idelvion® has been shown to provide Sanofi/Amunix). FVIII is linked to an Fc portion of immuno- effective haemostasis for surgery, with less total factor con- globulin (similar to Eloctate®), the FVIII binding D’D3 region sumption and prolonged dosing intervals [30]. of VWF as well as 2 flexible hydrophilic XTEN linkers. These Extension of half-life as well as reduction in immunogen- linkers are unstructured polypeptide sequences that pro- icity can be achieved by changes to the glycosylation vide a hydrophilic cloud around the protein and further re- patterns that are added to the FVIII protein during post- duce the clearance rate. This leads to an impressive and as translational modification (PTM) [31,32]. Reduced inhibitor yet unprecedented four-fold extension in FVIII half-life (37 rates and EHL have been reported with Nuwiq® hours), breaking through the VWF clearance ceiling and (Octapharma) and Eloctate® (Bioverativ/Sanofi). These are reducing the need for infusions to once weekly [41]. produced in a human embryonic kidney cell line (HEK-293) BIVV002 for haemophilia B is currently in clinical trials but and thus have a human epithelial cell-derived glycosylation expected to be effective when dosed weekly via the subcu- pattern [33,34]. Eloctate® is also fused with IgG1 Fc taneous route [42]. (fragment crystallisable region/constant domain of immu- Inspired by naturally occurring fully functional antibod- noglobulin G1) which, when infused, interacts with Fc re- ies from llamas and camels, nanobodies have heavy chains ceptors on immune cells, protecting the protein from degra- with a single variable domain but no light chains. In FVIII- dation in lysosomes [35]. Rapid declines in inhibitor titre KB013bv, the FVIII B domain has been replaced with a non- and reduced times to tolerisation have been observed when inhibitory nanobody that recognises the VWF D’D3 domain. Eloctate® is used to treat high risk inhibitor patients [36]. This results in a 25-fold increase in affinity for VWF. The Similarly, Alprolix® (Bioverativ/Sanofi) is a FIXFc fusion with nanobody is liberated when cleaves FVIII, thus a half-life of 86 hours licenced for all ages [37]. Kovaltry® preserving co-factor activity. This novel FVIII molecule (Bayer) is produced in a genetically engineered BHK cell demonstrates a 2-fold increase in half-life in murine studies line that co-expresses the gene for human heat shock pro- and reduced immunogenicity [43,44]. tein 70, which improves proper folding of FVIII. It has a Rational protein design of FIX was used to increase re- high degree of sialic acid capping of N-terminal glycans sistance to antithrombin (AT) inhibition & catalytic activity resulting in increased half-life [38,39]. Other advances and to improve affinity for FVIIIa in the therapeutic DalcA® with PTMs include modification with polysialic acid. BAX (Catalyst). DalcA® has a 22-fold enhanced potency over wild 826 (Shire/Takeda), is a polysialylated full length FVIII that is -type FIX thus allowing subcutaneous daily administration currently in clinical trials [40]. [45]. FVIII EHL has been further improved by incorporating A number of these novel FVIII products have been docu- four different modifications into BIVV001 (Bioverativ/ mented to show significant discrepancies between the

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New developments in haemophilia treatment

different functional FVIII assays. In most instances full- generation results that overlap with the normal. This strate- length FVIII activity generally measures ~20% lower with the gy would be useful in haemophilia A and B, with or without one-stage assay compared to the chromogenic substrate inhibitors, and potentially for other inherited bleeding dis- assay. With B-domain deleted products this discrepancy is orders. In the Fitusiran® clinical trial, a fatal thrombosis oc- up to 50%. Afstyla® (CSL Behring) is a novel B-domain trun- curred when clotting factor concentrate was co- cated FVIII with the heavy and light chains covalently fused administered for a presumed breakthrough bleed. Howev- to achieve a single polypeptide protein [46]. The product er, avoidance of further thrombotic episodes has been monograph for Afstyla® states that the one-stage assay re- achieved with guidance issued on the cautious manage- sults can be aligned to chromogenic substrate results by ment of concomitant replacement therapy [54]. multiplying the one-stage result by 2 [47]. Another approach to magnifying the thrombin burst is MarzAA® is a novel variant of activated human FVII with via blocking of tissue factor pathway inhibitor (TFPI). Mono- four substitutions. Although not an extended clonal antibodies are in development, including Conci- half-life product, it was developed to provide increased pro- zumab® (mAb 2021, Novo Nordisk) which is the furthest coagulant activity and a longer duration of action in people along in development, currently in phase II trials, BAY- with haemophilia A and B. It has been shown to shorten 1093884 (Bayer) and PF-06741086 (Pfizer) [55,56,57]. These the activated partial thromboplastin time and prothrombin drugs have been shown to significantly reduce plasma con- time, and increase peak thrombin generation [48]. centrations of TFPI after monthly subcutaneous administra- tion and improve thrombin generation for ≥14 days. Non-Factor Coagulation Products Activated Protein C (APC) is an that func- HemLibra® (Emicizumab, ACE 910, Roche) is a human- tions to down-regulate thrombin generation. In vivo APC is ised bispecific monoclonal antibody that partially mimics inhibited by protein C inhibitor (PCI) and α1-antitrypsin (α1- the co-factor function of FVIIIa. As it has an entirely differ- AT). Properties of both PCI and α1-AT have been combined

ent structure from that of wild-type FVIII, it is not expected into a mutated PCI therapeutic, KRK α1AT or SerpinPC66. In

to induce or be affected by pre-existing FVIII inhibitors. Sub- mice, KRK α1AT has a 5-7 day half-life, low immunogenicity cutaneous administration combined with a long half-life (of and can be delivered subcutaneously [58]. A monoclonal 30 days) makes it an attractive option for patients. It signifi- antibody approach (HAPC1573) is also being used to target cantly reduces bleeds both in paediatric and adult patients, APC which has been efficacious and safe in monkeys [59]. with or without inhibitors [49]. There are also other Protein S is also being investigated in animal models alt- bispecific antibodies in development with increased speci- hough there are concerns about potential immune side ficity to FIXa and FX [50]. However, there is concern regard- effects [60]. ing assigning bioequivalence as no assay investigated thus far has achieved appropriate sensitivity and parallelism with Gene Therapy existing FVIII assays or consistency with clinical action [51]. The treatment aim with gene therapy is to achieve a This is likely to be attributed to the differences in in vivo long-term cure of haemophilia A or B with a single injection, regulatory mechanisms between Emicizumab® and FVIII i.e. restoration of normal (>0.50 IU/mL) factor levels. This [52]. Nonetheless, laboratory assessment is important for contrasts with the historic prophylactic treatment goal of a this drug as neutralising drug antibodies have been report- maintained trough levels > 0.01 IU/mL. In addition, gene ed in a small number of patients [11]. Rare thrombotic therapy could also eradicate previous therapy-induced in- effects have also been observed in the Haven clinical trials. hibitors by inducing regulatory T cells towards FVIII or FIX These events have almost exclusively occurred in the con- [61]. Adeno Associated Virus (AAV) vectors are non- text of repeated high dose-activated prothrombin complex pathogenic and incite relatively minor host immune re- concentrate during the treatment of break-through bleed- sponses. However, approximately 50% of potential gene ing [49]. therapy recipients have pre-existing immunity that limits the use of this vector type (higher vector doses may be effi- Rebalancing Agents cacious in some cases and other types of viral vector are Rebalancing the haemostatic system can be achieved by also being investigated e.g. Lentivirus) [12]. Elevation of depletion of anticoagulant proteins in the presence of hae- liver transaminases has been documented in ~30% of recipi- mophilia. Fitusiran® (ALN-AT3, Alnylam) is an RNA interfer- ents, although this has been effectively managed with tran- ence therapeutic that targets AT mRNA in the liver, thus sient steroid treatment/immunosuppression [62]. Ultimate- significantly reducing AT protein synthesis and ultimately ly, AAV vectors have shown effective delivery of a FVIII or leading to sufficient thrombin generation to prevent bleed- FIX transgenes to human hepatocytes and have maintained ing [53]. Fitusiran® is in stage II trials at this time and its long-term (>6 years) expression with associated phenotype subcutaneous once-monthly dosing schedule has been conversion. In haemophilia B, a gain of function gene vari- shown to produce stable reductions of plasma AT levels, ant (FIX-Padua R338L) is being used for gene therapy in with AT reductions to levels of ~25% producing thrombin phase III trials (AAV5-hFIXco-Padua, AMT-061, UniQuire and

2019 Page 5 New developments in haemophilia treatment

SPK-9001, AAV8-coF9-Padua, Spark) [63,64]. Additional Additionally, pH-responsive anionic complexation hydrogels steps to improve gene therapy include the use of enhanced are in development that exploit the physiological differ- liver-specific promoters, codon optimisation and reduction ences within the gastro-intestinal tract [71]. These methods of CpG motifs (a pathogen-associated molecular pattern) to preserve the protein through pH changes and proteolytic reduce immunogenicity [65,66,67]. There are currently over enzymes of the stomach until it is released in the small in- a dozen ongoing clinical trials for gene therapy in haemo- testine. Unfortunately, poor bioavailability prevents the use philia [12]. of this route for treatment, but effective inhibitor suppres- Further to standard gene therapy approaches, a zinc sion has been demonstrated [72]. finger nuclease-mediated genome editing protocol (SB-FX, Sangamo, in phase I clinical trial) targets the FIX transgene Summary to the first intron of the native albumin locus which results Recent developments in the treatment of haemophilia in high-level hepatocyte FIX expression [68]. Pleightlet have the potential to revolutionise the patient’s quality of (MUT6) is due to enter phase 1 trials. It uses a lentiviral vec- life. This is being achieved through reduced frequency of tor ex vivo to introduce F8 gene into a patient’s own stem infusion, switching subcutaneous administration, decreased cells which have been removed by apheresis. Following re- incidence of inhibitors, eradication of previously untreata- infusion, these stem cells create platelets that produce and ble inhibitors, improved haemostatic control and even long- store FVIII [69]. lasting cure. Alongside these benefits there will inevitably be clinical and laboratory challenges in implementing thera- Orally Administered Therapeutics pies in this exciting clinical field. Importantly, translating The oral route of administration is being considered for traditional laboratory assays into this new era of complex immune tolerance induction using bio-encapsulated FVIII or therapeutics will impact on patient management and pro- FIX in lyophilised lettuce cells with a cholera toxin B subunit vide essential insights into the efficacy of these novel thera- adjuvant which acts as a transmucosal carrier (CTB-FIX) [70]. peutics.

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Efmoroctocog Alfa: A Review in Haemophilia A.; Drugs. 2017; 77, 1677–1686. 36. Malec, L. M., Ragni, M. V, Journeycake, J. M. & Alabek, M. Immune Tolerance Induction Using Rfviiifc (Eloctate).; Blood. 2015; 126, 3531 LP – 3531. 37. Hoy, S. M. Eftrenonacog Alfa: A Review in Haemophilia B.; Drugs. 2017; 77, 1235–1246. 38. Keating, G. M. BAY 81-8973 (Octocog Alfa; Kovaltry®): A Review in Haemophilia A.; BioDrugs. 2016; 30, 453–459. 39. Kavakli, K. et al. Prophylaxis vs. on-demand treatment with BAY 81-8973, a full-length plasma protein-free recombinant factor VIII product: Results from a ran- domized trial (LEOPOLD II).; J. Thromb. Haemost. 2015; 13, 360–369. 40. Turecek, P. L. et al. Development of BAX 826, a Polysialylated Full-Length rFVIII with Significantly Improved PK Properties.; Blood. 2015; 126, 3536 LP – 3536. 41. Konkle, B. A. et al. BIVV001: The First Investigational Factor VIII Therapy to Break Through the VWF Ceiling in Hemophilia A, with Potential for Extended Protec- tion for One Week or Longer.; Blood. 2018; 132, 636 LP– 636. 42. Liu, Z. et al. Evaluation of Recombinant Fixfc-Xten Bleeding Efficacy in Hemophilia-B Mouse Models.; Blood. 2016; 128, 3757 LP – 3757. 43. Lenting, P. J., Muczynski, V., Aymé, G., Denis, C. V & Christophe, O. D. Von Willebrand Factor Interaction with FVIII: Development of Long Acting FVIII Therapies. Blood. 2016; SCI-8 LP-SCI-8. 44. Muczynski, V. et al. A factor VIII–nanobody fusion protein forming an ultrastable complex with VWF: Effect on clearance and antibody formation.; Blood. 2018; 132, 1193–1197. 45. You, C. W. et al. Phase 1/2 trial of single and multiple dose subcutaneously administered Factor IX variant CB2679D / ISU304: pharmacokinetics and safety. PB159. 2679. 2018 Febr 09 Catalyst Biosciences, Inc. at 11th Annual Congress of the European Association for Haemophilia and Allied Disorders. 46. Mahlangu, J. et al. Efficacy and safety of rVIII-SingleChain: results of a phase 1/3 multicenter clinical trial in severe hemophilia A.; Blood. 2016; 128, 630–637 47. Amgen Canada Inc. Product Monograph Including Patient Information Afstyla. 59 (2015). 48. Gruppo, R. A. et al. Phase 1, single-dose escalating study of marzeptacog alfa (activated), a recombinant factor VIIa variant, in patients with severe hemophilia. Thromb. Haemost. 2018; 16, 1984–1993. 49. Oldenburg, J. et al. Emicizumab Prophylaxis in Hemophilia A with Inhibitors.; N. Engl. J. Med. 2017; 377, 809–818. 50. Leksa, N. C. et al. Intrinsic differences between FVIIIa mimetic bispecific antibodies and FVIII prevent assignment of FVIII-equivalence.; J Thromb Haemost. 2019; Jul;17(7):1044-1052. 51. Al-Samkari, H. & Croteau, S. E. Shifting landscape of hemophilia therapy: Implications for current clinical laboratory coagulation assays. Am. J. Hematol. 2018; 93, 1082–1090. 52. Lenting, P. J., Denis, C. V. & Christophe, O. D. Emicizumab, a bispecific antibody recognizing coagulation factors IX and X:How does it actually compare to factor VIII?; Blood. 2017; 130, 2463–2468. 53. Sehgal, A. et al. An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia.; Nat. Med. 2015; 21, 492. 54. Alnylam. Alnylam Alnylam Reports Patient Death in Fitusiran Clinical Study. National Hemophilia Foundation. Natl. Hemoph. Found. 2017. 55. Eichler, H. et al. A randomized trial of safety, pharmacokinetics and pharmacodynamics of concizumab in people with hemophilia A. J. Thromb. Haemost. 2018; 16, 2184–2195. 56. Chowdary, P. et al. Pharmacodynamics, Pharmacokinetics and Safety of Bay 1093884, an Antibody Directed Against Human TFPI, in Patients with Factor VIII or IX Deficiency (With and Without Inhibitors): A Phase 1 Study.; Blood. 2018; 132, 1176 LP – 1176. 57. Cardinal, M. et al. A first-in-human study of the safety, tolerability, pharmacokinetics and pharmacodynamics of PF-06741086, an anti-tissue factor pathway inhibitor mAb, in healthy volunteers.; J. Thromb. Haemost. 2018; 16, 1722–1731. 58. Huntington, J. A. et al. Design and characterization of an APC-specific for the treatment of hemophilia.; Blood. 2016; 129, 105–113. 59. Zhao, X.-Y. et al. Targeted Inhibition of Activated Protein C Anticoagulant Activity By Monoclonal Antibody HAPC1573 for Treatment of Hemophilia. Blood. 2016;128, 80 LP – 80. 60. Prince, R. et al. Targeting anticoagulant protein S to improve hemostasis in hemophilia.; Blood. 2018; blood-2017-09-800326. 61. Arruda, V. R. & Samelson-Jones, B. J. Gene therapy for immune tolerance induction in hemophilia with inhibitors.; J. Thromb. Haemost. 2016; 14, 1121–1134. 62. Nathwani, A. C. et al. Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B.; N. Engl. J. Med. 2014; 371, 1994–2004. 63. Simioni, P. et al. X-Linked Thrombophilia with a Mutant Factor IX (Factor IX Padua).; N. Engl. J. Med. 2009; 361, 1671–1675. 64. George, L. A. et al. Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.; N. Engl. J. Med. 2017; 377, 2215–2227. 65. Nathwani, A. C. et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.; Blood.2006; 107, 2653–61. 66. Sack, B. K. et al. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy.; PLoS One. 2012; 7(5):e37671. 67. Meijer, K. et al. Gene therapy with adeno-associated virus vector 5–human factor IX in adults with hemophilia B.; Blood.2017; 131, 1022–1031. 68. Park, C. Y., Lee, D. R., Sung, J. J. & Kim, D. W. Genome-editing technologies for gene correction of hemophilia.; Hum. Genet. 2016; 135, 977–981. 69. U.S. Nation Library of Medicine. Gene Therapy Trial for Platelet Derived Factor VIII Production in Hemophilia A. ClinicalTrials.gov. ClinicalTrials.gov 2019. https://clinicaltrials.gov/ct2/show/NCT03818763. 70. Daniell, H., Kulis, M. & Herzog, R. W. Plant cell-made protein antigens for induction of Oral tolerance.; Biotechnol Adv. 2019 Nov 15;37(7):107413. 71. Horava, S. D., Moy, K. J. & Peppas, N. A. Biodegradable hydrophilic carriers for the oral delivery of hematological factor IX for hemophilia B treatment. Int.J.Pharm. 2016; 514, 220–228. 72. Herzog, R. W. et al. Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce. Mol. Ther. 2017; 25, 512–522.

2019 Page 7 New developments in haemophilia treatment

Role of gene therapy in the future treatment of haemophilia patients

Wolfgang Miesbach, MD comes such as plasma coagulation factor activity and bleed- ing episodes, readily enabling an assessment of the impact Institute of Transfusion Medicine, University Hospital Frank- of gene therapy on the clinical profile of the disorder. How- furt, Germany. ever, as discussed below, additional endpoints, such as func- tional activity, quality of life and joint imaging, may be useful Introduction to further delineate any benefits of gene therapy compared In the history of haemophilia treatment, the major mile- with current coagulation factor replacement options. stones were the introduction of replacement therapy with Recombinant vectors based on AAV have been used in coagulation factors VIII (FVIII) and factor IX (FIX) in the early studies with adult patients. AAV are used as viral vectors in 1970s, the virus inactivation of coagulation factor concen- gene therapy in vivo because they are not associated with trates derived from blood plasma in the 1980s and the pro- diseases; they show a strong liver tropism depending on the duction of recombinant coagulation factors in the 1990s [1]. serotype; and the viral genome is not generally integrated Severe haemophilia is associated with frequent spontaneous into the genome of the host cell [7]. Transduction requires bleeding in the joints, muscles, and soft tissues that can re- the internalisation of the AAV containing the therapeutic sult in chronic synovitis and debilitating arthropathy. Affect- gene into the target cell, the entry of the therapeutic gene ed individuals may need to avoid activities such as sports and into the nucleus and the production of the therapeutic pro- experience mobility and functional impairments later in life, tein [7]. which may limit their social interactions and quality of life [2]. Results of clinical studies Recently, extended half-life FVIII and FIX concentrates Clinical gene transfer investigations have been conducted have simplified both prophylactic therapy and treatment on for over 25 years and gene therapies are now starting to be demand by reducing the frequency of dosing and maintain- used in medical practice [8]. Several clinical studies have ing elevated coagulation factor levels [3]. provided proof of concept evidence for the use of gene ther- The prophylactic use of the subcutaneously administered apy to treat haemophilia using different vectors and coagula- FVIII mimetic (bispecific monoclonal antibody Emicizumab) tion factor genes. enables the dosing frequency to be further extended in pa- In 2000, the first in-human AAV study with an AAV sero- tients with severe haemophilia A and in patients with hae- type 2 vector expressing human FIX (2e11 vector copies (vc)/ mophilia A with inhibitor [4]. kg) was used to transfect the skeletal muscle of three partici- In contrast to the therapeutic options described above, pants with haemophilia B [9]. The study revealed vector and gene therapy for haemophilia offers the prospect of a per- FIX expression in muscle biopsies, but only modest changes manent increase in coagulation factor levels. in circulating FIX levels and the clinical need for FIX replace- The aim of gene therapy for haemophilia is to provide ment. long-term expression of the missing or abnormal coagulation The first groundbreaking results in haemophilia B gene factor at sufficient and steady levels to reduce or even elimi- therapy were published in 2011 and 2014. Researchers from nate bleeding and the need for exogenous coagulation factor the St Jude Children’s Research Hospital and the University replacement. College London transfected an AAV8 vector containing a codon-optimised FIX transgene under the control of a liver- Why gene therapy for haemophilia? specific promoter (2e11, 6e11, or 2e12 vc/kg) into partici- Four main reasons why haemophilia is an optimal target pants with haemophilia B [10,11]. for gene transfer have been proposed. Firstly, it is a mono- After the administration of therapy, a consistent increase genic condition that is improved by achieving the expression in the FIX level of up to 7% was observed in all study partici- of functional coagulation factor. Secondly, a relatively small pants, even years later. The bleeding rate decreased by 90%, increase in coagulation factor activity from <1 IU/dL to 5-10 and thus some patients were able to stop regular prophylac- IU/dL substantially improves the bleeding risk and thus ex- tic replacement with a coagulation factor concentrate. No erts a major impact on the lives of affected individuals [5]. further bleeding occurred in these patients, even during Thirdly, the genetic transcript for B-domain-deleted FVIII sports activities. (with the same haemostatic efficacy as the full-length FVIII Further studies using a baculovirus-derived AAV5 con- molecule) and FIX is small enough to fit into an adeno- firmed the good response [12]. In both studies the response associated virus vector (AAV, maximum packing capacity of was dose-dependent with the highest factor levels in high- approximately 4.7 kb) [6]. Finally, haemophilia is well- dose cohorts. described and has established and easily monitored out- Using a highly effective variant of FIX (Padua variant),

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a greater than 30% increase in FIX activity was achieved, of individuals and many potentially eligible patients have protecting against bleeding even in the event of injury and nAbs to wild-type AAV, which may limit successful transduc- reducing the bleeding rate by 97% [13]. tion and long-term success [17]. The level of infection with Recently, the first results of a study on gene therapy for the different serotypes of wild-type AAV and their cross- haemophilia A were published. After only a few weeks, a reactivity in the human population varies [17]. Exposure to significant increase in FVIII activity was achieved [14]. Ina wild-type AAV begins very early in life and the prevalence of pilot study, 15 participants with severe haemophilia A were the virus increases with age. Therefore, individuals with pre- administrated the FVIII gene via an AAV5 vector. Coagulation existing nAbs are usually excluded from clinical trials of gene factor levels of 50 to 150 IU/dL, which are comparable to the therapy. Because humoral immunity to the AAV capsid is levels observed in healthy individuals, were achieved and likely to be generated upon the initial exposure, readmin- remained constant for one year. No spontaneous bleeding istration of the same AAV serotype is unlikely to be effective. was observed, and even after severe trauma or necessary This hypothesis has been confirmed in murine studies, show- surgery, except for one patient, no FVIII concentrate had to ing the inhibition of gene transfer following the repeated be substituted. administration of AAV1, AAV2 or AAV5 vectors [18]. Table 1 summarises the results of the Phase 1 studies on Furthermore, transient liver toxicity (particularly in- gene therapy for haemophilia A and B published to date. creased alanine aminotransferase, ALT) has been observed Currently, various Phase 3 studies are being conducted in in some patients after gene therapy in a dose-related man- larger patient populations. ner, which was associated with the activation of AAV capsid- specific T cells potentially leading to a loss of efficacy of the Long-term results therapy in some studies [10-14]. The increased levels of ALT The longest running human study of haemophilia B, were transient in all cases, presenting a varying duration and which used an AAV8 vector to transfer codon-optimised FIX decreasing upon the early administration of corticosteroids, transgene, has now collected data for more than 8 years but this liver toxicity is dose-limiting for the respective gene which show that participants have stable FIX expression and therapy approach. have been able to discontinue FIX prophylaxis over the long - Inhibitors which are neutralising to coagulation factors term [15]. Additionally, longer-term data have been collect- have not been reported so far. However, the studies only ed from canine models which support the continued safety included previously treated patients (PTPs) without a history and efficacy of liver-directed gene transfer for haemophilia B of inhibitors. [16]. According to the current understanding, AAV mostly do not integrate into the host DNA and remain extra- Safety chromosomal and as a result the risk of gene therapy-related According to these data, no major safety concern has genotoxicity is not increased [19]. Further studies, however, been noted but immunogenicity is the main limitation for are needed to demonstrate the long-term safety of gene systematically administered gene therapy. therapy. A challenge to gene therapy is the presence of pre- existing, neutralizing antibodies against different AAV sero- Laboratory challenges types (nAbs), which explains why not every patient is suita- Routine biomarkers in blood and urine, including blood ble for every AAV-based gene therapy approach. Up to 50 % count and haemoglobin levels, clinical chemistry variables,

Table 1. Results of Phase 1 gene therapy studies for treatment of haemophilia A and haemophilia B

Study/Sponsor Indication Vector Increase of the Reduction of annual Stop of prophylactic Number of study coagulation factor bleeding rate replacement of participants (n) coagulation factor concentrates SJCRH/UCL haemophilia B AAV2/8 dose-dependent increase of 15,5 (IQR10,3 – 19,3) 4/7 n = 10 [10,11] FIX to 1 – 6 % to 1,5 (IQR 1,0 – 4,0) Spark Therapeutics haemophilia B AAV-Spark-100 increase of FIX to 14 - 81 % 11,1 (0-48) 7/7 n = 10 [13] (bioengineered) to 0,4 (0 – 4) UniQure haemophilia B AAV5 dose-dependent increase of Highest dose cohort: 8/9 n =n 10 [12] FIX to > 5% 3,0 to 0,9 Biomarin haemophilia A AAV5 dose-dependent increase of 16 (IQR 1-24) 8/9 n = 9 [14] FVIII to > 50 % in 6/7 patients to 1 (IQR 1-2) of highest dose cohort

IQR: interquartile range 2019 Page 9 New developments in haemophilia treatment

including markers of kidney and liver function, and immuno- An unmet need for clinicians to manage expectations in logical parameters are measured in most clinical studies to terms of what individuals can expect following gene transfer monitor the safety of gene therapy. Liver enzymes are par- for haemophilia has been noted [21]. The likelihood of a re- ticularly important in gene therapy, as they are indicators of duction in the bleeding frequency and a reduction or cessa- anti-AAV immune responses since. Thus, liver-targeted ther- tion in the need for coagulation factor replacement should apy might cause liver toxicity. Additionally, humoral and cel- be discussed. Importantly, individuals should be made aware lular immune responses are regularly monitored using suita- that gene transfer is not a cure, bleeds may still occur and ble assays. For the detection of pre-existing neutralising anti- some continuing medical management will be necessary as bodies to AAV, different tests can be used, e.g. a luciferase- long as the coagulation factor has not been normalised. In based in vitro transduction assay. Moreover, the virus parti- addition, the field of gene transfer for haemophilia is still at cles themselves potentially represent a safety issue when an early stage. Thus, while clinicians are awaiting more clini- they leave the treated individual’s body, a process described cal evidence, a prudent strategy would be to inform candi- as viral shedding. dates for gene transfer that they may not respond, or might Variability in the results of assessments of the transgene respond poorly to treatment. clotting factor levels using one-stage and chromogenic as- Gene therapy is still an experimental technology and, says has also been observed [14], and therefore, we recom- therefore is highly regulated and carefully monitored to mend that the coagulation factor levels be measured using maximise patient safety. Depending on the type of gene both methods and to correlate the results to the clinical situ- therapy used, potential risks can include unwanted immune ation of the patient [20]. reactions or the incorporation of new genetic material into cells. Thus, every patient treated with gene therapy should Conclusions be included in international registries and receive life-long

Clinical trials have successfully used adeno-associated follow-up care to monitor late toxicity of the gene construct. viral vectors to transfer functional coagulation factors as Further studies are needed to obtain a better under- treatments for haemophilia A and B, and this therapy has standing of the limitations of gene therapy and to enable the potential to transform the lives of affected individuals. patients to be treated with gene therapy in order to induce Gene transfer increases coagulation factor activity to func- the long-term expression of the transgene with the lowest tional levels, reduces the bleeding frequency and decreases possible risks of side-effects. or abrogates the need for coagulation factor replacement.

References 1. Schramm W. The history of haemophilia - a short review. Thromb Res. 2014 Nov; 134 Suppl 1:S4-9. 2. Oldenburg J: Optimal treatment strategies for haemophilia: achievements and limitations of current prophylactic regiments. Blood 2015; 125: 2038-2044. 3. Collins P, Chalmers E, Chowdary P, et al: The use of enhanced half-life coagulation factor concentrates in routine clinical practice: guidance from UKHCDO. Hae- mophilia 2016; 22: 487-98. 4. Weyand AC, Pipe SW. New therapies for hemophilia. Blood. 2019 Jan 31;133(5):389-398. 5. White, G.C., 2nd, et al., Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardiza- tion committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost, 2001. 85(3): p. 560. 6. Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): p. 80-6. 7. Flotte TR, Carter BJ: Adeno-associated virus vectors for gene therapy. Gene Ther 1995; 2: 357-62. 8. Spencer, H.T., B.E. Riley, and C.B. Doering, State of the art: gene therapy of haemophilia. Haemophilia, 2016. 22 Suppl 5: p. 66-71. 9. Kay, M.A., et al., Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet, 2000. 24(3): p. 257-61. 10. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011 Dec 22;365(25):2357-65. 11. Nathwani AC, Reiss UM, Tuddenham EG, Rosales C, Chowdary P, McIntosh J, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med. 2014 Nov 20;371(21):1994-2004. 12. Miesbach W, Meijer K, Coppens M, et al. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B. Blood 2018; 131: 1022 -1031. 13. George LA, Sullivan SK, Giermasz A, et al. Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant. N Engl J Med 2017; 377: 2215-2227. 14. Rangarajan S, Walsh L, Lester W, et al. Factor VIII Gene Transfer in Severe Hemophilia A. N Engl J Med 2017; 377: 2519-2530. 15. Nienhuis AW, Nathwani AC, Davidoff AM. Gene Therapy for Hemophilia. Mol Ther. 2017 May 3;25(5):1163-1167. 16. Niemeyer, G.P., et al., Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy. Blood, 2009. 113(4): p. 797-806. 17. Louis Jeune, V., et al., Pre-existing anti-adeno-associated virus antibodies as a challenge in AAV gene therapy. Hum Gene Ther Methods, 2013. 24(2): p. 59-67. 18. Boutin S, Monteilhet V, Veron P, et al: Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 2010; 21: 704-12. 19. Balakrishnan B, Jayandharan GR. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy. Curr Gene Ther 2014;14:86-100. 20. Peyvandi F, Garagiola I.Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia. Haemophilia. 2019 Jul 8. 21. Miesbach W, O'Mahony B, Key NS, Makris M. How to discuss gene therapy for haemophilia? A patient and physician perspective. Haemophilia. 2019 Jul;25 (4):545-557. Page 10 Special Issue 9

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Personalising therapy in haemophilia Pharmacokinetic- guided dosing of factor VIII and factor IX concentrates Tine M.H.J. Goedhart MD and Marjon H. Cnossen, MD, PhD Figure 1. Interindividual variation in FVIII levels after a dose

of FVIII concentrate in haemophilia A patients with FVIII Department of Pediatric Hematology, Sophia Children’s levels <0.02 IU mL-1 (n=2053 blood samples) Hospital, Erasmus University Medical Center, The Nether- lands

Prophylactic replacement therapy Severe haemophilia patients have been treated prophy- lactically since 1965, on the basis of the observation by Ahl- berg [1] that moderate haemophilia patients with factor VIII (FVIII) or factor IX (FIX) levels above 0.01 IU mL-1 have far fewer joint bleeds and less arthropathy than patients with severe haemophilia and levels below 0.01 IU mL-1. Moreo- ver, time spent below this trough level is associated with an increased number of bleeding events [2]. Although the target level of 0.01 IU mL-1 is generally accepted, there is growing consensus that higher trough levels may be necessary to protect patients more optimally from invalidating joint bleeds and other miscellaneous bleeding Data was extracted from three clinical trials containing 236 PK datasets from 152 patients ranging from 1-6 and 10-65 years of events. This perspective is supported by Den Uijl et al. who age [10, 11]. Figure adapted from Bjorkman et al. (2012) [4]. showed that annual bleeding frequency only declined drastically in moderate haemophilia A patients [3] when Figure 2. Differences in half-life after infusion of FVIII (30 IU -1 patients’ baseline levels were above 0.03 IU mL . To kg-1) concentrate in patients between 10 and 65 years old achieve these trough levels, prophylaxis with factor concentrates is still dosed per kilogram of body weight, usually two to three times weekly when standard half-life factor (SHL) concentrates are used, dependent on haemophilia type and bleeding frequency. However, in current practice these trough levels are rarely monitored. Generally, dose and frequency of prophylactic infusions are only adjusted when (spontaneous) bleeding events occur. This approach, does not take the interindividual variation in pharmacokinetics (PK) of factor concentrates into account, which can be significant as demonstrated by Bjorkman et al. [4].

Interindividual variability in pharmacokinetics of factor These differences may be explained by differences in volume of concentrates distribution and FVIII clearance. Young haemophilia A patients The interindividual differences in PK parameters, most have a larger volume of distribution relative to their total body importantly clearance (CL), volume of distribution (Vd), area weight and a higher FVIII clearance rate than older haemophilia A patients who have a smaller volume of distribution and a lower under the curve (AUC), half-life (t1/2) of a FVIII or FIX con- FVIII clearance rate (Figure adapted and adjusted from Collins, centrate are partly explained by a variety of patient-specific 2011) [12]. factors such as age, body weight, activity levels, and other both known and unknown factors [4-9]. Bjorkman et al. years. This may lead to a time difference of 59 hours be- reported this variation over time in haemophilia A patients tween patients, when a trough FVIII level of 0.01 IU mL-1 is when evaluating FVIII levels after FVIII concentrate infusion reached (Figure 2). (Figure 1) [4,8]. As a result of this interpatient variability, extreme differences in FVIII concentrate clearance and Population pharmacokinetics therefore half-life (t1/2 = Ln2 x Vd/ CL) are observed, rang- To address interindividual differences in necessary dos- ing between 6 and 25 hours after similar FVIII SHL concen- ing regimen, PK-guided therapy can be implemented. trate doses (30 IU kg-1) in haemophilia patients aged 10-65 2019 Page 11 New developments in haemophilia treatment

Figure 3. Creating an individual FVIII PK profile based on population data to determine patient-specific FVIII clearance.

(A) Black lines represent the collected PK population data on FVIII clearance. (B) Next, an individual FVIII PK profile is constructed by measuring FVIII levels in the individual patient at three consecutive time points (4, 24 and 48 hours after infusion). (C) When comparing data from the indi- vidual PK profile to PK population data, the model can predict the patient-specific clearance of FVIII concentrate by Bayesian analysis.

PK is described as the process by which a drug is absorbed, average values for PK parameters derived from such a mod- distributed, metabolised and eliminated by the body. Until el, but also their inter- and interindividual variability in PK recently, the PK of FVIII or FIX concentrates in a specific due to various covariates i.e. specific patient characteristics individual was not easily determined due to the necessity of or circumstances. For example, the most important covari- frequent blood samples (>10 samples required) [13]. When ate in FVIII population models for haemophilia A patients is Bayesian analysis is applied however, information from the most likely von Willebrand factor (VWF), due to its role as a individual patient, e.g. dose, timing of dose and FVIII levels chaperone to FVIII, thus protecting FVIII from proteolysis in at documented time points, is combined with PK data from the circulation. In other words, lower VWF levels will lead to a large population with similar disease and treatment i.e. higher FVIII clearance. As patients with blood group O have also called a population PK model, as depicted in Figure 3, 25% lower VWF levels, blood group is also an important thus, leading to the need for less blood sampling in the indi- covariate influencing PK as shown by Hazendonk et al. [2]. vidual (”limited sampling”). In a population PK model the Pioneering work was performed by Bjorkman, and Collins relationship between dose and plasma concentration of the has made PK-guided prophylactic dosing in haemophilia medication involved is described on the basis of the PK pa- possible with only three samples (with rFVIII-sampling just rameters clearance and volume of distribution. Not only are before and 24 and 48 hours after FVIII dose administration) without a washout period, which has significantly reduced Table 1. Limited blood sampling strategies to construct indi- patient burden of PK-guided dosing as frequency of sam- vidual PK curves.[14-16] pling and risk of bleeding is minimalised (Table 1) [14]. Application of PK-guided dosing will lead to the optimal dosing regimen for each individual and make it possible to target specific trough and peak levels reliably. More specifi- cally, a patient with a more pronounced bleeding pheno- type or more intensive activity level and therefore a greater risk of bleeding can be dosed to achieve high trough levels according to the preference of the physician.

PK-guided dosing in the perioperative setting Besides the need to target trough levels during prophy- laxis, it can also be necessary to target peak levels in the perioperative setting or during acute bleeding. In the peri- Bolus operative setting, these factor levels are often monitored. infusion FVIII or FIX measurements During acute bleeding however, they are often estimated, -1 (IUkg ) on the basis of in vivo recovery (IVR) estimates and the dose Factor VIII (FVIII) 50 T=4, T=24, T=48 h of factor concentrate units infused per kilogram of body (Björkman et al.) weight. This IVR-based dosing originates from studies that showed that each infused unit of factor concentrate per Plasma derived Factor 50 T=48, T=72 h or kilogram will result in a mean increase of 0.02 IU mL-1 for IX (FIX) (Brekkan et al.) T=54, T=78 h FVIII and 0.01 mL-1 for FIX SHL concentrates. However, Recombinant Factor IX 50 One sample post-infusion, these estimates do not take PK into account and can there- (FIX) (Preijers et al.) two samples between fore lead to inadequate target trough and peak levels. A T=72 and T=80 h perioperative study demonstrated that during standard

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treatment, 45% of the FVIII measurements were below the Future research target range within the first 24h after surgery and 75% The OPTI-CLOT research programme (Patient-tailOred above the target range during hospitalisation more than six PharmacokineTIc-guided dosing of CLOTting factor concen- days after surgery [2]. trate and desmopressin in bleeding clotting disorders) is a multicentre initiative in which all six Dutch Academic Hae- Benefits of PK-guided therapy mophilia Treatment centres participate. The OPTI-CLOT There are several benefits to be gained by using PK- studies aim to implement PK-guided dosing of clotting fac- guided therapy. Firstly, as described before, PK-guided dos- tor replacement therapy and desmopressin in prophylactic, ing is based on FVIII and FIX trough and peak levels as pre- on demand- and perioperative settings in all bleeding disor- dicted by population PK modelling, instead of FVIII and FIX ders by proving its implications, by the construction of PK estimates based on IVR-based dosing [2]. Secondly, this population models and by evaluating possible cost reduc- intervention may be more cost-effective for society. Hae- tion. A randomised controlled trial comparing PK-guided mophilia is expensive to treat, mainly due to the excessive perioperative treatment of clotting factor concentrates with costs of clotting factor concentrates [17]. Carlsson et al. standard treatment in severe and moderate haemophilia A compared PK-guided prophylactic dosing to standard patients, in order to analyse the amount of clotting factor prophylactic dosing and reported a dose and cost reduction concentrates administered, time spent to achieve targeted of 30% in FVIII clotting factor concentrate in a small group FVIII levels, as well as staff investment and costs, has now of 14 patients. The number of reported bleedings was simi- almost been completed [20, 21]. Currently, a non- lar in both treatments arms. Such a reduction in dose can randomised, prospective cohort study has been initiated to have a significant financial impact, as annual costs of re- investigate the reliability and feasibility of PK-guided placement therapy with factor concentrates in the Nether- prophylactic dosing of both SHL and EHL factor concen- lands amounts to approximately 130 million euros [7]. trates in haemophilia A and B patients. This study is neces- Thirdly, with PK guidance, knowledge will increase about sary to prove the reliability and feasibility of PK-guided the relationship between FVIII and FIX levels and the risk of prophylactic dosing according to the population PK models bleeding in individual patients and categories of patients. In constructed and to enrich models with real-world data, ever addition, an increase in dosing intensity will not only de- improving models and enlarging the diversity of the pa- pend on actual bleeding, and a reduction of dosing intensity tients included in the population. can be considered by the treating professional in consulta- Finally, it is obligatory for the hemophilia community to tion with patients and parents on the basis of predicted further facilitate PK-guided dosing for constructing a treat- factor levels. Over time, more exact targeting of FVIII and ment regime involving physicians, nurses and patients and FIX levels may also lead to reliable lowering of target levels their families. PK-guided dosing requires close collaboration of treatment. Especially in haemophilia B, studies and clini- between a clinical pharmacologist with expertise in PK mod- cal experience suggest that lower FIX target levels may be elling using NONMEM® software and the treatment team as acceptable to prevent bleeding. Fourthly, PK-guided dosing implemented within OPTI-CLOT. Solutions to improve and is able to facilitate individualisation of dosing according to simplify this innovation are the availability of web portal- individual lifestyle and activity level, thereby achieving true based consultancies for PK-guided dosing advice, as estab- personalisation of treatment according to personal needs lished by Iorio et al. and as developed by one of the phar- and characteristics. Patients and families should be aware maceutical companies for their own concentrates [21]. of time periods when factor concentrate levels are low or Transparency and reliability of the data used to construct high and will be able to consider additional dosing when the underlying population models are of crucial importance in bleeding risk is significant [2]. Lastly, PK-guided therapy can such settings. be helpful when switching from an SHL concentrate to an extended half-life (EHL) concentrate. When switching, the Conclusion prediction of the factor levels can be facilitated by PK- We believe PK-guided therapy of clotting factor concen- guided dosing based on PK models for EHL concentrates trates in prophylactic, on demand and perioperative [18]. settings is promising and an important goal for the near It is important to realise that PK-guided dosing should future. More optimal dosing regimens for the individual only be performed using factor activity level measurements patient based on an individual PK profile, while targeting set with the type of assay either one-stage assay (OSA) or trough and peak levels on the basis of individual bleeding chromogenic assay (CSA) used to construct the population tendency and risk, will lead to the actual tailoring of treat- PK model, as assays may give varying results [19]. Especially ment with increased quality of care and potential decrease in the EHL concentrates, but also in some SHL B domain- of societal costs. deleted FVIII concentrates, the choice of the type of assay used for PK profiling and modelling is of significant im- portance.

2019 Page 13 New developments in haemophilia treatment

References 1. Ahlberg A. Haemophilia in Sweden. VII. Incidence, treatment and prophylaxis of arthropathy and other musculo-skeletal manifestations of haemophilia A and B. Acta Orthop Scand Suppl 1965: Suppl 77:3-132. 2. Hazendonk H, van Moort I, Mathot RAA, Fijnvandraat K, Leebeek FWG, Collins PW, et al. Setting the stage for individualized therapy in hemophilia: What role can pharmacokinetics play? Blood Rev 2018; 32: 265-71. 3. Den Uijl IE, Mauser Bunschoten EP, Roosendaal G, Schutgens RE, Biesma DH, Grobbee DE, et al. Clinical severity of haemophilia A: does the classification of the 1950s still stand?Haemophilia 2011; 17: 849-53. 4. Bjorkman S, Oh M, Spotts G, Schroth P, Fritsch S, Ewenstein BM, et al. Population pharmacokinetics of recombinant factor VIII: the relationships of pharmaco- kinetics to age and body weight. Blood 2012; 119: 612-8. 5. Aronstam A MD, Wassef M & Mbatha PS. Effect of height and weight on the in vivo recovery of transfused factor VIII C. Journal of Clinical Pathology 1982; 35: 289-91. 6. Bjorkman S, Carlsson M, Berntorp E, Stenberg P. Pharmacokinetics of factor VIII in humans. Obtaining clinically relevant data from comparative studies. Clin Pharmacokinet 1992; 22: 385-95. 7. Carlsson MB, E; Björkman, S; Lethagen, S; Ljung,R. Improved cost-effectiveness by pharmacokinetic dosing of factor VIII in prophylactic treatment of haemo- philia A. Haemophilia 1997; 3: 96-101. 8. Bjorkman S, Folkesson A, Jonsson S. Pharmacokinetics and dose requirements of factor VIII over the age range 3-74 years: a population analysis based on 50 patients with long-term prophylactic treatment for haemophilia A. Eur J Clin Pharmacol 2009; 65: 989-98. 9. Matucci M, Messori A, Donati-Cori G, Longo G, Vannini S, Morfini M, et al. Kinetic evaluation of four Factor VIII concentrates by model-independent methods. Scand J Haematol 1985; 34: 22-8. 10. Blanchette VS, Shapiro AD, Liesner RJ, Hernandez Navarro F, Warrier I, Schroth PC, et al. Plasma and albumin-free recombinant factor VIII: pharmacokinetics, efficacy and safety in previously treated pediatric patients. J Thromb Haemost 2008; 6: 1319-26. 11. Tarantino MD, Collins PW, Hay CR, Shapiro AD, Gruppo RA, Berntorp E, et al. Clinical evaluation of an advanced category antihaemophilic factor prepared using a plasma/albumin-free method: pharmacokinetics, efficacy, and safety in previously treated patients with haemophilia A.Haemophilia 2004; 10: 428-37. 12. Collins PW, Fischer K, Morfini M, Blanchette VS, Bjorkman S, Group IPSGPEW. Implications of coagulation factor VIII and IX pharmacokinetics in the prophylac- tic treatment of haemophilia. Haemophilia 2011; 17: 2-10. 13. Bjorkman S, Collins P, ISTH PoFVaISSCot. Measurement of factor VIII pharmacokinetics in routine clinical practice. J Thromb Haemost 2013; 11: 180-2. 14. Bjorkman S. Limited blood sampling for pharmacokinetic dose tailoring of FVIII in the prophylactic treatment of haemophilia A. Haemophilia 2010; 16: 597- 605. 15. Brekkan A, Berntorp E, Jensen K, Nielsen EI, Jonsson S. Population pharmacokinetics of plasma-derived factor IX: procedures for dose individualization.J Thromb Haemost 2016; 14: 724-32. 16. Preijers T, Hazendonk H, Fijnvandraat K, Leebeek FWG, Cnossen MH, Mathot RAA. In silico evaluation of limited blood sampling strategies for individualized recombinant factor IX prophylaxis in hemophilia B patients. J Thromb Haemost 2017; 15: 1737-46. 17. Zhou ZY, Koerper MA, Johnson KA, Riske B, Baker JR, Ullman M, et al. Burden of illness: direct and indirect costs among persons with hemophilia A in the Unit- ed States. J Med Econ 2015; 18: 457-65. 18. Nederlof A, Mathot RAA, Leebeek FWG, Fijnvandraat K, Fischer K, Cnossen MH, et al. Positioning extended half-life concentrates for future use: a practical proposal. Haemophilia 2018; 24: e369-e72. 19. Preijers T, Schutte LM, Kruip M, Cnossen MH, Leebeek FWG, van Hest RM, et al. Strategies for individualized dosing of clotting factor concentrates and desmo- pressin in hemophilia A and B. Ther Drug Monit 2019. 20. Hazendonk HC, van Moort I, Fijnvandraat K, Kruip MJ, Laros-van Gorkom BA, van der Meer FJ, et al. The "OPTI-CLOT" trial. A randomised controlled trial on periOperative PharmacokineTIc-guided dosing of CLOTting factor concentrate in haemophilia A. Thromb Haemost 2015; 114. 21. Hazendonk HC, Fijnvandraat K, Driessens MH, van der Meer FJ, Meijer K, Kruip MA, et al. Population pharmacokinetics in hemophilia A: towards individualiza- tion of perioperative replacement therapy. Presented at ISTH 2015, Toronto, 2015.

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New developments in the Treatment of Haemophilia: Implications for Laboratory Testing

Armando Tripodi, PhD introduction of these drugs. Most of the information so far available is concerned with emicizumab, the first drug to be Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlini- introduced and licensed by regulatory authorities for co, Angelo Bianchi Bonomi Hemophilia and Thrombosis prophylaxis in patients with haemophilia A with or without Center and Fondazione Luigi Villa, Milano, Italy. inhibitors to FVIII. Therefore, this review will mainly concern analysis in patients using emicizumab. Introduction Haemophilia is an X-linked haemorrhagic disorder char- Emicizumab and the laboratory acterised by low or dysfunctional factor VIII (FVIII) According to current indications, emicizumab treatment (haemophilia A) or factor IX (FIX) (haemophilia B), sponta- does not require dose-adjustment based on laboratory neous or posttraumatic bleeding events and arthropathy. testing and can be administered by fixed body weight ad- During their lifetime about 30% of haemophiliacs may de- justed doses [1, 2]. However, there may be situations when velop alloantibodies directed against FVIII or FIX, which clinicians need to know the activity/concentration of circu- make these patients unresponsive to the infusion of FVIII/ lating drugs or they need to know the titre of the inhibitor FIX concentrates. Historically, haemophilias were treated on to FVIII or the activity of FVIII when concentrates containing demand by infusion of the missing factor (FVIII or FIX), this factor are used in combination with emicizumab (see which may be plasma-derived, recombinant or modified to below). Furthermore, being emicizumab a FVIII mimetic extend the half-life. Patients with inhibitors were treated agent, it is (unsurprisingly) able to affect many of the coagu- with bypassing agents such as activated prothrombin com- lation parameters, whose measurement is based on the plex concentrate (aPCC) or recombinant activated FVII intrinsic pathway of coagulation. There are many publica- (rFVIIa). Recently, regular prophylaxis with relatively low tions dealing with the reported effects of emicizumab on concentrate dose was introduced in order to prevent ar- coagulation measurements [3-7] and others which really thropathy. More recently, drugs not based on coagulation demonstrate the interference caused by spiking haemo- factors concentrates were developed. They include emici- philic or non-haemophilic plasma with emicizumab [8-10]. zumab, fitusiran and concizumab. Their characteristics, mode of action and administration are summarised in Table Measurement of emicizumab (Table 2) 1. The introduction of these new drugs will challenge the Being a mimetic FVIII agent, emicizumab can be meas- laboratory monitoring of haemophiliacs. This review is ured by the regular one-stage clotting assay for FVIII, based aimed at addressing the changes that are expected in the on the activated partial thromboplastin time (APTT) of the clinical laboratory for monitoring haemophilia following the FVIII-deficient plasma, which in this manuscript will be called here “FVIII-surrogate” activity. However, this activity Table 1. Novel drugs, not based on coagulation factors that are measurement would be too responsive to the procoagulant or will be shortly used to treat haemophilia action elicited by emicizumab. In fact, a FVIII-surrogate ac- tivity of 100% would be obtained at a relatively small (5 µg/ Drug Mechanism of action mL) emicizumab plasma concentration, which would then Emicizumab Monoclonal humanised bi-specific antibody reach a plateau (e.g. 150%) at concentrations typically en- that binds FIXa and FX, thus enhancing FX acti- countered in patients on regular prophylaxis (i.e. 40-50 µg/ vation in the absence of FVIII. mL) [10]. This measurement is therefore not useful in prac- It is currently used to treat haemophilia A. tice. There are however alternatives which will be described Concizumab Monoclonal antibody that neutralises the tissue in the following paragraphs. factor pathway inhibitor (TFPI), thus enhancing thrombin generation. Modified one-stage FVIII clotting assays. In principle, it could be used for the treatment One-stage clotting assays for FVIII can be modified in of haemophilia A and B. such a way as to decrease the responsiveness to emici- Fitusiran Fitusiran an RNA agent interfering with anti- zumab, thus allowing a linear and dose-response relation- thrombin translation, thereby silencing anti- ship between clotting times and activity. The laboratory can thrombin gene expression and reducing anti- prepare dose-response calibration curves by testing using thrombin synthesis. these methods the plasma calibrators at certified emici- In principle, it could be used for the treatment zumab concentrations, which are commercially available (r2 of haemophilia A and B. Diagnostics Inc. South Bend, IN). The clotting time ofthe

2019 Page 15 New developments in haemophilia treatment

Table 2. Methods to measure emicizumab in plasma

Method Principle and application Modified one-stage clotting assays Based on the APTT and FVIII-deficient plasma. They can be used in combination with emicizumab calibrators to measure the plasma con- centration with results expressed as µg/mL. Modified two-stage chromogenic assays Based on the FVIII mimetic effect of emicizumab to activate human-derived FX, which will (with human-derived reagents) then be measured by specific synthetic peptide. They can be used in combination with emicizumab calibrators to measure the plasma con- centration with results expressed as µg/mL. Modified two-stage chromogenic assays Based on the FVIII effect to activate bovine-derived FX, which will then be measured by (with bovine-derived reagents) specific synthetic peptide. Being insensitive to emicizumab, these methods can be used to measure the titre of inhibi- tor to FVIII or to measure FVIII in the plasma of the patient in the presence of emicizumab.

patient plasma can be interpolated from these calibration factor reagents of bovine origin, which are completely in- curves to derive the emicizumab plasma concentration, sensitive to emicizumab (see left). which will be expressed as µg/mL. Influence of emicizumab on common haemostasis parame- Modified two-stage FVIII chromogenic assays. ters Another alternative for measuring the plasma concen- As mentioned, as emicizumab is a FVIII mimetic agent, trations of emicizumab is the modification of the regular its presence in plasma is likely to affect the results of some FVIII chromogenic assay. As it is known, this method is com- of the most common haemostatic parameters that are prised of two stages. In the first, FVIII of the patient plasma based on the intrinsic pathway of coagulation. Here are is the limiting factor to activate exogenous FX in the pres- some details (Table 3). ence of optimal (exogenously added) concentrations of Activated partial thromboplastin time (APTT). those factors that are upstream from FX in the coagulation The APTT of the haemophilic plasma is completely nor- cascade. In the second step, activated FX is measured by malised at emicizumab concentrations (i.e. 5 µg/mL), which means of a specific synthetic chromogenic peptide. Dose- are far smaller than those achieved in patients on regular response linear calibration curves can be obtained by prophylaxis (i.e., 40-50 µg/mL) [10]. Caution should there- testing with these methods a set of emicizumab calibrators fore be taken when interpreting results of the APTT in trau- to derive emicizumab concentrations in patient plasma. ma patients or in an emergency. It is mandatory to learn However, when using these methods it is essential that the from patients or relatives about the patient’s pharmacologi- chromogenic assays used for testing employ human-derived cal history, bearing in mind that the effect of emicizumab is coagulation factors as reagents [10]. In fact, coagulation long-lasting (weeks or months after the last injection). The factors from bovine origin are completely insensitive to APTT could be useful in helping detect anti-drug antibodies emicizumab [10]. that may develop in patients following treatment with emi- cizumab. If these antibodies occur, the APTT will be pro- Measurement of FVIII or of the inhibitor to FVIII in the longed. presence of emicizumab Measurement of individual coagulation factors. Although regular emicizumab prophylaxis in patients As expected the measurement of coagulation factors with or without inhibitor to FVIII is able to prevent sponta- (other than FVIII) that are based on the APTT-derived assays neous bleeding events, it is unable to prevent bleeding fol- are strongly affected by emicizumab. As an example, the lowing trauma or surgery. On these occasions patients must activity of FIX, FXI or FXII are increased even in the presence be additionally treated with FVIII concentrates or bypassing of relatively small amounts of emicizumab (10). In contrast, agents (aPCC or rFVIIa) depending on whether or not they the measurement of coagulation factors based on the pro- have inhibitors. Consequently, clinicians need to know the thrombin time (PT)-derived or those based on the chromo- titre of inhibitors to FVIII or the activity of FVIII elicited at genic-derived technique are not affected by emicizumab post infusion. Both of the above-mentioned measurements [10]. are heavily affected by the presence of emicizumab. In par- Activated clotting time (ACT). ticular, emicizumab give false-negative results for the inhibi- The ACT is mainly used in cardio-surgery to monitor hep- tors when they are measured by the regular Bethesda as- arin during extracorporeal circulation. The ACT is likely to be say. The logical solution for both situations is the modified shortened by emicizumab. On this occasion, anti-FXa activi- two-stage FVIII chromogenic assay employing coagulation ty assays should be used instead of ACT.

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Table 3. Interference to be expected when measuring haemostatic parameters in plasma containing emicizumab

Parameter Expected interference Activated partial thromboplastin time (APTT) The APTT of haemophilic plasmas will be completely normalised by concentrati- ons of emicizumab far smaller than those found in patients under treatment. Completely useless, with the possible exception of helping to detect anti-drug antibodies in patients on treatment with emicizumab. Activated coagulation time (ACT) Shortened Prothrombin time (PT) Slightly prolonged Protein C anticoagulant activity based on APTT Underestimation Protein S anticoagulant activity based on APTT Underestimation Activated protein C resistance based on APTT Overestimation (underestimation of the APC-ratio) (PT-derived) Underestimation Fibrinogen (Clauss) No effect Antithrombin No effect Thrombin time No effect Von Willebrand factor No effect (antigen and ristocetin-cofactor activity) D dimer No effect

Prothrombin time (PT). measurement of functional fibrinogen (i.e. Clauss methods For reasons that are unclear, the PT and consequently based on thrombin time) are not affected by emicizumab the international normalised ratio (INR) is slightly prolonged [10]. by emicizumab. This effect seems not to be dependent on Assays that are not affected by emicizumab. the thromboplastin used for testing and negligible in prac- The following haemostatic parameters are not affected tice, but the interpretation of the results requires by emicizumab. (i) Thrombin clotting time; (ii) antithrombin knowledge of this effect [10]. activity and antigen; (iii) von Willebrand factor antigen and Protein C (PC) anticoagulant activity. ristocetin co-factor activity; (iv) D-dimer and the measure- Those assays that measure the anticoagulant activity of ment of individual coagulation factors based on PT-derived PC and are based on the APTT are likely to underestimate or chromogenic-derived technique [10]. the activity in the presence of emicizumab. Methods based on PC chromogenic activity or those based on the measure- Concizumab, fitusiran and the laboratory ment of the antigen are not affected by emicizumab [10]. As mentioned, scanty information is available on the Protein S (PS) anticoagulant activity. role played by the clinical laboratory in the management of Those assays that measure the anticoagulant activity of these drugs. They are still undergoing clinical trials and re- PS and are based on the APTT are likely to underestimate sults are not yet fully available. It is expected that like emici- the activity in the presence of emicizumab (10). Methods zumab, concizumab and fitusiran will also eventually be based on the measurement of the free or total antigen are used at fixed dose without further adjustment by laboratory not affected by emicizumab. testing. One might speculate that since the mechanistic Resistance to activated protein C (APC). action of concizumab is mediated by the neutralisation of Those assays that measure the APC resistance due or the tissue factor pathway inhibitor (TFPI), some sort of not due to the FV Leiden mutation and based on the assess- measurement of TFPI levels during treatment could be re- ment of the APTT prolongation with and without APC, are quired. Similarly, one might speculate that antithrombin likely to underestimate the APC-ratio (without/with APC) measurement could be useful for checking the levels of this [10]. The genetic analysis of the FV Leiden mutation is not important naturally occurring anticoagulant that is de- affected by emicizumab. pressed by fitusiran. Fibrinogen. Those assays that estimate the concentrations of fibrin- Concluding remarks ogen from the coagulation curve of the PT (collectively It is reasonable to assume that the way clinical laborato- known as PT-derived) are likely to underestimate fibrinogen ries has been dealing with haemophilia in the past will un- in the presence of emicizumab [10]. Methods based on the dergo dramatic changes in the near future when the new 2019 Page 17 New developments in haemophilia treatment

drugs, not based on the infusion of coagulation factors, will gents) will be added to the resources available to clinical be largely used. Time-honoured methods such as the one- laboratories. Finally, it is also worth considering that global stage clotting assays for FVIII/FIX based on the APTT and procedures such as thrombin generation or thromboelas- factor-deficient plasma will still assist patients treated with tography/thromboelastometry that are responsive to plasma-derived, recombinant or modified long-acting FVIII/ thrombin, the crucial enzyme of the coagulation cascade, FIX, but other methods such as the modified one-stage could play a role in the management of haemophilia pa- clotting or chromogenic assays (with human or bovine rea- tients.

References

1. Oldenburg J, Mahlangu JN, Kim B, et al. Emicizumab Prophylaxis in Hemophilia A with Inhibitors. N Engl J Med 2017; 377: 809-818. 2. Mahlangu J, Oldenburg J, Paz-Priel I, et al. Emicizumab Prophylaxis in Patients Who Have Hemophilia A without Inhibitors. N Engl J Med 2018; 379: 811-822. 3. Müller J, Pekrul I, Pötzsch B, Berning B, Oldenburg J, Spannagl M. Laboratory Monitoring in Emicizumab-Treated Persons with Hemophilia A. Thromb Haemost. 2019 Jun 16. doi: 10.1055/s-0039-1692427. [Epub ahead of print] PubMed PMID: 31203578. 4. Brophy DF, Martin EJ, Kuhn J. Use of global assays to monitor emicizumab prophylactic therapy in patients with haemophilia Awith inhibitors. Haemophilia. 2019 Mar;25(2):e121-e123. doi: 10.1111/hae.13689. Epub 2019 Feb 12. PubMed PMID: 30748061. 5. Lippi G, Favaloro EJ. Emicizumab (ACE910): Clinical background and laboratory assessment of hemophilia A. Adv Clin Chem. 2019;88:151-167. doi: 10.1016/ bs.acc.2018.10.003. Epub 2018 Nov 16. Review. PubMed PMID: 30612605. 6. Al-Samkari H, Croteau SE. Shifting Landscape of Hemophilia Therapy: Implications for Current Clinical Laboratory Coagulation Assays. Am J Hematol. 2018 Jun 8. doi: 10.1002/ajh.25153. [Epub ahead of print] Review. PubMed PMID: 29884997. 7. Tripodi A, Chantarangkul V, Novembrino C, et al. Advances in the treatment of hemophilia: Implications for laboratory testing. Clin Chem 2019; 65:254-262. 8. Calatzis A, Kotani N, Levy G, Adamkewicz J. Effect of emicizumab–a humanized bispecific antibody mimicking FVIII cofactor function– on a variety of assay sys- tems. Amsterdam: European Congress on Thrombosis and Hemostasis; 2016. p. 32. 9. Adamkewicz J, Soeda T, Kotani N, Calatzis A, Levy G. Effect of emicizumab (ACE910) a humanized bi-specific antibody mimicking FVIIIa cofactor function on coagulation assays commonly in use for monitoring hemophilia A patients [Abstract]. Haemophilia 2017;23:S3. 10. Adamkewicz JI, Chen DC, Paz-Priel I. Effects and Interferences of Emicizumab, a Humanised Bispecific Antibody Mimicking Activated Factor VIII Cofactor Func- tion, on Coagulation Assays. Thromb Haemost. 2019. doi:10.1055/s-0039-1688687.

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Potency labelling of extended half-life FVIII and FIX products Elaine Gray, PhD cates that 1 IU/kg of therapeutic concentrate for FVIII would be expected to raise FVIII activity on average by 2 IU/ Haemostasis Section, Biotherapeutics Group, National Insti- dL [3, 4], while 1 IU/kg of FIX would raise FIX activity by 1 tute for Biological Standards Control, South Mimms, Potters IU/dL [5, 6]. Table 1 shows the traceability of the IU for FVIII Bar UK. concentrate from the WHO 1st IS established in 1970 [7] to the 8th IS established in 2009 [8]. The availability of commercial coagulation factor con- centrates (CFC) in the late 1960s revolutionised the treat- Potency labelling strategy is through agreement be- ment of haemophilia and greatly enhanced the quality of tween regulators and the manufacturer as part of the mar- life for these patients [1]. The importance of potency stand- keting authorisation process and this can lead to different ard for compariing the activity of different products was strategies for the same product marketed in different coun- illustrated as early as 1964 by Pool and Shannon [2]. They tries. In Europe, regulators follow the specifications of the employed a pooled plasma standard obtained from the Di- European Pharmacopoeia shown in Table 2 and are legally vision of Biologics Standards, National Institute of Health in binding in the EU. In the US, potency labelling of the prod- the USA to value assign the potency of high potency FVIII ucts is agreed between the manufacturer and the US FDA, concentrates produced from cryoprecipitates. All factor VIII usually on the basis of the supporting data submitted by the (FVIII) and factor IX (FIX) products in use since the 1960s, manufacturer. This is not an issue if similar IU/vial can be from the plasma-derived intermediate purity preparations obtained by the different potency-labelling methods when and highly purified single component concentrates to prod- assayed against the current IS. For plasma-derived and ucts manufactured by recombinant technology are labelled some full-length recombinant products, because of their and dosed in International Unit (IU) supported by the World similarity to the plasma derived IS for FVIII or FIX, similar Health Organization (WHO) International Standards (IS) for potencies are obtained by either chromogenic (CH) or one- therapeutic concentrates. In parallel, there is also WHO IS stage clotting (OSC) assays. for activity in plasma and there is a close relationship be- tween the “concentrate” and “plasma” IU. The plasma IU However marked assay discrepancies for some products harmonises the measurement of activity in plasma and aids when assayed against the IS have resulted in substantial the correct diagnosis of deficiency of coagulation factors. In differences in the labelled potency of the same product for addition, it provides an important link between dosing the therapeutic concentrates and post-infusion monitoring of Table 2. European Pharmacopoeia monographs for patients. For coagulation factors, 1 IU is defined as the ac- FVIII and FIX, with potency labelling methods and tivity of the analyte found in 1 mL of normal human plasma, specification for potency i.e. 1 IU/mL (100 IU/dL) is equivalent to 100% normal. In general, a summary of product characteristics (SmPCs) indi- Monograph Potency Limits for esti- labelling mated potency Table 1. The traceability of the IU of FVIII from the 1st IS established in method (% of stated 1970 to the current 8th IS potency) Human Coagulation Chromogenic 80 – 120 WHO NIBSC Year of Purity Value Factor VIII standard code establishment assigned 07/2013:0275 IU/ampoule Human Coagulation Chromogenic 80 – 125 1st 67/19 1970 Intermediate 2.6 Factor VIII (rDNA) 2nd 73/552 1976 Intermediate 1.1 01/2008:1643 rd 3 80/556 1983 Intermediate 3.9 Human Prothrombin One-stage 80 – 125 4th 88/804 1989 Intermediate 6.3 Complex Clotting 01/2011:0554 5th 88/640 1994 High (Mab) 5.4 Human Coagulation One-stage 80 – 125 th 6 97/616 1998 Recombinant 8.5 Factor IX Clotting (full length) 01/2011:1223 7th 99/678 2003 High 11.0 Human Coagulation One-stage 80 – 125 (gel chromatography) Factor IX (rDNA) Clotting 8th 07/350 2009 High 9.4 Concentrated Soluti- (gel chromatography) on 01/2016:2522 2019 Page 19 New developments in haemophilia treatment

different countries. , aB-domain deleted substance published by the European Medicine Agency FVIII, marketed in Europe as Refacto AF and as Xyntha in the (EMA) and effective from September 2104 indicated that US, is an early example where clotting and chromogenic potency assay for each modified coagulation factor product assay discrepancy lead to global potency disparity. Both should be based on in vitro and in vivo clinical studies of brands are labelled relative to the IS for FVIII concentrate assay methods involving multiple methods and reagents which is plasma-derived, with Xyntha labelled by one stage and that labelling could be in IU-based on statistical validity clotting assay and Refacto AF by chromogenic assay. The when assayed against the relevant IS [16]. This guidance assay discrepancy is approximately 40% with chromogenic was paralleled to the recommendation by the Scientific and assays giving lower values than the one-stage clotting as- Standardisation Committee of the International Society on says. The Refacto AF product insert states ”1 IU of the Xyn- Thrombosis and Haemostasis on potency labelling of factor tha product is approximately equivalent to 1.38 IU of the VIII and factor IX concentrates [17]. A workshop involving ReFacto the EMA and manufacturers also illustrated the concerns on AF product". As product monitoring is routinely carried assay discrepancies of modified and EHL products and pro- out by one-stage clotting assay, this assay discrepancy is vided information and advice to manufacturers and clinical managed by using the Refacto laboratory standard available laboratories on approaches to potency labelling and clinical to clinical laboratories [9, 10]. monitoring [18].

The introduction of the modified and extended half-life To conclude, accurate measurement of CFC activity for (EHL) products offers similar or better efficacy with fewer potency labelling is important to ensure consistency of pro- infusions but presented standardisation challenges that duction and the efficacy of a product. The assay discrepan- impact on potency labelling and post-infusion monitoring of cies issue-related to the measurement of extended half-life these products. Assay discrepancies for EHF products are FVIII and FIX products could impact on the accuracy of po- well documented by a number of publications [11 – 15]. tency labelling of these products. Potency labelling method Recommendations are available from regulators as regards for each product has been carefully considered and selected potency labelling. The guideline on the declaration of the by the manufacturers and regulators, with the labelled po- quantitative composition / potency labelling of biological tency verified by results of clinical trials. medicinal products that contain modified proteins as active

References 1. Chtourou S. Production and clinical profile of human coagulation factor VIII. In Production of plasma proteins for therapeutics use. First Edition. Eds. Bertolini J, Goss N, Curling J. John Wiley & Sons, Inc., 2014, pp. 31 - 40. 2. Pool JG, Shannon AE. Production of high-potency concentrates of antihemophilic globulin in a closed-bag system. N Engl J Med 1965; 273: 1443-7. 3. https://www.medicines.org.uk/emc/medicine/30681 4. https://www.medicines.org.uk/emc/product/6422/smpc 5. https://mri.cts-mrp.eu/Human/Downloads/DE_H_0483_001_FinalPI_3of17.pdf 6. https://www.medicines.org.uk/emc/product/6422/smpc 7. Bangham DR, Biggs R, Brozović M, Denson KW, Skegg JL. A biological standard for measurement of blood coagulation Factor VIII activity. Bull World Health Organ. 1971;45(3):337-51. 8. https://apps.who.int/iris/bitstream/handle/10665/70137/WHO_BS_09.2117_eng.pdf;sequence=1 9. Ingerslev J, Jankowski MA, Weston SB, Charles LA. Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDD Factor VIII:C activity in hemophilic plasma using the one-stage clotting assay. J Thromb Haemost 2004; 2; 623-628. 10. Pouplard C, Caron C, Aillaud MF, Ternisien C, Desconclois C, Dubanchet A, Sobas F. the use of the new ReFacto AF laboratory standard allows reliable measure- ment of FVIII:C levels in ReFacto AF mock plasma samples by a one stage assay. Haemophilia 2011; 17: e958-e962 11. 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 hemostasis laboratories. Haemophilia 2014; 20: 294-300. 12. Hillarp A, Bowyer A, Ezban M et al. Measuring FVIII activity of glycopegylated recombinant factor VIII, N8-GP, with commercially available one-stage clotting and chromogenic assay kits: a two-centre study. Haemophilia. 2017; 23:458-465. 13. Church N, Leong L, Katterle Y, et al. Factor VIII activity of BAY 94-9027 is accurately measured with most commonly used assays: Results from an international laboratory study. Haemophilia. 2018; 24:823-832. 14. Sommer JM, Buyue Y, Bardan S, et al. Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion pro- tein (rFIXFc) activity Thromb Haemost. 2014;112: 932-940. 15. Tiefenbacher S, Bohra R, Amiral J, et al. Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol. J Thromb Haemost. 2017;15:1901-1912. 16. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-declaration-quantitative-composition/potency-labelling-biological-medicinal- products-contain-modified-proteins-active-substance_en.pdf 17. Hubbard AR, Dodt J, Lee T, Mertens K, Seitz R, Srivastava A, Weinstein M;Factor VI I I and Factor IX Subcommittee of The Scientific and Standardisation Com- mittee of The International Society on Thrombosis and Haemostasis. Recommendations on the potency labelling of factor VIII and factor IX concentrates. J Thromb Haemost. 2013 May;11(5):988-9. 18. Dodt J, Hubbard AR, Wicks SJ, Gray E, Neugebauer B, Charton E, Silvester G. Potency determination of factor VIII and factor IX for new product labelling and postinfusion testing: challenges for caregivers and regulators. Haemophilia. 2015 Jul;21(4):543-9. doi: 10.1111/hae.12634. Epub 2015 Jan 27. PubMed PMID: 25623631. Page 20 Special Issue 9

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Laboratory measurement of extended half-life FVIII products

Stefan Tiefenbacher, PhD1 and Robert C Gosselin, CLS2 recently approved. The challenges facing clinical laborato- ries, when monitoring these new-generation FVIII products 1Colorado Coagulation, Laboratory Corporation of America and when using their established factor-activity assays ,will Holdings, Englewood, CO USA be reviewed. 2Thrombosis & Hemostasis Center, University of California, Davis Health System, Sacramento, CA USA EHL products A number of EHL rFVIII products have recently entered Introduction the market in the United States and Europe for the treat- Congenital haemophilia A (HA) is a relatively rare (1 in ment of haemophilia A (Table 1). Approaches to extend the 5,000 males) X-linked disorder secondary to molecular half-life (e.g. reduce the number of infusions required to changes (inversions, point mutations, small and large dele- maintain adequate FVIII trough levels) as well as to de- tions, insertions, or duplications) in the factor VIII (FVIII) crease immunogenicity of the administered factor product, gene causing a factor VIII deficiency [1]. The severity of the include changes to protein expression systems (e.g. the use deficiency is related to the patients’ bleeding risk, with se- of human instead of animal cell lines), as well as modifica- vere FVIII deficiencies (<0.01IU/mL) having a greater risk tions to the actual therapeutic protein, including random than moderate (0.01 – 0.05IU/mL) or mild deficiencies (0.05 modification (i.e. ADYNOVATE®/ADYNOVI®, Takeda) [9] or – 0.40IU/mL) [2]. The one-stage clot assay (OSA) remains site-specific modification (i.e. JIVI®, Bayer) [10] PEGylation, the standard FVIII activity assay used by the clinical labora- glyco-PEGylation (i.e. ESPEROCT®, Novo Nordisk) [11], fusion tory to diagnose HA as well as monitor therapy and inhibi- of the FVIII protein to the fragment crystallisable (Fc) region tor development in HA [3,4]. However, to ensure the cor- of human immunoglobulin G1 (i.e. ELOCTATE®/ELOCTA®, rect diagnosis and classification of haemophilia A, both the Sanofi) [12] or single-chain modification of FVIII (i.e. AFSTY- FVIII OSA and the chromogenic substrate assay (CSA) are LA®, CLS Behring) [13] (Table 1). Half-life extensions required to arrive at the correct diagnosis and classification achieved with these modifications have been limited to 1.4- of disease severity [5]. This recommendation was due to 1.6 fold, compared to existing rFVIII products, which has observed discrepant results between the OSA and the CSA been attributed to the clearance of the EHL rFVIII products for a subset of patients with haemophilia A, likely due to being largely dependent on the clearance of von Willebrand the responsiveness of the assays to certain mutations in the Factor (vWF) [14]. More recently, novel fusion proteins de- FVIII gene [6,7]. signed to overcome this limitation such as BIVV001 (rFVIIIFc As HA results in FVIII deficiency, the common treatment -VWF-XTEN, Sanofi) [15] a rFVIII protein that among other for the past several decades has been FVIII replacement modifications includes the D’D3 domain of vWF (to provide therapy. With improving manufacturing processes (e.g. re- protection and stability of vWF while evading half-life limi- combinant FVIII, rFVIII), the safety profile for HA patients tation of endogenous vWF), have entered clinical studies. with this treatment has also improved with a reduced risk Available data on the behaviour of this “next generation” of blood-borne viral infections, in particular hepatitis C and EHL rFVIII product [15] in clinical assays used to monitor human immunodeficiency virus (HIV) [8]. However, the lim- factor replacement therapy in the clinical laboratory are not iting issue for HA treatment was the relatively short half-life yet available.

(t1/2) of FVIII (~12 hours) which requires multiple treatment For information regarding the prescribing of rFVIII EHL, doses over short periods of time to maintain adequate FVIII of particular interest to laboratorians with regard to labora- levels in order to avoid spontaneous bleeding. More recent- tory testing (Table 2), these data are usually related to dose ly, molecular modifications of the FVIII protein have in- (usually section 2 of prescribing information) and any rec-

creased the t1/2 of FVIII. These modified FVIII molecules are ommended laboratory monitoring concerns or require- commonly referred to as extended half-life (EHL) rFVIII ments (usually section 5 of prescribing information), with products. additional information available in the clinical pharmacolo- While this has been considered an improvement for HA gy section (section 12) indicating the EHL pharmacokinetics treatment, the laboratory measurement of EHL rFVIII prod- and pharmacodynamics properties [9-13]. Of particular ucts has created challenges in the clinical laboratory com- note, are the EHL prescribing recommendations indicating munity tasked with the monitoring of these factor replace- the use of a ‘validated’ method. As validation of any labora- ment products in the patient. This manuscript will provide tory method prior to clinical use is a requirement by regula- an overview of the one-stage and chromogenic FVIII activity tory agencies, this description suggests the expectation of data published for the EHL FVIII products that have been regulators for each laboratory to evaluate the performance

2019 Page 21 New developments in haemophilia treatment

Table 1. Recently Approved EHL rFVIII Products [9-13]

Trade Name Manufacturer Modification for Approval Date Other names t1/2 extension

ELOCTATE®/ELOCTA® Sanofi Fusion to Fc domain of IgG1 FDA Jun 2014 rFVIII-Fc (BDD) EMA Nov 2015 efmoroctocog alfa AFSTYLA® CSL Behring Single-chain with truncated BD FDA May 2016 CSL627 (BDD) EMA Nov 2015 lonoctocog alfa ADYNOVATE®/ADYNOVI® Takeda 20-kDa branched PEG FDA Dec 2016 BAX 855/TAK 660 EMA Jan 2018 rurinoctocog alfa pegol Jivi® Bayer Site-specific 60-kDa PEG FDA Aug 2018 BAY 94-9027 (BDD) EMA Nov 2018 damoctocog alfa pegol ESPEROCT® Novo Nordisk 40-kDa glycoPEGylation FDA Feb 2019 N8-GP (BDTrunc PEGlyated) EMA Apr 2019 turoctocog alfa pegol

BDD – B-domain deleted; BD: B-domain; PEG: Polyethylene Glycol

Figure 1A. One-stage clot-based assay method. Diluted patient Figure 1B. Chromogenic FVIII method. Patient plasma is add- plasma is mixed with FVIII-deficient plasma (DP), then standard ed to reagent, then reagents containing factor IX, APTT performed. Clotting times are compared to multipoint calcium (Ca) and phospholipids (PL). After a short incubation calibration curve using same dilution scheme. Diluent may be period for factor Xa (FXa) generation, a substrate specific for with saline, buffer or FVIII DP. Samples are typically analysed factor Xa (FXa) is added, resulting in peptide cleavage and using 2-3 dilutions to check for parallelism. Non-parallelism yellow colour formation. The amount of FXa generated is would suggest inhibitor presence. proportional to FVIII present in patient plasma.

of each particular EHL product in their local OSA and/or CSA most coagulation instruments have applications for in vitro assay system. FVIII diagnostic use, although typically the default program is reagent specific. A two-stage clot assay also exists, though Laboratory considerations for EHL rFVIII monitoring/ it is not often used as it is more complex to perform, cannot testing easily be automated, and there are no commercial kits and/ I. OSA and CA FVIII methods or instrument applications available. Fewer than 1% per The OSA is a modified, calibrated activated partial cent of clinical laboratories participating in ECAT proficiency thromboplastin time (aPTT) assay, containing the additional survey perform this test [3]. For a number of the EHL rFVIII step of taking diluted patient plasma and adding it to FVIII- products, the APTT reagent-dependent recovery in the FVIII deficient plasma (Figure 1A). It is simple, rapid, inexpensive, one-stage clot assay has been reported [16-18]. Therefore, relatively easy to automate; a large number of approved depending on the EHL rFVIII product and the aPTT reagent aPTT reagents, differing in classes of contact activators and used for FVIII testing, there is potential for either over- or phospholipid sources are available (Table 3). Additionally, under-estimating an EHL treatment using some FVIII OSA,

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Table 2. EHL prescribing information related to laboratories and laboratory testing [9-13].

EHL Select Section 2 Notes Select Section 5 Notes

ELOCTATE® Potency assignment is determined using a Monitor plasma Factor VIII activity by performing a validated test chromogenic substrate assay. A field study (e.g. one-stage clotting assay), to confirm that adequate Factor has indicated that plasma Factor VIII levels VIII levels have been achieved and maintained. can be monitored using either a chromoge- nic substrate assay or a one-stage clotting assay routinely used in US clinical laborato- ries. AFSTYLA® Each vial of AFSTYLA states the actual Monitor plasma Factor VIII activity in patients receiving AFSTYLA amount of Factor VIII activity in International using either the chromogenic assay or the one-stage clotting Units (IU) as determined by chromogenic assay, which is routinely used in US assay. One IU corresponds to the activity of clinical laboratories. The chromogenic assay result most accu- Factor VIII contained in 1 millilitre (mL) of rately reflects the clinical haemostatic potential of AFSTYLA and normal human plasma. is preferred. The one-stage clotting assay result underestimates Plasma Factor VIII levels can be monitored the Factor VIII activity level compared to the chromogenic assay using either a chromogenic assay or a one- result by approximately one-half. If the one-stage clotting assay stage clotting assay – routinely used in US is used, multiply the result by a conversion factor of 2 to determi- clinical laboratories. If the one-stage clotting ne the patient’s Factor VIII activity level. Incorrect interpretation assay is used, multiply the result by a con- of the Factor VIII activity obtained by the one-stage clotting version factor of 2 to determine the patien- assay could lead to unnecessary additional dosing, higher chronic t’s Factor VIII activity level dosing, or investigations for an inhibitor. ADYNOVATE® Potency assignment is determined using a Monitor plasma factor VIII activity by performing a validated one- one-stage clotting assay. Plasma factor VIII stage clotting assay to confirm the adequate factor VIII levels levels can be monitored clinically using a have been achieved and maintained. one-stage clotting assay. Jivi® Monitor the Factor VIII activity of Jivi in If monitoring of Factor VIII activity is performed, use a validated plasma using either a validated chromogenic chromogenic assay or a selected validated one-stage clotting as- substrate assay or a validated one-stage say [see Dosage and Administration (2.1)]. clotting assay. Laboratories intending to measure the Factor VIII activity of Jivi should check their procedures for accuracy. For Jivi, select silica- based one-stage assays may underestimate the Factor VIII activity of Jivi in plasma samples; some reagents, e.g. with kaolin-based activators, have the potential for overestimation. Therefore, the suitability of the assay must be ascertained. If a validated one- stage clotting or chromogenic assay is not available locally, then use of a reference laboratory is recommended. ESPEROCT® If monitoring of Factor VIII activity is perfor- If monitoring of Factor VIII is performed, use a chromogenic or med, use a chromogenic or one-stage one-stage clotting assay appropriate for use with ESPEROCT® clotting assay appropriate for use with [see Dosage and Administration (2)]. Factor VIII activity levels ESPEROCT®. can be affected by the type of activated partial thromboplastin time (aPTT) reagent used in the assay. Some silica-based aPTT reagents can underestimate the activity of ESPEROCT® by up to 60%; other reagents may overestimate the activity by 20%. If an appropriate one-stage clotting or chromogenic assay is not available locally, then use a reference laboratory.

which may have significant impact on patient management mogenic substrate assay (CSA), in particular with the recent when the patient receives EHL rFVIII products [19]. While approval of several of the EHL rFVIII products, has gained FVIII OSA assay protocols are available on most coagulation acceptance and is utilised in clinical laboratories around the instruments, the FVIII CSA method (Figure 1B) may not be world, and has been recommended both for diagnosis and pre-programmed on an analyser, and if available, is more the monitoring of FVIII replacement therapy in haemophilia likely specific for a particular reagent kit/platform which [20]. However, as most of the coagulation instruments are may not readily be validated or suitable for alternative rea- open systems, CSA FVIII methods can be programmed, albe- gent sources. Based on the two-stage clot assay, the chro- it with more difficulty than OSA methods, but the availabil-

2019 Page 23 New developments in haemophilia treatment

ity of standardised test protocols for commonly used coagu- Table 3. Commercial APTT reagents, activator and phospholipid lation analysers is still lacking. As suggested in recently pub- (PL) source lished factor-replacement product field studies [17,18,21] Activator PL source and ECAT proficiency surveys [3], although the implementa- tion of the CSA in clinical laboratories has increased sub- Diagnostica Stago Polyphenol Cephalin stantially, in particular in laboratories associated with the Cephascreen® (rabbit brain tissue) measurement of factor replacement therapy, as recom- Diagnostica Stago C.K.Prest Kaolin Cephalin mended by the Medical and Scientific Advisory Council (rabbit brain tissue) (MASAC) of the National Hemophilia Foundation, its utilisa- Diagnostica Stago PTT A Silica Cephalin± tion in standard clinical laboratories performing FVIII activi- ty measurements is still limited [16,17,20,22]. Hyphen Biomedical Cephen Silica Cephalin±

1 II. FVIII testing: diagnostics versus monitoring Hyphen Biomedical Cephen Silica Cephalin± Prior to the advent of EHL therapy, the use of FVIII 1 LS testing was primarily used as an aid for diagnosis of a bleed- Instrumentation Laboratory Colloidal Synthetic PL ing patient, monitoring replacement therapy, and some- HemosIL® APTT-SP silica times to assess inflammatory response (increased FVIII) and Instrumentation Laboratory Ellagic acid Synthetic PL thrombophilia evaluations (persistently elevated FVIII). All HemosIL® SynthAFax of these intended uses of the FVIII were adequately validat- ed using regional regulatory requirements including accura- Instrumentation Laboratory Non-settling Synthetic PL cy, precision, linearity (CSA) and reference interval determi- HemosIL® SynthASil Colloidal nation. The laboratory validation of the lower limit of detec- silica tion (LOD) for FVIII is critical in order to properly character- Pacific Hemostasis® 0.003% Rabbit brain PL ise the diagnosis of HA, whereas the LOD may be less critical APTT-XL Ellagic acid for factor replacement monitoring. The additional consider- Sekisui Coagpia Ellagic acid Rabbit brain PL ation of the sensitivity of the FVIII method and its applicabil- APTT-N ity to modified replacement products was first elucidated with moroctocog alfa (ReFACTO®, Pfizer) which required a Siemens Dade® 1.0 x 10-4 M Cephalin Actin® ellagic acid (rabbit brain extract) specialised calibrator to assure accurate FVIII recovery [23].

Siemens Dade® 1.0 x 10-4 M Soy phosphatides III. EHL Monitoring Actin® FS ellagic acid Field studies are EHL manufacturer-sponsored studies Siemens Dade® 1.0 x 10-4 M Soy and rabbit brain where contrived EHL samples (e.g. congenital HA- deficient Actin® FSL ellagic acid phosphatides plasma spiked with the EHL FVIII product of interest) are Siemens Pathromtin® SL Silicon Plant PL blinded, distributed and tested at select clinical laboratories dioxide (typically those that routinely perform HA testing and moni- toring) with different reagent and instrument platforms in Technoclone Siron Ellagic acid Synthetic PL§ order to gauge the recovery and responsiveness of the par- LS (aPTT Liquid) ticular EHL FVIII product in these commonly used FVIII activ- § Technoclone Siron LIS (aPTT Ellagic acid Synthetic PL ity assays. For all of these EHL rFVIII field studies, FVIII OSA Liquid) and CSA were evaluated. Results for the recovery are usual- Technoclone Dapttin TC Silica and Synthetic PL§ ly expressed as a percentage of the expected assigned or Sulphatide target value established by the EHL manufacturer. An ac- ceptable reagent has been reported to be one that recovers TriniCLOTTM Micronized PF3 reagent (rabbit Automate APTT silica brain phosphatides) within 20%-30% of expected target FVIII activity [24,25]. Upcoming recommendations from the United Kingdom TriniCLOTTM aPTT HS Micronized Porcine and chicken Haemophilia Centre Doctors’ Organisation guideline will silica phospholipids recommend ±20% recovery for FVIII activity >0.30IU/mL and TriniCLOTTM aPTT S Micronized Porcine and chicken ±30% recovery for FVIII between 0.10 – 0.30IU/mL [26]. silica phospholipids Although concerns have been raised by some to the degree of commutability of results obtained using contrived ± Cephalin is a generic term for tissue derived phospholipids – no partic- ular source disclosed;[5] Package insert states “highly purified phospho- (product-spiked) versus post-infusion samples, a recent lipids” whereas Bowyer, et al (IJLH 2010) cites “synthetic phospholipids” publication comparing contrived samples to HA post ELOC-

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Table 4. EHL rFVIII Products International, Multi-Center Field Study Results for One-Stage and Chromogenic FVIII Activity Assays [16-18,22,28]

Name Manufacturer One-Stage (aPTT) Assay Chromogenic Assay rFVIII-Fc (BDD) – ELOCTATE®/ELOCTA® Sanofi √ √

CSL627 (BDD) – AFSTYLA® CSL Behring all aPTT reagents ↓ √ (multiply OSA result x2) Bax 855 (FL) – ADYNOVATE®/ADYNOVI® Takeda √ √

BAY 94-9027 (BDD) - Jivi® Bayer APTT-SP ↓, STA-PTT-A ↓ √ CK Prest ↑, Actin FS ↑ N8-GP (BDtrunc PEGylated) – ESPEROCT® Novo Nordisk APTT-SP ↓, STA-PTT-A ↓ √A TriniCLOT™ ↓

A at upper acceptable limit of ±30%; ↓indicates under-recovery; ↑ indicates over-recovery; √ indicates acceptable recovery; BDtrunc = B-domain truncated

TA® infusion treatments demonstrated this is not likely to be more exploration [30]. For the AFSTYLA® field study, the the case [27]. OSA was found to consistently under-recover FVIII activity, The first published comparative field study for ELO- with a fairly consistent underestimation by approximately CATE®/ELOCTA® reported no significant differences between 50% [16]. As such, the prescribing information indicates FVIII:C results obtained using a number of different aPTT that when any OSA is being used, a correction factor of 2 reagents and reported acceptable although slightly higher should be utilised to estimate AFSTYLA® FVIII activity, as the recovery in the CSA [22]. In the years following this pub- study showed that with a correction factor, most laborato- lished field study, data using post-infusion samples have ries achieved the desirable recovery of AFSTYLA® within 20% confirmed these findings and suggest that ELOCTATE®/ [13,16]. The CA method suitably recovered AFSTYLA® and ELOCTA® can be successfully monitored in the clinical labor- data showed that no correction factor is required when atory using either the one-stage or the chromogenic factor using this method for monitoring. With the ESPEROCT® field activity assay. For the ADYNOVATE® field study [28], on av- study, the authors observed that most APTT reagents recov- erage slightly higher than target FVIII activities were report- ered close to expected FVIII levels, except for three silica- ed for both the OSA and CSA (OSA mean 116%, range and based reagents (APTT-SP, TriniCLOTTM, and PTT-Automate) CSA mean 114%, range 94 -124%). The authors noted that which underestimated ESPEROCT® recovery by 40-60% [17]. slightly higher recovery was measured with ellagic acid/ All chromogenic assays demonstrated comparable and ac- polyphenolic type APTT reagents compared to silica/kaolin ceptable recovery, albeit at the higher upper limit of the reagents, but the differences were not statistically signifi- acceptable ± 30% range. 17 A summary of field studies is cant [28]. In a publication by Swiss-centred laboratories, the provided in Table 4. confirmation of increased recovery using either OSA or CSA, demonstrated improved recovery using a product-specific IV. EHL FVIII testing: Other considerations calibration versus local calibration methods using commer- Although existing chromogenic assays currently in use in cial calibrators [29]. In a comparative field study published clinical laboratories have been suggested in field studies to for Jivi®, it was demonstrated that most OSA and CSA meth- recover most if not all of the currently approved EHL rFVIII ods adequately recovered expected FVIII concentrations, products within acceptable limits [16,18,22,28], the availa- but two reagents with silica activators (PTT-A and APTT-SP) bility and implementation of the chromogenic assay in clini- substantially underestimated Jivi® recovery and thus were cal laboratories have been relatively slow. This is partly due considered not to be acceptable reagents that should be to the relatively higher complexity of the assay (compared avoided [18]. Currently, a larger Jivi® field study is being to the one-stage clot assay) and the limited number of as- undertaken in conjunction with ECAT to provide any inter- say manufacturer-validated and approved applications for ested laboratory with an open-label kit containing samples many of the commonly used coagulation analysers. As a of varying Jivi® concentrations. The preliminary results re- result, many laboratories develop their own in-house appli- cently presented at the American Society of Hematology cation, likely contributing to the observed variability for this meeting confirm previous findings when using the two silica assay in field studies, that at times can exceed that of the aPTT reagents (APTT-SP and APTT-A), but also revealed an one-stage FVIII activity assay [17,21] For the laboratory, unsuitable chromogenic method (Trinichrome) that requires there is a real perception of the chromogenic assay being

2019 Page 25 New developments in haemophilia treatment

more costly, despite a recent publication suggesting other- calibrator, or alignment of the clinical assay with the assay wise [31]. In our experience, the OSA is still cheaper to use, system utilised by the manufacturer to assign the potency given that most manufacturers provide substantial cost re- to the EHL rFVIII product or the use of a chromogenic assay. ductions for FVIII-deficient plasma when coupled with the Although either of the first two approaches would theoreti- purchase of large-volume reagents (e.g. prothrombin time cally result in accurate measurement of the EHL rFVIII prod- and aPTT tests). Although CSA cost can be substantially re- uct, both would require the laboratory to know which par- duced with reagent aliquoting and freezing, instrument ticular EHL rFVIII product is received and measured and dead-space volume (amount of reagent lost due to pi- would require the use of product-specific test code for each petting limitations) can be excessive (as much as 0.3mL) per of the EHL rFVIII products being measured, therefore not aliquot, thus resulting in reagent wastage or loss. Whether presenting a reasonable or feasible option for most clinical one may argue that OSA is cheaper than CSA, the focus laboratories. Most chromogenic assays on the other hand, should be the potential institutional savings on EHL usage if based on available field study data appear to be a viable appropriate testing is provided. While the laboratory com- option, though as mentioned previously its use is currently ponent may be measured in tens of dollars, EHL costs and limited to more experienced laboratories, due to the lack of potential savings would equate to thousands of dollars, and available CSA applications for most coagulation analysers. thus makes the laboratory OSA versus CSA costs seem ra- ther trivial. Conclusions A common limitation to all field studies is that observa- The emergence of recombinant EHL rFVIII replacement tions and conclusions for particular reagent systems are products for the treatment of HA has created challenges for typically based on average responses observed across all clinical laboratories that monitor these patients. The chal- the participating laboratories, even though a substantial lenges may include the local use of any or all EHL products number of laboratories reporting EHL FVIII recovery for a with sub-optimal OSA available in the laboratory, and the particular assay reagent system may have reported results lack of instrument-ready CA kits. Those challenges with- outside (above or below) the expected threshold standing, for those clinical laboratories that provide HA ser- [16,18,22,28]. As such, these studies are useful for identify- vices, providing the optimal test method with correspond- ing particular reagent or assay systems that result in gross ing EHL product would better serve the patient and the in- over- or under-estimation of a particular EHL FVIII product. stitution. At this time, the CSA appears to be the optimal Additionally, not all APTT reagents were tested in field stud- test method that is able to accurately measure all of the ies, and reagents with similar activators could imply a par- currently marketed and approved EHL products, although ticular EHL FVIII response, so each laboratory should never- quality program results indicate an unexpected variability theless carefully evaluate their in-house FVIII measurement between laboratories, which suggests that some standardi- methods for EHL products to be used by their patient popu- sation of this testing method needs further attention. We lation. External Quality Assurance (EQA) programs (e.g. recommend the following considerations when providing ECAT) are working to provide EHL EQA samples to assist FVIII testing for EHL rFVIII monitoring: laboratories in establishing local verification of performance - Each laboratory should validate their in-house OSA of their FVIII methods in monitoring EHL-treated HA pa- or CSA method for each EHL rFVIII product ex- tients. In the absence of local validation, EHL manufacturers pected to be used by their HA patient population are developing post-market approval testing programs for - Most CSA methods appear to be suitable for meas- laboratories to send samples to a central or designated la- uring currently available EHL rFVIII products boratory for EHL testing and method comparison. A current - If these are available, laboratories should enrol in project is in place in the United States for ELOCTATE®, and EQA programs that specifically provide EHL rFVIII others EHL programs are being currently designed. material for laboratory assessment or A number of different approaches have been proposed - Laboratories should consider utilising post-market in the literature to achieve accurate measurement of EHL approval testing programs as they become availa- rFVIII products in post-infusion samples in the laboratory. ble for select EHL rFVIII products. Proposed approaches include the use of a product-specific

References:

1. Oldenburg J, Pezeshkpoor B, Pavlova A. Historical review on genetic analysis in hemophilia A. Semin Thromb Hemost. 2014;40(8):895-902. doi:10.1055/s-0034- 1395161. 2. Srivastava, A., A.K. Brewer, E.P. Mauser-Bunschoten, N.S. Key, S. Kitchen, A. Llinas, et al., Guidelines for the management of hemophilia. Haemophilia, 2013; 19: e1-47. 3. ECAT Foundation, External Quality Control for Assays and Tests Report, Survey 2018-M4, Main Labcode 9021 4. Van den Bossche D, Peerlinck K, Jacquemin M. New challenges and best practices for the laboratory monitoring of factor VIII and factor FIX replacement. Int J Lab Hem 2018; 40(Suppl 1): 21-29.

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5. Duncan EM, Rodgers SE, McRae SJ. Diagnostic testing for mild hemophilia a in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays. Semin Thromb Hemost. 2013 Apr;39(3):272-82. doi: 10.1055/s-0033-1334863. 6. Oldenburg J, Pavlova A. Discrepancy between the one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A pheno- type. Hamostaseologie 2010; 30: 207-11. 7. Pavlova A, Delev D, Pezeshkpoor B, et al. Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays. J Thromb Haemost 2014; 111: 851-61. 8. Samuelson Bannow B, Recht M, Négrier C, Hermans C, Berntorp E, Eichler H, Mancuso ME, Klamroth R, O'Hara J, Santagostino E, Matsushita T, Kessler C. Factor VIII: Long-established role in haemophilia A and emerging evidence beyond haemostasis. Blood Rev. 2019 May;35:43-50. doi: 10.1016/j.blre.2019.03.002. 9. Adynovate® prescribing information. Baxalta US Inc. Lexington, MA Issued 05/2018. 10. Jivi® prescribing information. Bayer Healthcare LLC. Whippany, NJ. Revised 08/2018. 11. Esperoct® prescribing information. Novo Nordisk Inc. Plainsboro, NJ. Revised 02/2019. 12. Eloctate® prescribing information. Bioverativ Therapeutics Inc. Waltham, MA. Revised 09/2019. 13. Afstyla® prescribing information. CSL Behring LLC. Kanakee, IL Revised 09/2017. 14. Pipe SW, Montgomery RR, Pratt KP, Lenting PJ, Lillicrap D. Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A. Blood 2016; 128(16): 2007-16. 15. Brown K, Green G. The hemophilia drug market. Nat Rev Drug Discov 2018; 17(8):541-2. 16. St Ledger K, Feussner A, Kalina U, et al. International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemo- stasis laboratories. J Thromb Haemost 2018; 16(3): 555-64. 17. Tiefenbacher S, Clausen WHO, Hansen M, et al. A field study evaluating the activity of N8-GP in spiked plasma samples at clinical haemostasis laboratories. Hae- mophilia 2019; 25(5): 893-901. 18. Church N, Leong L, Katterle Y, et al. Factor VIII activity of BAY 94-9027 is accurately measured with most commonly used assays: Results from an international laboratory study. Haemophilia 2018; 24(5): 823-32. 19. Van den Bossche D, Peerlinck K, Jacquemin M. New challenges and best practices for the laboratory monitoring of factor VIII and factor FIX replacement. Int J Lab Hem 2018; 40(Suppl 1): 21-29. 20. Medical and Scientific Advisory Council (MASAC) Document #228: MASAC statement regarding use of various clotting factor assayts to monitor factor replace- ment therapy. National Hemophilia Foundation. June 2014. https://www.hemophilia.org/sites/default/files/document/files/masac-228.pdf. Last accessed Dec 10, 2019. 21. Tiefenbacher S, Albisetti M, Baker P, et al. Estimation of Nuwiq® (simoctocog alfa) activity using one-stage and chromogenic assays – results from an internation- al comparative field study. Haemophilia 2019; 25(4): 708-17. 22. 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. 23. Pouplard C, Ternisien C, Desconclois C, Lasne D, Aillaud MF, Caron C. Discrepancies between one stage assay and chromogenic substrate assay in patients treat- ed with recombinant or plasma-derived FVIII and usefulness of a specific standard in ReFacto AF(®) -treated patients. Haemophilia. 2016;22(2):e101-e103. doi: 10.1111/hae.12867. 24. Peyvandi F, Oldenburg J, Friedman KD. A critical appraisal of one-stage and chromogenic assays of factor VIII activity. J Thromb Haemost. 2016 Feb;14(2):248- 261. 25. Bowyer AE, Hillarp A, Ezban M, Persson P, Kitchen S. Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chro- mogenic assay kits: a two-center study. J Thromb Haemost. 2016;14(7):1428-1435 26. Gray E, Kitchen S, Bowyer A, Chowdary P, Jenkins PV, Murphy P, Platton S, Riddell A, Lester W. Laboratory Measurement of Factor Replacement Therapies in the Treatment of Congenital Haemophilia A United Kingdom Haemophilia Centre Doctors’ Organisation guideline Authors. Haemophilia (in press) 27. Jennings I, Kitchen D, Kitchen S, Woods T, Walker I. The importance of commutability in material used for quality control purposes. Int J Lab Hematol. 2019;41 (1):39-45. doi: 10.1111/ijlh.12918. 28. Turecek PL, Romeder-Finger S, Apostol C, et al. A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variabil- ity of ADYNOVATE and OBIZUR in comparison with ADVATE. Haemophilia 2016; 22(6): 957-65. 29. Bulla O, Poncet A, Alberio L, Asmis LM, Gähler A, Graf L, Nagler M, Studt JD, Tsakiris DA, Fontana P. Impact of a product-specific reference standard for the measurement of a PEGylated rFVIII activity: the Swiss Multicentre Field Study. Haemophilia. 2017;23(4):e335-e339. doi: 10.1111/hae.13250. 30. MeijerP, Marlar RA, Teare JM, Adcock D. Inter-Laboratory Evaluation of the Recovery of Bay 94-9027 [Jivi®] with One-Stage Clotting and Chromogenic Assays. American Society of Hematology, San Diego 2019. 31. Kitchen S, Blakemore J, Friedman KD, et al. A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromo- genic activity assays.J Thromb Haemost 2016; 14: 757-64.

2019 Page 27 New developments in haemophilia treatment

Laboratory measurement of extended half-life FIX products

Annette E. Bowyer, PhD and Steven Kitchen, PhD ment of FIX:C is the OSA which is based on the activated par- tial thromboplastin time (APTT). This method is well de- Coagulation Department, Royal Hallamshire Hospital, scribed in the literature but briefly, the assay determines the Sheffield, UK degree of correction to the APTT of a plasma known to be deficient in FIX by the addition of diluted patient plasma, Introduction quality control (QC) plasma or reference plasma [10]. The Haemophilia B (HB) is a haemostatic disorder caused by a clotting times of the patient or QC plasma are compared to reduction of clotting factor IX activity (FIX:C). HB is classified those of the reference (calibrator) plasma to quantify FIX:C. according to the measureable level of FIX:C in plasma; se- OSA performed worldwide are not homogeneous vere HB <1 IU/dL FIX:C, moderate HB 1-5 IU/dL FIX:C and between laboratories. APTT reagents vary greatly in their mild HB 5-40 IU/dL FIX:C [1]. The risk of bleeding to the constituents; the phospholipid source, type and patient increases with decreasing FIX:C. Patients with severe concentration and the activator (ellagic acid, silica, kaolin and moderate HB may require regular suppletion of FIX with etc) used to initiate contact activation of the coagulation concentrate to prevent bleeding; this is known as process may be specific to each reagent [11]. Calcium prophylactic treatment. The half-life of FIX in plasma is 18-24 chloride concentration and plasma dilutions may also vary. A hours [2] thus prophylactic treatment is administered number of different reference plasmas are used to perform intravenously approximately every 3 to 4 days. Alternatively, the calibration curves however, these should all be patients may only be treated for active bleeding episodes; referenced to the current international standard for FIX in this is known as on-demand or therapeutic treatment. FIX plasma [12]. Different analyser systems are used to measure concentrates derived from human plasma (plasma derived the APTT of the assay incorporating several end-point FIX, pdFIX) were the initial treatment of choice from the detection systems [13]. FIX-deficient plasma may be sourced 1970s until the introduction of the first recombinant FIX from patients with severe congenital HB or can be artificially (rFIX) concentrate in 1997 [3]. Over the last decade, a num- depleted [14]. There are guidelines available for the ber of approaches have been taken to modify rFIX and in- recommended best practice for analysing OSA and crease the length of time that FIX is present in the plasma. chromogenic assays (CSA). These include testing a minimum rFIX has been combined with albumin, the Fc portion of IgG of three dilutions of the test plasma for OSA and ensuring or polyethylene glycol (PEG) [4-6] moieties. Pharmaceutical that a stored calibration curve uses the same batch of company clinical trial data show a 3-5 fold extension of reagents as the test samples [15,16]. plasma FIX half-life with these modifications [7-9]. Patients Chromogenic or amidolytic assays (CSA) for FIX:C are with HB who require prophylactic treatment with these ex- fairly new to the haemostasis laboratory but are slowly tended half-life (EHL) FIX products, may now only require increasing in popularity, mainly for the monitoring of some intravenous injections every 7-14 days which greatly increas- EHL rFIX concentrates [17,18]. Their implementation is es quality of life. generally restricted to tertiary haemostasis laboratories although they are not approved for use in some countries. Laboratory measurement of FIX:C The principle of CSA focuses on the tenase complex in which The one-stage clotting assay (OSA) is the assay required factor X (FX) is converted to FXa by activated FIX (FIXa) and by both the Food and Drug Administration (FDA) and Euro- activated FVIII (FVIIIa) in the presence of calcium ions and pean Medicines Agency (EMA) to assign the potency of each phospholipids. Commercial CSA kits contain FX, FVIII, FXIa FIX concentrate. It is this potency that is used to determine and either FV/prothrombin (Rossix, Molndal, Sweden) [19] the appropriate volume of treatment to administer to pa- or thrombin (Biophen, Neuville-sur-Oise, France) [20]. FIX is tients. Some pharmaceutical companies provide specific de- provided by the patient plasma, QC plasma or reference tails of which reagents were used in the potency assignment, plasma. Activated FX cleaves a chromophore (para- others choose to omit this useful information. nitroaniline, pNA) specific to FXa and the resultant yellow In HB patients, laboratory measurement of FIX:C is un- colour is determined by absorbance or change in delta dertaken to assess the response to a particular FIX concen- optical density (dOD) at 405-410 nm. This absorbance is trate. This information may then be used to calculate the directly proportional to the concentration of FXa which is half-life of the product in the patient, whether anti-FIX anti- directly proportional to the FIX:C in the plasma. The dOD of bodies to the product are forming, patient compliance and the patient and reference plasma are compared to quantify whether trough levels are adequate to prevent spontaneous the FIX:C. joint bleeds in severe HB. The routine method for measure-

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Table 1. Currently licensed EHL rFIX and the suitability of different one-stage and chromogenic FIX assays in the monitoring of these products.

EHL FIX OSA tested and acceptable OSA tested and not acceptable CSA tested

rFIXFc, Alprolix Actin, Actin FS, Actin FSL, APTT SP, CK Prest underestimates [29] Yes; Hyphen and Rossix Cephascreen, Pathromtin SL, STA acceptable [18, 29] PTT A, Synthafax Synthasil, [18, 29] rIX-FP, Idelvion Actin, Actin FSL, APTT SP Cephas- Actin FS, CK Prest underestimate Overestimation reported creen, Pathromtin SL, STA PTT A, [14, 18] with Hyphen and Rossix in a Synthafax, Synthasil [18, 32] single study [18] N9-GP, Refixia/Rebinyn Cephascreen and Synthafax [17, 35] Actin, Actin FS, Actin FSL and Yes; Hyphen and Rossix Synthasil underestimate acceptable [17, 35] APTT SP, Pathromtin SL overestimate [17]

Chromogenic FIX assays demonstrate acceptable bleeding. The degree of difference between the expected reproducibility in automated analysers [21], are less (target) FIX:C and measured FIX:C that is considered clinical- affected than OSA by interference with heparin or lupus ly significant has not been universally agreed. Some studies anticoagulant antibodies due to the higher plasma dilutions have considered as acceptable up to 30% difference be- used but are often perceived as complicated and costly tween the target and measured FIX:C in spiked plasma [17, compared to OSA. Historically, FVIII CSA were performed in 24]; however this would confer 60% disparity if one APTT a microtitre plate but many automated haemostasis reagent underestimated by 30% and a second APTT reagent analysers have protocols for testing both FVIII and FIX CSA overestimated by 30% in the sample plasma. The under- or [21]. A recent reported concluded that with careful reagent overestimation may not be to the same degree at trough handling including aliquoting and freezing of reagents, the levels as it is at peak FXI:C levels [24]. costs of CSA may be close to comparable with OSA [22]. There are three EHL FIX concentrates currently available Although in an emergency setting, where the CSA results for use worldwide although treatment use is variable are required urgently, it may not be possible to test a between countries. A summary of currently licensed EHL number of patients in a single run and the cost per sample rFIX concentrates and the acceptability of monitoring these will be increased. products with different one-stage and chromogenic FIX:C assays is shown in table 1. Measurement of plasma derived and recombinant FIX Fc fusion FIX products rFIXFc (Alprolix, Biogen, Cambridge, USA) is human FIX Despite the many variables in the OSA, measurement of covalently linked to the dimeric Fc domain of human pdFIX or rFIX can be accurately performed using most APTT immunoglobulin G1 (IgG1) with a 3-4 fold increase in half- reagents currently available on the market. This means that life [5]. Field studies of plasma artificially spiked with rFIXFc laboratories do not require specific details of FIX measured in laboratories worldwide concluded that kaolin- concentrate treatment prior to testing of FIX:C. CSA FIX:C activated APTT reagents in OSA underestimated rFIXFc by can be used to monitor pdFIX:C though underestimation of approximately 50% whereas silica and ellagic acid activated some rFIX concentrates has been reported with this method APTT reagents and one CSA (Hyphen Biomed) recovered [23]. close to the expected FIX:C [24,28]. Separate studies in spiked plasma and plasma from HB patients treated with Measurement of EHL FIX products rFIXFc confirmed acceptable FIX:C results with a number of It became apparent during the initial pharmaceutical APTT reagents and two chromogenic kits (Hyphen and drug trials of EHL rFIX concentrates that the modification to Rossix) and verified the underestimation with CK Prest, a rFIX may affect the recovery of FIX in OSA or CSA [24-26]. kaolin-activated APTT reagent [18,21,29]. The UK National Not all APTT reagents could accurately measure FIX:C in External Quality Assurance Scheme (NEQAS) distributed plasma artificially spiked with EHL products; some samples from severe HB plasma artificially spiked with significantly underestimated the FIX:C, whilst others rFIXFc to 76 predominately UK centres. Laboratories significantly overestimated the activity [27]. reporting results using either Rossix or Hyphen CSA, Actin Underestimation can lead to overtreatment of the patient, FS, Actin FSL and Synthasil OSA were close to the expected wasting money and concentrate. Conversely, overestima- FIX:C of 60 IU/dL however small numbers of laboratories tion can leave patients undertreated and at potential risk of returning results with CK Prest, Pathromtin SL and PTT Auto

2019 Page 29 New developments in haemophilia treatment

(n=5, 4, 3 respectively) reported medians of 48 IU/dL, 46.9 Conclusion IU/dL and 40 IU/dL [30]. It has been reported that APTT reagents that have a common activator may not recover equivalent FIX:C in the Albumin fusion FIX same EHL FIX concentrate [17,32]. In contrast to EHL FVIII rIX-FP (Idelvion, CSL Behring, King of Prussia, USA) is a concentrates, where CSA have been demonstrated to be fusion of recombinant FIX and albumin [4] with a mean suitable for monitoring all currently licensed products, there reported half-life of 102 hours [7]. During the PROLONG- is no easy solution to the issue of how to monitor EHL rFIX 9FP trial, underestimation of rIX-FP of up to 50% was products. Laboratories that cannot readily access treatment observed with Actin FS and CK Prest in the OSA [31]. This information from referring centres may have difficulty in has subsequently been confirmed in independent studies in reporting accurate results. In some centres it is possible both rIX-FP spiked plasma [32] and HB patients treated with that the routinely used APTT reagents are unacceptable for this product [18]. The aforementioned 2017 UK NEQAS measuring EHL rFIX therapies, consequently unfamiliar exercise also distributed plasma spiked with rFIX-FP at reagents or methods must be locally validated or verified nominally 60 IU/dL. They also concluded that OSA with for use. This is costly in time and resources and less Actin FS and CK Prest CSA underestimated FIX:C (median experienced laboratories may struggle to implement new 31.5 IU/dL and 32 IU/dL respectively) and CSA significantly assays. Even a laboratory that routinely uses reagents that overestimated FIX:C (median 99.4 IU/dL with Hyphen and are reported to be acceptable for use with a particular 116 IU/dL with Rossix) [30]. In a single centre study, CSA concentrate, must still perform local validation with the EHL was reported to overestimate by more than 30% the FIX:C rFIX products available in their country. Wide inter- in patients treated with rIX-FP [18]. More data is needed in laboratory variability has been reported with some reagents HB patient plasma to confirm these findings in CSA. and reliance on published data may result in local under- or overestimation [24,35]. A number of pharmaceutical GlycoPEGylated FIX companies recognise these difficulties and have N9-GP (Refixia, Rebinyn, Novo Nordisk A/S, Bagsvaerd, commissioned laboratory testing kits of plasma spiked to Denmark) is a glycoPEGylated rFIX with a reported half-life various concentrations with their EHL rFIX product of approximately 93 hours [33]. Initial pharmaceutical (Precision Biologic, Dartmouth, Canada). Laboratories can company data highlighted a variability in recovery of N9-GP use these kits to assist with confirmation of acceptability of spiked plasma with 16 APTT reagents in OSA, 30-50% their existing APTT reagents or for local verification of new underestimation of FIX:C was observed with 4 reagents and APTT or CSA reagents in the monitoring of rFIXFc and N9- overestimation of greater than 400% with 8 reagents [26]. GP. FIX:C by CSA was acceptable using 2 commercial assays. A Over the last five years there has been an subsequent two-centre study in plasma spiked with N9-GP unprecedented period of change in treatment of observed that FIX CSA and a limited number of APTT haemophilia which has impacted on haemostasis reagents could accurately measure N9-GP [17]. Rosen and laboratories. Real world studies are in progress and data is colleagues investigated the cause for the significant eagerly awaited for all three EHL rFIX concentrates. Good overestimation observed with some silica-activated APTT communication is vital between clinical colleagues and reagents and demonstrated that the PEG colocalised FIXa, laboratory staff to ensure that HB treatment is monitored prekallikrein and N9-GP on some silica surfaces [34]. This using the appropriate assay. The onus is on each laboratory enabled premature activation of N9-GP to FIXa during the to recognise which EHL rFIX products are in use within their contact activation phase of coagulation, shortened the APTT geographical area and whether their routine OSA and CSA and thus overestimated FIX:C. It is recommended that N9- reagents are able to accurately measurement them. GP is monitored by OSA using either Synthafax (Werfen) or Table 1. Currently licensed EHL rFIX and the suitability of Cephascreen (Diagnostica Stago) APTT reagents or by CSA different one-stage and chromogenic FIX assays in the using kits from Rossix or Hyphen Biomed [35,36]. monitoring of these products.

References

1. Blanchette, V, Key, NS, Ljung, R, et al., Definitions in hemophilia: communication from the SSC of the ISTH, J Thromb Haemost, 2014. 12: p. 1935-1939. 2. Bolton-Maggs, P and Pasi, K, Haemophilias A and B, Lancet, 2003. 261: p. 1801-1809. 3. Shapiro, A, Ragni, M, Lusher, J, et al., Safety and efficacy of monoclonal antibody purified factor IX concentrate in previously untreated patients with hemophilia B, Throm Haemost, 1996. 75(1): p. 30-35. 4. Metzner, HJ, Weimer, T, Kronthaler, U, Lang, W and Schulte, S, Genetic fusion to albumin improves the pharmacokinetic properties of factor IX, Thromb Haemost, 2009. 102(4): p. 634-44. 5. Peters, RT, Low, SC, Kamphaus, GD, et al., Prolonged activity of factor IX as a monomeric Fc fusion protein, Blood, 2010. 115(10): p. 2057-2064. 6. 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): p. 2333-41.

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7. Santagostino, E, Martinowitz, U, Lissitchkov, T, et al., Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemphilia B: results of a phase 3 trial, Blood, 2016. 127(14): p. 1761-1769. 8. Shapiro, AD, Ragni, MV, Valentino, LA, et al., Recombinant factor IX-Fc fusion protein (rFIXFc) demonstrates safety and prolonged activity in a phase 1/2a study in hemophilia B patients, Blood, 2012. 119(3): p. 666-672. 9. Collins, PW, Moss, J, Knobe, K, et al., Population pharmacokinetic modeling for dose setting of nonacog beta pegol (N9-GP), a glycoPEGylated recombinant factor IX, J Thromb Haemost, 2012. 10: p. 2305-12. 10. Langdell, RD, Wagner, RH and Brinkhous, KM, Effect of antihaemophilic factor on one-stage clotting tests. A presumptive assay for haemophilia and a simple antihaemophilic factor assay procedure., J Lab Clin Med, 1953. 41: p. 637-47. 11. Cartwright, IJ, Higgins, JA, Wilkinson, J, et al., Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes, J Lipid Res, 1997. 38: p. 531-545. 12. Gray, E, Barson, H, Hockley, J and Rigsby, P, Collaborative study for value assignment of the 4th international standard for factors II, VII, IX, X, plasma, 2010. Accessed. 11/6/19 13. Rathod, NN, Nair, SC, Mammen, J and Singh, S, A comparison study of routine coagulation screening tests (PT and APTT) by three automated coagulation analyzers, Int J Med Sci Publ Health, 2016. 5(8): p. 1563-1568. 14. Chantarangkul, V, Ingram, GIC, Thorn, MB and Darby, SC, An artificial haemophilic plasma for one-stage factor VIII assay, Br J Haematol, 1978. 40: p. 471-488. 15. Mackie, IJ, Cooper, PC, Lawrie, AS, et al., Guidelines on the laboratory aspects of assays used in haemostasis and thrombosis, Int J Lab Hematol, 2013. 35: p. 1- 13. 17. Bowyer, AE, Hillarp, A, Ezban, M, Persson, P and Kitchen, S, Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two centre study, J Thromb Haemost, 2016. 14: p. 1428-1435. 18. Bowyer, AE, Shepherd, MF, Kitchen, S, Maclean, RM and Makris , M, Measurement of extended half-life recombinant factor IX products in clinical practice, Int J Lab Hematol, 2019. 41: p. e46-e49. 21. Kershaw, GW, Dissanayake, K, Chen, VM and Khoo, T, Evaluation of chromogenic FIX assays by automated protocols, Haemophilia, 2018. 24(3): p. 492-501. 22. Kitchen, S, Blakemore, J, Friedman, KD, et al., A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays, J Thromb Haemost, 2016. 14(4): p. 757-64. 23. Wilmot, HV, Hogwood, J and Gray, E, Recombinant factor IX: discrepancies between one-stage clotting and chromogenic assays, Haemophilia, 2014. 20(6): p. 981-7. 24. Sommer, JM, Buyue, Y, Bardan, S, et al., Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity, Thromb Haemost, 2014. 112(5): p. 932-40. 25. St Ledger, K, Fuessner, A, Kalina, U, et al., Characteristics of rVIII-SingleChain in the one-stage and the chromogenic substrate assay: Results of an international field study, Haemophilia, 2016. 22 (Suppl. 4): p. 59. 26. Holm, PK, Sørensen, MH, Hermit, BM and Ezban, M, The activity of GlycoPEGylated recombinant FIX (N9-GP) can be measured in two-stage chromogenic and one-stage clotting assays, J Thromb Haemost, 2013. 11(s2, abstract PB3.49-1): p. 828. 27. Kitchen, S, Tiefenbacher, S and Gosselin, R, Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies, Semin Thromb Haemost, 2017. 43(3): p. 331-337. 29. Sinegre, T, Trayaud, A, Tardieu, M, Talon, L and Lebreton, A, Measuring rFIX-Fc with 19 different combinations coagulometers-reagents: A single centre study, Haemophilia, 2019. 25(S1): p. 53. 30. Kitchen, S, Jennings, I, Makris, M, et al., Chromogenic and One Stage FIX Assays in the Presence of Idelvion (rFIXFP), Alprolix (rFIXFc), Benefix and Replenine: Data from a UK NEQAS for Blood Coagulation Survey (OC 65.2), Res Pract Thromb Haemost, 2017. 1(S1): p. 124-5. 31. Santagostino, E, Voigt, C, Wolko, D, et al., Efficacy and Safety Results of Prolong-9FP Clinical Program of Recombinant Fusion Protein Linking Coagulation Factor IX with Albumin (rIX-FP) in Previously Treated Patients with Hemophilia B, Blood, 2015. 126(548): p. 32. Horn, C, Négrier, C, Kalina, U, Seifert, W and Friedman, KD, Performance of a recombinant fusion protein linking coagulation factor IX with recombinant albumin in one-stage clotting assays, J Thromb Haemost, 2019. 17(1): p. 138-148. 33. Negrier, C, Knobe, K, Tiede, A, Giangrande, P and Moss, J, Enhanced pharmacokinetic properties of a glycoPEGylated recombinant FIX: a first human dose trial in patients with hemophilia B, Blood, 2011. 118: p. 2695-701. 34. Rosen, P, Rosen, S, Ezban, M and Persson, E, Overestimation of N-glycoPEGylated factor IX activity in a one-stage factor IX clotting assay owing to silica- mediated premature conversion to activated factor IX, J Thromb Haemost, 2016. 14: p. 1420-7. 35. Tiefenbacher, S, Bohra, R, Amirel, J, et al., Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol, J Thromb Haemost, 2017. 15: p. 1-12. 36. Ezban, M, Hermit, MB and Persson, E, FIXing postinfusion monitoring: Assay experiences with N9-GP (nonacog beta pegol; Refixia® ; Rebinyn® ), Haemophilia, 2019. 25(1): p. 154-161.

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Quality Assurance of replacement therapy and future perspectives

Piet Meijer, PhD1 and Moniek P.M. de Maat, PhD2 therapy. Kitchen et al have published a study where they compared the test results of samples of patients treated 1ECAT Foundation, Voorschoten, The Netherlands with different FVIII products (Advate, Kogenate FS and 2Department of Hematology, Erasmus MC, University Medi- Refacto AF) in 52 haemophilia centres in the United Kingdom cal Center Rotterdam, The Netherlands [10]. Differences in test results have been reported, depend- ing either on the use of one-stage clotting assay or chromo- Introduction genic assay or the type of reagent used in the one-stage External Quality Assurance (EQA) is an important and clotting assay. Also the type of calibrator used may affect the indispensable part of the total quality management system test result. Another study showed that for B-domain deleted in the laboratory and addresses the analytical quality of la- recombinant FVIII (Refacto AF) the results with the one-stage boratory testing. The main objective of EQA is to establish assay were approximately 30% higher than the results ob- the trueness of measurement. However, EQA can also play tained with chromogenic assays [11]. This difference could an important role in revealing different types of problems in be reduced by using a B-domain deleted FVIII calibrator. laboratory testing, such as, for instance, differences between Kitchen et al also reported a post-infusion study on FIX reagents in detecting specific abnormalities or medication replacement therapy [12]. Even for FIX, differences between effects [1-3]. one-stage clotting reagents as well as differences between In this contribution we focus on the current role of one-stage and chromogenic assays have been reported.

quality assurance for replacement therapy for Factor VIII These differences also depend on the type of FIX product. (FVIII) and Factor IX (FIX) and future perspectives in EQA with These studies emphasise the need for regular external respect to the new developments in haemophilia treatment. quality assessment for post-infusion monitoring. This may advantage laboratories by providing insight into their accura- Diagnostic testing cy of measurement in post-infusion samples as well as re- Haemophilia patients are classified according to their vealing problems with particular laboratory methods and/or level of FVIII or FIX (table 1) [4]. reagents. Because of the relationship between the classification of a haemophilia patient and treatment, accurate Extended Half-life products measurement of FVIII and/or FIX is important. It has been With the introduction of extended half-life FVIII and FIX demonstrated that there is a wide variation in laboratory products for the treatment of haemophiliac patients new test results when samples of severe, moderate or mild challenges for accurate laboratory measurement arose. haemophilia patients are distributed in EQA surveys [5-8]. These are extensively discussed elsewhere in this Special Between-laboratory variation in test results may vary Issue. between 15% and 40% for mild haemophilia patient samples and up to 80 – 100% for severe patient samples. This may Factor IX also have an impact on the correct classification of patients In a joint survey by the ECAT Foundation with the [9]. Further improvement and standardisation in laboratory UKNEQAS Blood Coagulation (United Kingdom) and the RCPA testing is therefore needed. Quality Assurance Program (Australia) 3 different extended half-life FIX products (Idelvion, Alprolix and Refixia) were, Monitoring of treatment together with Benefix, distributed to laboratories worldwide Laboratory monitoring of factor replacement therapy is [13]. For each of the products samples of 0.06 IU/mL and essential for establishing the appropriate dosing strategy [4]. 0.60 IU/mL were prepared by spiking congenital-deficient FIX Also here accuracy in measurement is essential. So far only plasma with the respective products. The concentrations limited data is available on the inter-laboratory were established on the basis of the potency of the product comparability of laboratory test results for replacement given by the respective pharmaceutical companies. In total 172 participants returned results. The overall results for the Table 1. Classification of haemophilia patients one-stage assays (table 2) are given below. Differences were observed between the results of the Severity Clotting Factor Level (IU/mL) one-stage assay for APTT reagents used. No specific pattern Severe < 0.01 could be observed in the response of the different reagents Moderate 0.01 – 0.05 to the extended half-life products. This implies that for none of the APTT reagents a similar recovery could be observed Mild 0.05 – 0.40 for all included extended half-life FIX products. Table 3 shows a summary of the comparison of the median value

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Table 2. Summary table one-stage clotting assay result

One-stage Clotting Assay Results Product Benefix Idelvion Alprolix Refixia Assigned FIX level (IU/mL) * 0.06 0.60 0.06 0.60 0.06 0.60 0.06 0.60 N 161 164 160 164 161 164 162 74 Median (IU/mL) 0.08 0.64 0.06 0.39 0.07 0.50 0.20 1.32 Range (IU/mL) 0.04 – 0.21 0.49 – 0.90 0.01 – 0.11 0.18 – 0.77 0.01 – 0.19 0.27 – 0.80 0.01 – 1.45 0.13 – 10.6 CV(%) 25.2 12.4 25.2 29.8 35.8 19.1 168.7 178.8

* Based on potency

Table 3. Summary table one-stage clotting assay result

Benefix Idelvion Alprolix Refixia Assigned FIX level (IU/dL) * 6.0 60 6.0 60 6.0 60 6.0 60 Reagent IL HemosIL APTT-SP ▲ ≈ ≈ ▼ ≈ ▼ ▲▲▲ ▲▲▲ IL HemosIL Synthasil ▲ ≈ ≈ ▼ ▲ ▼ ▼ ▼ Siemens Actin FS ▲ ≈ ▼▼ ▼▼ ▲ ≈ ▼▼ ▼▼ Siemens Actin FSL ▲▲ ▲ ≈ ▼ ▲▲ ▲ ≈ ▼ Siemens Pathromtin SL ▲ ≈ ▲ ▼ ▼ ▼ ▲▲▲ ▲▲▲ Stago Cephalin/Kaolin/CK Prest ▲ ▲ ▼ ▼▼ ▼ ▼ ▼▼ ▼▼ Stago Cephascreen ▲ ▲ ▲ ≈ ▲ ≈ ≈ ▼ Stago PTT (automate) ▲ ▲ ▼ ▼ ▼ ▼ ▲▲▲ ▲▲▲ Tcoag TriniCLOT APTT HS ▲ ≈ ▼ ▼ ▼ ▼ ▲▲▲ ▲▲▲ Tcoag TriniCLOT APTT S ▲ ▲ ≈ ▼ ≈ ▼ ▲▲▲ ▲▲▲

Legend: ▼/▲: > 10 and < 50% under- or overestimation; ▼▼/▲▲: > 50 and < 100% under- or overestimation; ▲▲▲: > 100% overestimation

obtained for each of the most frequently used APTT The highest between-laboratory variation was observed for reagents and the target value based on the potency the one-stage assay for Refixia (169 - 179%), due to the labelling. large variation in test results between different APTT rea- In this survey the participants had also the possibility of gents. The between-laboratory variation for the chromo- reporting results for chromogenic testing. Table 4 shows the genic assay (22.8 – 25.7%) with Refixia is quite similar to overall results for the chromogenic assays. those of the other products. From these data on the be- For the chromogenic assays a summary is given of the tween-laboratory variation it becomes clear that, even comparison of the median value and the target value based when on average a recovery close to 100% can be observed, on the potency labelling (table 5). the variation between laboratories using the same reagent For both the chromogenic assays that are currently can be substantial. It is therefore important that individual used, differences between the products were observed. laboratories investigate their accuracy of measurement for But In general with the chromogenic assays fewer differ- each product in use in their hospital. ences were observed than with the one-stage assays. Another aim of this survey was the assessment of the Factor VIII between-laboratory variation. Overall, the between- Another approach was chosen for investigating in a mul- laboratory variation for the chromogenic assay is lower than ticentre survey the recovery of BAY 94-9027 (damoctocog for the one-stage assay. The lowest between-laboratory alfa pegol; Jivi®). ECAT set up a study in which FVIII-deficient variation was observed for Benefix (OSA: 12.4 – 25.2%; CA: plasma was spiked with different levels of BAY 94-9027, 17.6 – 25.4%). The between-laboratory variations for ranging from 0.05 – 1.5 IU/mL. The spiked samples were Idelvion (OSA: 29.8 – 40.5%; CA: 21.8 – 22.2%%) and Alpro- lyophilised. The target values were established using the lix (OSA: 19.1 – 35.8%; CA: 19.7 – 26.5%) are comparable. same assay procedure used by Bayer for potency labelling

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Table 4. Summary table chromogenic assay result

Chromogenix Assay Results Product Benefix Idelvion Alprolix Refixia Assigned FIX level (IU/mL) * 0.06 0.60 0.06 0.60 0.06 0.60 0.06 0.60 N 40 40 40 40 40 40 40 40 Median (IU/mL) 0.04 0.45 0.07 0.73 0.04 0.45 0.05 0.54 Range (IU/mL) 0.03 – 0.08 0.31 – 0.68 0.03 – 0.12 0.23 – 1.22 0.03 – 0.08 0.33 – 0.74 0.02 – 0.09 0.23 – 0.97 CV(%) 25.4 17.6 21.8 22.2 26.5 19.7 25.7 22.8

* Based on potency

Table 5. Summary table chromogenic assay result

Benefix Idelvion Alprolix Refixia FIX level (IU/dL) * 6.0 60 6.0 60 6.0 60 6.0 60 Reagent Hyphen ▼ ▼ ≈ ▲ ▼ ▼ ▼ ▼ Rossix ▼ ▼ ▲ ▲ ≈ ▼ ≈ ≈

Legend: ▼/▲: > 10 and < 50% under- or overestimation

of the product. Laboratories were asked to measure the On the basis of published field studies it was the percep- samples according to their standard procedures for OSA and tion that extended half-life products could be reliably meas- CA. Currently, samples were distributed to 139 laboratories ured with chromogenic assays (see for an overview the con- in 13 different countries with 80 laboratories returning re- tribution of Stefan Tiefenbacher and Robert Gosselin in this sults which are included in the current data analysis. For Special Issue). However, this international survey demon- each of the samples, the recovery with respect to the target strates that with the Trinichrom method a significant under- value was calculated. estimation was observed, while the Siemens method Figure 1 and 2 show the recovery for the most frequently showed a concentration-dependent recovery pattern. Sam- used one-stage clotting reagents and chromogenic assays. ples in the low range showed an underestimation. From these results it is clear that a significant under- The between-laboratory variation varied for the different recovery is obtained with the Stago PTT-A reagent. methods by between 13% and 22%. The between-laboratory variation ranged for different This approach with a sample set containing a wide range reagents from 12% to 36%. The results were similar for the of FVIII or FIX products can be very helpful for laboratories FIX survey; this indicates that even though the mean recov- for investigating whether their one-stage or chromogenic ery for a particular reagent can be close to 100%, the recov- assay produces the appropriate recovery of a certain extend- ery for an individual laboratory can still deviate significantly ed half-life product. from 100%.

Figure 1. The Recovery by FVIII level of the BAY 94-9027 Figure 2. The Recovery by FVIII level of the BAY 94- Spiked Samples for the most frequently used One-Stage 9027 Spiked Samples for the different Chromogenic Assays in the Study. Assays in the Study.

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even when the consensus model on the level of reagents is Figure 3. The distribution of FIX results with a single APTT used, this consensus value can differ significantly from the reagent and one of the products (example taken from [13]). target value on the basis of the potency labelling. This is demonstrated in the example shown in figure 3. The consensus value for this reagent is 50 IU/dL, while the target value on the basis of the potency labelling is 60 IU/dL. If we take as an example result 1 (R1), 40 IU/dL, this result deviates 20% from the consensus value and 33% from the target value. If the same calculation is made for sample 2 (R2), 60 IU/dL, the deviation is respectively 20% and 0%. This indicates that the performance assessment of an indi- vidual laboratory can be strongly influenced by the type of target value used. We propose for external quality control for drug moni- toring the use of the target values assessed with the same method used for the potency labelling of the drug.

Future perspective The current new developments in the treatment of hae- External Quality Control for drug monitoring mophiliacs have significant implications for the laboratory To establish the assigned value in external quality con- [15]. New technologies or modified assays may need to be trol surveys the most widely used concept is the calculation introduced. This has also implications for the range of EQA of the consensus value on the basis of the participants’ re- services. So far, most of the EQA programmes focus in their sults [14]. Also in the surveys of the ECAT Foundation this regular services on laboratory tests used for the diagnosis concept is used. The question is whether this concept is also of patients. Only in special surveys is attention paid to drug suitable for external quality control on the monitoring of monitoring for haemophilia patients. Because of the extended half-life products. From table 2 and 4 it is clear importance of accurate measurement not only in the that the overall median value (= consensus value) can differ diagnosis of patients but also in monitoring treatment, EQA significantly from the target value on the basis of the organisations should also introduce EQA services for this on potency labelling. However when the results are evaluated a regular basis. EQA organisations may play also a role in on the level of the individual reagents the consensus value the provision of sample sets spiked with different drug lev- can be similar or close to the target value (table 3 and 5). els to be used for the validation and/or verification of labor- This demonstrates that only assigning a consensus value for atory tests. each individual reagent could be a suitable model. However In addition, it important that EQA organisers pay atten- this can only be done when a sufficient number of tion to the interference of new drugs into regular laborato- participants are using the same reagent. In our ECAT ry tests by organising special educational surveys. surveys the minimum number of 10 participants is used. But

References 1. Libeer, J.C., H. Baadenhuijsen, C.G. Fraser, P.H. Petersen, C. Ricos, D. Stockl, et al., Characterization and classification of external quality assessment schemes (EQA) according to objectives such as evaluation of method and participant bias and standard deviation. External Quality Assessment (EQA) Working Group A on Analytical Goals in Laboratory Medicine. Eur J Clin Chem Clin Biochem, 1996; 34: 665-78. 2. Sciacovelli, L., S. Secchiero, L. Zardo and M. Plebani, External Quality Assessment Schemes: need for recognised requirements. Clin Chim Acta, 2001; 309: 183-99. 3. Jones, G.R., The role of EQA in harmonization in laboratory medicine - a global effort. Biochem Med (Zagreb), 2017; 27: 23-29. 4. Srivastava, A., A.K. Brewer, E.P. Mauser-Bunschoten, N.S. Key, S. Kitchen, A. Llinas, et al., Guidelines for the management of hemophilia. Haemophilia, 2013; 19: e1-47. 5. Jennings, I., D.P. Kitchen, T.A. Woods, S. Kitchen, I.D. Walker and F.E. Preston, Laboratory performance in the World Federation of Hemophilia EQA programme, 2003- 2008. Haemophilia, 2009; 15: 571-7. 6. van Moort, I., P. Meijer, D. Priem-Visser, A.J. van Gammeren, N.C.V. Pequeriaux, F.W.G. Leebeek, et al., Analytical variation in factor VIII one-stage and chromogenic assays: Experiences from the ECAT external quality assessment programme. Haemophilia, 2019; 25: 162-169. 7. Mammen, J., S.C. Nair and A. Srivastava, External quality assessment scheme for hemostasis in India. Semin Thromb Hemost, 2007; 33: 265-72. 8. Favaloro, E.J., P. Meijer, I. Jennings, J. Sioufi, R.A. Bonar, D.P. Kitchen, et al., Problems and solutions in laboratory testing for hemophilia. Semin Thromb Hemost, 2013; 39: 816-33. 9. van Moort, I., M. Joosten, M.P. de Maat, F.W. Leebeek and M.H. Cnossen, Pitfalls in the diagnosis of hemophilia severity: What to do? Pediatr Blood Cancer, 2017; 64. 10. Kitchen, S., I. Jennings, M. Makris, D.P. Kitchen, T.A. Woods and I.D. Walker, Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII. Haemophilia, 2016; 22: 806-12. 11. Ingerslev, J., M.A. Jankowski, S.B. Weston, L.A. Charles and P. ReFacto Field Study, 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: 623-8. 12. Kitchen, S., E. Gray and K. Mertens, Monitoring of modified factor VIII and IX products. Haemophilia, 2014; 20 Suppl 4: 36-42. 13. Nederlof, A., S. Kitchen, P. Meijer, M.H. Cnossen, N.A. Pour, G. Kershaw, et al., Performance of FIX Extended Half-life product measurements in External Quality Control Assessment programmes.) Submitted). 14. Coucke, W. and M.R. Soumali, Demystifying EQA statistics and reports. Biochem Med (Zagreb), 2017; 27: 37-48. 15. Tripodi, A., V. Chantarangkul, C. Novembrino and F. Peyvandi, Advances in the Treatment of Hemophilia: Implications for Laboratory Testing. Clin Chem, 2019; 65: 254- 262. 2019 Page 35 New developments in haemophilia treatment

List of corresponding authors:

Julie L Tarrant MBChB, MRCP(UK) FRCPath(Haem) Department of Pathology & Molecular Medicine, Queen’s University 99 University Ave, Kingston, Ontario K7L 3N6, Canada Email: [email protected]

Wolfgang Miesbach, MD Medical Clinic 2, Institute of Transfusion Medicine, University Hospital Frankfurt Theodor-Stern-Kai 7, 60590 Frankfurt Am Main, Germany Email: [email protected]

Marjon Cnossen, MD, PhD Department of Pediatric Hematology, Sophia Children’s Hospital, Erasmus University Medical Center, The Netherlands Postbus 2060, 3000 CB Rotterdam, The Netherlands. Internal postal address Sp 2435 Email: [email protected]

Armando Tripodi, PhD Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa Via Pace 9, 20122 Milano, Italy. Email: [email protected].

Elaine Gray, PhD Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards Control South Mimms, Potters Bar UK Email: [email protected]

Annette Bowyer, PhD Department of Coagulation, Section Lead Assays and Haemophilia, Royal Hallamshire Hospital S10 2JF, Sheffield, UK Email: [email protected]

Stefan Tiefenbacher, PhD Colorado Coagulation 8140 Upland Drive, Suite 100, Englewood CO 80112, USA Email: [email protected]

Piet Meijer, PhD ECAT Foundation P.O. Box 107, 2250 AC Voorschoten, The Netherlands Email: [email protected]

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