S. A. Bevan

A survey of one-stage and chromogenic potencies in therapeutic factor VIII concentrates.

Methods-based potency discrepancies in factor VIII (FVIII) concentrates can lead to confusion when the method used for product labelling differs from the method used for regulatory batch release or the testing of post-infusion plasma samples. A previous survey reported substantial discrepancies (‡ 15%) between one-stage and two-stage clotting potencies, in 7 out of 13 concentrates tested (Barrowcliffe et al, 1990). The present survey was undertaken as there have been changes in the products, standards and assay technology over the last 10 years. The use of recombinant products has increased considerably although there is continued use of both intermediateand high-purity plasma-derived products. Recently, the choice of recombinant FVIII concentrates has widened with the introduction of the B-domain-deleted product (Sandberg et al, 2001). The methodology for potency estimation has also evolved with the publication of recommendations for the assay of highpurity FVIII concentrates (Barrowcliffe, 1993) and the replacement of the two-stage clotting method with the recommended chromogenic method (European Pharmacopoeia (EP), 2002). In the present survey we tested several batches of 10 different products licensed for use in the UK using both the one-stage clotting and chromogenic methods (Table I). Seven of the products were plasma-derived (codes 1–7) and three were recombinant (codes 8, 9, 10). One-stage clotting assays were carried out using the Instrumentation Laboratory activated partial thromboplastin time (APTT) reagent (APTT-SP liquid) and artificially depleted FVIIIdeficient plasma containing normal levels of von Willebrand Factor (Organon Teknika Ltd, Cambridge, UK). Chromogenic assays were carried out using the Chromogenix Coatest FVIII:C ⁄ 4 kit. All potencies were estimated relative to the EP Human Coagulation Factor VIII concentrate standard biological reference preparation (batch 1), which consists of intermediate purity FVIII concentrate and has good agreement between the one-stage clotting potency (mean 6Æ30 IU per vial) and the chromogenic potency (mean 6Æ22 IU per vial) (Barrowcliffe, 1996). In all assays both the standard and test concentrates were prediluted in FVIII-deficient plasma (Organon Teknika Ltd) and further diluted in buffer containing human albumin (10 mg per ml), in accordance with the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC) recommendations (Barrowcliffe, 1993). Potencies between methods differed by £ 7% for 7 out of the 10 products, although there were significant differences (paired t-test; P < 0Æ05) for 3 of these products (coded 2, 4, 7). The largest discrepancies were found with two immunopurified products (coded 5 and 6), manufactured using Method M technology (Addiego et al, 1992), in which the mean one-stage potency exceeded the mean chromogenic potency by 33% and 24% respectively. A large discrepancy was also found with the B-domain-deleted recombinant product (coded 10) in which the mean one-stage potency was 22% lower than the mean chromogenic potency. In comparison with the previous survey (Barrowcliffe et al, 1990), the present survey found generally closer agreement between the different assay methods for the majority of products. This was most striking with the results

Activation profiles of factor VIII in concentrates reflect one-stage/chromogenic potency discrepancies

Summary.  We have investigated the possibility that differences in the profile of factor VIII (FVIII) activation, by thrombin, may help to explain the one‐stage/chromogenic potency discrepancies in two therapeutic concentrates. A Method M concentrate and a recombinant B‐domain‐deleted (B‐DD) concentrate were found to have one‐stage/chromogenic ratios of approximately 1·15 and 0·70, respectively, relative to the World Health Organization (WHO) 6th International Standard (IS) FVIII concentrate, whether pre‐diluted in FVIII‐deficient plasma or buffer (± von Willebrand factor, VWF). The activation of FVIII, by thrombin, was followed in a buffer medium (± VWF) and all three concentrates showed similar times to reach peak FVIII coagulation (FVIII:C) activity. However, despite the use of equivalent amounts of FVIII:C for all three concentrates, the B‐DD concentrate reached a higher peak level and maintained higher FVIII:C compared with the WHO 6th IS throughout the incubation period, whereas the Method M concentrate reached a lower peak level and maintained lower FVIII:C throughout the incubation period. We propose that the higher levels of FVIII:C found with the B‐DD concentrate and the lower levels with the Method M concentrate, following activation, may be reflected in the potencies obtained by the chromogenic method and may be consistent with one‐stage/chromogenic ratios of < 1·0 and > 1·0 respectively.

A collaborative study to establish the 7th International Standard for Factor VIII Concentrate

Summary.  A candidate concentrate, preparation N (99/678), was assayed and calibrated, as a potential replacement, against four established factor (F) VIII concentrate standards: the current WHO 6th International Standard (IS) (97/616), the previous 5th IS (88/640), the Mega 1 standard and Ph. Eur. BRP Batch 2 standard, in a collaborative study involving 38 laboratories. All laboratories were instructed to use the ISTH/SSC recommendations, including predilution of concentrates in FVIII‐deficient plasma. Several laboratories performed more than one assay method and altogether there were 27 sets of assays with the one‐stage method, 31 with the chromogenic method, and 18 with both methods. There was good agreement between laboratories using each of the two methods for comparison of preparation N against the four established standards, with overall potencies by one‐stage and chromogenic methods differing only by less than 2%. However, there were significant differences in potencies relative to the different standards, ranging from 10.1 IU per ampoule against the Ph. Eur.BRP2 to 11.4 against the WHO 6th IS. Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity per year of less than 0.001% at the recommended storage temperature of −20 °C. Various options for potency of preparation N were considered by the participants and by members of the ISTH/SSC FVIII/FIX Subcommittee. In November 2003, preparation N (NIBSC 99/678) was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 7th International Standard for Factor VIII Concentrate with an assigned potency of 11.0 IU per ampoule.

Factor VIII heavy chain polypeptides in plasma and concentrates

Summary. Factor VIII polypeptides in plasma and FVIII concentrates have been analysed by an electrophoretic technique based on that of Weinstein et al (1981). Samples were complexed with radiolabelled anti‐FVIII Fab, and the immunocomplexes visualized by SDS‐polyacrylamide electrophoresis. The technique visualized FVIII heavy chain polypeptides in all types of samples, including plasma, without further purification.

Value assignment of the WHO 6th International Standard for blood coagulation factor VIII and von Willebrand factor in plasma (07/316)

Harmonization in the diagnosis andmonitoring of the bleeding disorders hemophilia A and von Willebrand disease (VWD) relies on the availability of the World Health Organization International Standard (WHO IS) for factor (F)VIII and von Willebrand factor (VWF) in plasma, which provides the primary definition of the International Unit (IU) for five analytes in human plasma [factor VIII:coagulant activity (FVIII:C), factor VIII:antigen (FVIII:Ag), VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCo) and VWF:collagen binding, (VWF:CB)]. The WHO IS provides a common traceable source of calibration in IU for secondary plasma standards throughout the world and the annual demand exceeds 800 ampoules. Depletion of the stocks of the WHO 5th IS (02/150), which was established in 2003 [1], made it necessary to prepare a replacement standard and this report describes the value assignment of the WHO 6th IS. The candidate WHO 6th IS (07/316) was prepared from recovered plasma from 80 normal healthy donors, pooled and buffered with HEPES, to a final concentration of 40 mmol L, before freeze drying under conditions suitable for the preparation of international biological standards [2]. Value assignment of the proposed WHO 6th IS was achieved through assays relative to the WHO 5th IS in an international multi-center study involving 44 laboratories from 14 countries which comprised 20 clinical laboratories, 21manufacturers and three regulators. Estimates for FVIII:C, relative to the WHO 5th IS, produced similar mean values for all three methods (0.67 IU per ampoule by 1-stage clotting, n = 31; 0.72 IU per ampoule by 2-stage clotting, n = 1; 0.70 IU per ampoule by chromogenic, n = 20) and there was good agreement between laboratories for the overall combined mean of 0.68 IU mL [n = 52, geometric coefficient of variation (GCV) 4.06%] (Table 1). Estimates for FVIII:Ag, relative to theWHO 5th IS, showed good agreement between laboratories with a GCV of 4.24% and an overall mean of 1.04 IU mL (n = 10). Estimates for VWF:Ag, relative to theWHO 5th IS, showed excellent agreement between the ELISA (mean 1.00 IU per ampoule, n = 21) and immuno-turbidimetric (mean 1.00 IU per ampoule, n = 10) methods and produced an overall mean of 1.00 IU mL (n = 31) with low inter-laboratory variability (GCV 4.29%) (Table 1). Estimates for VWF:RCo relative to the WHO 5th IS by the automated aggregometric methods were less variable (mean 0.86 IU per ampoule, GCV 6.52%) but not significantly different to estimates by manual visual agglutination (mean 0.90 IU per ampoule, GCV 12.0%). A combination of all estimates relative to the 5th IS produced a mean of 0.87 IU mL (n = 28) with acceptable inter-laboratory variability (GCV) of 8.13%. Estimates for VWF:CB, relative to the WHO 5th IS, were associated with very good agreement between laboratories using type 3 (mean 1.04 IU per ampoule) and type 1/3 mix collagen reagents (mean 1.02 IU per ampoule) and produced an overall mean of 1.03 IU mL (n = 21) with an inter-laboratory variability (GCV) of 5.80%. Estimates relative to local normal plasma pools were also calculated for the proposedWHO 6th IS as a check on possible drift of the IU away from the plasma unit where 1 IU mL represents the amount of analyte in 1 mL of fresh, normal pooled plasma (Table 1). However, these estimates have a limited value as the normal pools differ between laboratories and between collaborative studies. Moreover, there was a significant difference (P < 0.0001) for FVIII:C estimates in the proposed WHO 6th IS where mean values of 0.57 and 0.72 IU mL were calculated relative to fresh and frozen local pools, respectively. This was consistent with the loss of FVIII:C on freeze-thawing of the local pools. Estimates for all analytes, calculated relative to the locally collected normal plasma pools, Correspondence: Anthony R. Hubbard, Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. Tel.: +44 1707 641318; fax: +44 1707 641050. E-mail: anthony.hubbard@nibsc.hpa.org.uk Journal of Thrombosis and Haemostasis, 9: 2100–2102 DOI: 10.1111/j.1538-7836.2011.04471.x

Value assignment of the WHO 2nd International Standard von Willebrand factor, concentrate (09/182)

To cite this article: Hubbard AR, Hamill M, Beeharry M, Bevan SA, Heath AB, on behalf of the SSC sub‐committee on von Willebrand factor of ISTH. Value assignment of the WHO 2nd International Standard von Willebrand factor, concentrate (09/182). J Thromb Haemost 2011; 9: 1638–40.DOI: 10.1111/j.1538‐7836.2011.04365.x.

Long-term stability of the Scientific and Standardization Committee Secondary Coagulation Standard (SSC Lot no. 3)

Under the auspices of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (SSC/ISTH) a secondary plasma coagulation standard has been made available to manufacturers with the objective of improving the harmonization of labeled analytes in commercial plasma calibrants. The SSC/ISTH Secondary Coagulation Standard Lot no. 3 (SSC Lot no. 3) consists of vials each containing 1 mL of pooled normal human plasma, lyophilized, and has been assigned values for 19 coagulationrelated analytes by assay relative to the relevant World Health Organisation International Standards (WHO IS) [1]. The present report describes the stability of SSC Lot no. 3 based on studies of accelerated degradation at elevated temperatures and real-time storage at the bulk storage temperature of )20 C which have been undertaken over a 6-year period in order to support the continued validity of the assigned values as despatch commenced in July 2006. The standard (SSC Lot no. 3) was prepared from a plasma pool comprising 86 donations from normal, healthy donors, collected by plasmapheresis, each tested and found negative for viral markers for hepatitis B virus, hepatitis C virus and human immune-deficiency virus 1 and 2. The pooled plasma was filled in 1mL aliquots into 55 000 rubber-sealed, screw-capped glass vials and lyophilized. Mean residual moisture after freezedrying was 0.101%. Four analytes [factors (F)V, VII, VIII and XI] underwent testing in two different laboratories (NIBSC, Potters Bar, UK and Royal Hallamshire Hospital, RHH, Sheffield, UK). FV and VII were estimated using thromboplastin-based one-stage clotting methods and FXI was estimated using activated partial thromboplastin time (APTT)-based one-stage clotting methods; FVIII was measured using the two-stage clotting method (RHH) and the chromogenic method (Chromogenix Coatest SP4 kit) (NIBSC). Stability was assessed through an accelerated degradation studywhich involved storage of vials of SSCLot no. 3 at elevated temperatures (+4,+20, +37,+45 C) since December 2003 with periodic sampling and residual potency estimation relative to vials stored at the reference temperature ()20 C) after 2.2, 4.2 and 6.2 years storage. Each testing timepoint involved four independent potency estimates for each analyte by each laboratory using different vials in each assay. Predictions of degradation rate were calculated according to the Arrhenius equation (ACDWin software version 8.18, P K Phillips) based on the assumption that degradation results from a uni-molecular decay process where the degradation at higher temperatures only differs from degradation at lower temperatures in terms of rate [2]. Real-time stability was assessed by the comparison of vials of SSCLot no. 3 stored at)70 Crelative to vials stored at)20 C after 6.2 years (eight independent estimates for each analyte). Results from the two laboratories for samples stored at elevated temperatures agreed very closely in the estimates of residual potency for all four factors. Combined mean relative residual potency for vials stored at +4 C for 6.2 years exceeded 90% for FV, FVII and FXI with the greatest loss observed for FVIII with 83% residual potency (Table 1a). For the higher storage temperatures (20, 37, 45 C) the greatest loss was consistently found for FV and FVIII with a combined residual potency of 61% and 57%, respectively, after 6.2 years at 20 C and 6% and 17%, respectively, after 4.2 years at 37 C. All data points for the residual relative potencies fitted the Arrhenius model extremely well with good agreement between the observed and predicted loss. The predicted mean percent loss per year for vials stored at temperatures ranging from )20 to+37 C is given in Table 1b. All four factors were associated with a mean predicted loss of < 0.1% per year at )20 C. Predicted losses for storage at all of the higher temperatures confirmed that FV and FVIII were less stable than FVII and FXI. An estimate of the precision of the predictions can be obtained from the upper 95% CI of loss as Correspondence: Anthony R. Hubbard, Haemostasis Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK. Tel.: +44 1707 641318; fax: +44 1707 641050. E-mail: anthony.hubbard@nibsc.hpa.org.uk Journal of Thrombosis and Haemostasis, 9: 1246–1248 DOI: 10.1111/j.1538-7836.2011.04290.x

An international collaborative study to calibrate the WHO 2nd International Standard for Ancrod (15/106) and the WHO Reference Reagent for Batroxobin (15/140): communication from the SSC of the ISTH

Ancrod and batroxobin are both thrombin-like snake venom serine proteases produced by Calloselasma rhodostoma and Bothrops atrox moojeni, respectively. Details of the mechanism of fibrinogen binding and action have been published for batroxobin [1] and ancrod [2]. Because ancrod and batroxobin produce fibrin that is readily degraded they promote fibrinogen depletion and have been investigated as potential treatments to reduce normal clot formation under various circumstances. Both enzymes have been investigated as treatments for ischemic stroke; however, clinical trials over several decades have shown mixed results [3,4]. Ancrod has other indications and is currently in clinical trials for treatment of sudden sensorineural hearing loss (SSHL). Because batroxobin is insensitive to plasma inhibitors, it has found uses in clinical laboratories as a diagnostic reagent to measure batroxobin clotting time (Reptilase time) [5], which is useful when plasma samples contain heparin for example. Many batroxobin products are available globally under a variety of brand names, for example Reptilase, Hemocoagulase and Defibrase. The WHO 1st International Reference Preparation (IRP) for Ancrod (74/581) was established in 1977 by the WHO Expert Committee on Biological Standardization (ECBS) [6]. Preparation 74/581 was first established as the British Standard for ancrod with an activity of 55 units per ampoule and part of this batch was made available for use as an IRP. The WHO ECBS adopted the national unit, defining the international unit (IU) as the activity contained in one ampoule of 74/581. Efforts to standardize batroxobin activity at NIBSC began in 1978 with the establishment of the 1st British Standard (BS), 75/527. The original unit was based on the plasma clotting unit (PCU), where 2 PCU were defined as ‘the amount of enzyme in 0.1 mL which clots 0.3 ml of citrated plasma in 19 0.2 s’. The 2nd BS was calibrated against the 1st BS [7], but it was decided to develop a WHO Reference Reagent (RR) for batroxobin as a step towards making a WHO IS. The calibration of a candidate WHO RR for batroxobin and a candidate replacement WHO IS for ancrod were included in the same collaborative study.

An international collaborative study to establish the World Health Organization 2nd International Standard for Factor VII Concentrate: communication from the SSC of the ISTH

The 1st International Standard (IS) for factor VII concentrate (97/592) was established by the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) in 1998 [1]. A potency of 6.3 IU was assigned to the preparation with clotting and chromogenic methods, relative to the 2nd IS FII, FVII, FIX and FX plasma (94/746) and normal plasma pools. This reference material has been used for potency assignment to human coagulation FVII concentrate preparations used to treat FVII deficiency, and for FVIIcontaining prothrombin complexes used for reversal of anticoagulant treatment. As stocks of the 1st IS are low, an international collaborative study was organized to calibrate a replacement.