Potency labeling of novel factor VIII and factor IX concentrates: past experience and current strategy.

Various strategies to produce "longer-lasting" factor VIII and factor IX concentrates through chemical and genetic modifications are currently under evaluation. It is now clear that these new molecules are amenable to testing using conventional methods for biological activity (one-stage clotting and chromogenic) and there is a preference to maintain labeling in International Units (IU) traceable to the WHO International Standard Concentrates. This is an achievable goal; however, many of the new molecules are associated with potency discrepancies both between methods and also within methods, for instance, when different activated partial thromboplastin time reagents are used. In the interests of global harmonization, it is important for licensing authorities to reach agreement on the choice of the potency labeling method. This choice should be supported by a thorough characterization of product potency, both in vitro and in vivo, to anticipate future issues and with a view to maintaining equivalence of the IU compared with existing licensed products. In cases where the product potency is defined using specific reagents, the robustness of the manufacturer's product standard will be crucial for product consistency. The sensitivity of measured potency to different methodologies will require manufacturers to provide guidance to clinical laboratories on suitable postinfusion testing approaches.

International reference standards in coagulation.

Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.

International biological standards for coagulation factors and inhibitors.

The use of international biological standards during the last 30 years has proved extremely successful in promoting global harmonization of estimates between laboratories and methods. Experience has led to the identification of physical criteria essential for standards to be suitable for long-term use. High precision of liquid filling coupled with low residual moisture and oxygen and the use of sealed glass ampoules have been found consistent with homogeneous and stable International Standards (ISs). Most plasma coagulation factors and inhibitors are calibrated in International Units (IU), which are defined as the amount of analyte in 1 mL of normal pooled plasma. Adoption of the IU has provided clarity in the definition of normal and abnormal states and has facilitated dose calculation for replacement therapy. The assay of like-versus-like materials (e.g., concentrate versus concentrate) has been found to improve interlaboratory agreement and there are now both plasma and concentrate ISs available for many coagulation factors and inhibitors. Studies into the assay of recombinant factor VIII have indicated that additional measures, such as modifications to assay methodology, are necessary to reduce interlaboratory variability. This experience may prove valuable in the future, when we have to deal increasingly with the challenges to standardization associated with the products of bioengineering.

von Willebrand factor standards for plasma and concentrate testing.

The wide range of plasma von Willebrand factor (vWF) levels in the normal population makes vWF an ideal candidate for calibration in World Health Organization International Standards (WHO IS). Calibration of the WHO IS Plasma for vWF:antigen (vWF:Ag), vWF:ristocetin cofactor activity (vWF:RCo), and vWF:collagen-binding activity (vWF:CB) addresses the need to reconcile the original definition of the International Unit (IU) in fresh normal plasma with continuity between replacement freeze-dried preparations, whereas calibration of the WHO IS Concentrate aims to maintain equivalence between the IU used for the estimation of vWF in plasma (diagnosis) and the IU used for the potency labeling of therapeutic concentrates (replacement therapy). The latter objective was achieved through the calibration of the WHO 1st IS vWF Concentrate, for vWF:Ag and vWF:RCo, by assay directly relative to the WHO IS Plasma. Calibration of the WHO IS Concentrate for vWF:CB was not possible due to large interlaboratory and intermethod discrepancies, and resolution of this problem may require the use of recommended methodology. There is evidence that the use of the WHO IS Concentrate leads to considerable reduction in interlaboratory variability for vWF estimates in therapeutic concentrates and hence the "assay like versus like" principle should be applied.

A multi-centre collaborative study on the potency estimation of ReFacto.

Seven laboratories estimated factor VIII coagulant activity in recombinant B-domain-deleted (ReFacto) and plasma-derived FVIII concentrates (Octonativ-M) using chromogenic methods relative to the WHO 6th International Standard FVIII Concentrate (WHO 6th IS), European Pharmacopoeia BRP#2 (EP#2) and the ReFacto Laboratory Standard (RLS). Significantly higher estimates were obtained for all batches of product when calculated relative to the RLS in comparison with estimates vs WHO 6th IS and EP#2. Mean estimates for two batches of ReFacto product vs the RLS were within 10% of the labelled potency whereas estimates vs WHO 6th IS and EP#2 ranged from 21 to 31% lower than the label. Conversely, mean estimates for Octonativ-M relative to WHO 6th IS and EP#2 were within 10% of the label whereas the mean estimate vs RLS was 117% of label. Mean estimates for the ReFacto product, vs the WHO 6th IS and EP#2, varied considerably between the different chromogenic kits whereas estimates vs the RLS showed good agreement between kits. Mean estimates for the RLS vs the WHO 6th IS (8.10 IU/vial) and the EP#2 (7.66 IU/vial) were lower than the assigned value of 9.4 IU/vial. The results are consistent with ReFacto and full-length FVIII responding differently to variations in assay methodology and also indicate that the assigned value on the RLS may be too high. Since this study the unitage on the RLS has been adjusted to effectively increase the amount of ReFacto in the product by 20%.

Standardisation of von Willebrand Factor in therapeutic concentrates: calibration of the 1st International Standard for von Willebrand Factor concentrate (00/514).

An international study involving 26 laboratories assayed two candidate von Willebrand Factor (VWF) concentrates (B and C) for VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCo) and VWF:Collagen binding (VWF:CB) relative to the 4th International Standard Factor VIII/VWF Plasma (4th IS Plasma) (97/586). Estimates of VWF:Ag showed good agreement between different methods, for both candidates, and the overall combined means were 11.01 IU/ml with inter-laboratory variability (GCV) of 10.9% for candidate B and 14.01 IU/ml (GCV 11.8%) for candidate C. Estimates of VWF:RCo showed no significant difference between methods for both candidates and gave overall means of 9.38 IU/ml (GCV 23.7%) for candidate B and 10.19 IU/ml (GCV 24.4%) for candidate C. Prior to the calibration of the candidates for VWF:CB it was necessary to calibrate the 4th IS Plasma relative to local frozen normal plasma pools; there was good agreement between different collagen reagents and an overall mean of 0.83 IU per ampoule (GCV 11.8%) was assigned. In contrast, estimates of VWF:CB in both candidates showed large differences between collagen reagents with inter-laboratory GCV's of 40%. Candidate B (00/514) was established as the 1st International Standard von Willebrand Factor Concentrate by the WHO Expert Committee on Biological Standardisation in November 2001 with assigned values for VWF:Ag (11.0 IU/ampoule) and VWF:RCo (9.4 IU/ampoule). Large inter-laboratory variability of estimates precluded the assignment of a value for VWF:CB.

Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.

Factor VIII (FVIII) is assayed by one-stage and two-stage clotting methods and by chromogenic methods, although the chromogenic method has largely replaced the two-stage clotting assay. Clinical plasma samples are assayed mostly by one-stage assays, but most manufacturers of concentrates use the chromogenic method, which is more precise and is the reference method of the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). For most plasma-derived concentrates, assays against the World Health Organization (WHO) concentrate standard give similar results with the one-stage and chromogenic methods, but for products produced by the "method M" monoclonal antibody process, the one-stage potency is 25 to 30% higher than the chromogenic potency. For full-length recombinant products assayed against a plasma-derived concentrate standard, one-stage potencies are about 10% lower than chromogenic potencies, but for the B-domain deleted recombinant product ReFacto, the discrepancy is larger-from 20 to 50%. These discrepancies emphasize the need for an international methodology for labeling of concentrates. In ex vivo assays of hemophilic plasmas after infusion of concentrates, large discrepancies are found among laboratories and with different assay methods when a plasma standard is used. In most studies, the chromogenic potencies are higher than the one-stage potencies, and the discrepancy is highest for recombinant products. This discrepancy can be largely eliminated by the use of concentrate standards, diluted in FVIII-deficient plasma, to assay postinfusion plasma samples.

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

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.