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 intermediate- and 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 high-purity 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 FVIII-deficient 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 6·30 IU per vial) and the chromogenic potency (mean 6·22 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).
|Product||Description||Assay method for labelling||n||1-stage/chromogenic potency (%)|
|3||Ion exchange||1-stage clotting||9||103|
|8||Recombinant – full chain||1-stage clotting||4||101|
|9||Recombinant – full chain||1-stage clotting||3||101|
|10||Recombinant – B-domain-deleted||Chromogenic||4||78*|
Potencies between methods differed by ≤ 7% for 7 out of the 10 products, although there were significant differences (paired t-test; P < 0·05) for 3 of these products (coded 2, 4, 7). The largest discrepancies were found with two immuno-purified 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 on the full chain recombinant concentrates in which discrepancies of 20% and 35%, respectively, were previously reported, whereas, in the present survey, we report excellent agreement between the one-stage and chromogenic potencies. These differences in results relate to the implementation of the SSC recommendations in the present survey rather than any change in the characteristics of the concentrates (Barrowcliffe et al, 1998). The large discrepancies found with concentrates 5, 6 and 10, despite compliance with the SSC recommendations, could reflect true differences in FVIII at the molecular level rather than the effect of assay procedure. In the case of concentrates 5 and 6, the discrepancy could be indicative of some degree of FVIII activation induced during manufacture whereas, with concentrate 10, the discrepancy may reflect different behaviour induced by the B-domain-deletion mutation. In summary, the present survey indicated that 3 out of 10 products used in the UK display method-based potency discrepancies that exceeded 20%. The effect of assay method on measured potency needs to be recognized when such products are assayed. Moreover, the potency that more closely reflects clinical effectiveness has still to be established.