The other four FVIII products displayed pronounced inter-assay variability. less potent in the APTT (83% of standard) and product PH-797804 C (contains plasma FVIII) was less potent in FXase assays (66%). The ELISA immunoassay revealed that the specific activity of FVIII proteins in products A-C and E varied over a wide range (3,900-13,200 U/mg) and was higher for most lots than that for the standard (5,000 U/mg), whereas the specific activity of product D (contains plasma FVIII) was lower than expected (3,200-4,800 U/mg). Conclusions 1) FVIII potency estimated in different assays gives dissimilar results; 2) the specific activity of FVIII in various FVIII products is different and inconsistent. Thus, the administration of an equal FVIII potency in models means the administration of different amounts of FVIII protein, which may partly explain apparent discrepancies in product performance. hemostasis. The chromogenic assay, in turn, uses relatively high concentrations of thrombin for FVIII activation at high (nM) preexistent FIXa concentrations. Under physiological conditions, however, factor VIII is activated by extremely low amounts of thrombin produced [22] in the presence of competing substrates and low (pM) concentrations of FIXa. In this study, we evaluated five pharmacologic FVIII products (3 lots of each) made PH-797804 up of both natural and recombinant FVIII proteins in one commercially available (APTT) and in two in-house (FXase and synthetic coagulation proteome [23,24]) activity-based assays. The FVIII protein content in these products was estimated using an in-house immunoassay (ELISA), which allows the quantitation of FVIII antigen in human plasma and in pharmacologic products [25]. The comparison of FVIII products thus entails both potency and specific activity based quantitative analyses. Materials PH-797804 and Methods Materials Human coagulation factors VII, X, IX, and prothrombin were isolated from fresh frozen plasma using the methods of Bajaj [26] and FXI using the monoclonal anti-FXI antibody -FXI-2. All preparations were purged of trace contaminants and traces of active enzymes as described [24]. Human FV and AT-III were isolated from freshly frozen citrated plasma [27,28]. Citrated plasma for protein purification and von Willebrand factor (vWF) were purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Albumin-free rFVIII and FVIII products Rabbit Polyclonal to ACSA A-E were commercially acquired and provided by Baxter Healthcare Corp. (Duarte, CA). Products A and B contain full-length rFVIII, products C and D contain plasma-derived FVIII and product E contains truncated rFVIII. The 7th International Standard FVIII Concentrate (NIBSC code:99/678) was purchased from the National Institute for Biological Standards and Control (Hertfordshire, UK). In all experiments, recombinant full-length albumin-free FVIII produced in Chinese Hamster Ovary cell line was used as a standard. The concentration of this product (0.62 mg/ml) was established by the absorbance at 280 nm using an extinction coefficient E0.1% value of 1 1.3 [29]. rTF1-242 (expressed in [23] and van ‘t Veer [24]. Relipidated TF at 5 pM final concentration was added to the mixture of factors V, VII, VIIa, IX, X, and XI, prothrombin, TFPI and AT-III (all at mean physiologic concentrations) and FVIII at either 1 U/ml (indicated by manufacturer) or 0.7 nM (established by ELISA) in HBS, 2 mM CaCl2 made up of 2 M PCPS. Thrombin generation over time was measured in a chromogenic assay using PH-797804 Spectrozyme TH and a Molecular Devices (Sunnyvale, CA) THERMOmax microplate reader. Results Properties and quantitation of the rFVIII standard The molar concentration of the full-length albumin-free rFVIII (rFVIII standard) based upon absorbance at 280 nm was quantitated by functional activity, which provided 1:1 stoichiometry with FIXa decided in the intrinsic FXase assay (Physique 1A). Titration of this rFVIII standard in substrate plasma at concentrations ranging from 21 pM (0.03 U/ml) to 0.63 nM (0.9 U/ml) gave APTT clotting occasions similar to those observed for corresponding concentrations of the 7th International Standard FVIII Concentrate (Determine 1B). It was also established by gel electrophoresis and western blotting that this rFVIII standard is of a high purity, made up of no degradation products beyond the routine heterogeneity of the heavy chain [33] (Physique 1C). Open in a separate window Open in a separate window Open in a separate windows Fig. 1 (A) Binding stoichiometry.