Pharmacokinetics of two pasteurized Factor VIII concentrates by different and multicenter assays of Factor VIII activity

We assessed the pharmacokinetic characteristics of a new high-purity pasteurized FVIII concentrate in comparison with an intermediate purity pasteurized concentrate, produced by the same manufacturer. The study was designed as a cross-over single-dose pharmacokinetic investigation in 8 non-bleeding patients with severe hemophilia A. All patients were given 25 IU/kg of each of the two concentrates, with an interval of at least one week between the two administrations. Decay curves were assessed by collecting 10 serial blood samples over 36 hours following the end of infusion. The concentration of Factor VIII in blood samples was determined in triplicate in three different laboratories using each of the following assay methods: a one-stage clotting assay, a two-stage clotting assay, and a two-stage chromogenic-peptide substrate assay. All pharmacokinetic parameters were calculated by model-independent methods. The two products were found to differ significantly both in the clearance, which was on average 13.8% lower for Haemate P, and in the in-vivo recovery, which was 11.7% lower for Factor VIII:C P on the average. In comparison with previous pharmacokinetic data obtained from other heated Factor VIII concentrates, the clearance of Haemate P was found to be significantly slower, while the half-life of both products was longer. No differences were observed in the Vd-area. These findings indicate that the purification procedures to which both products are subjected do not increase the in-vivo rate of plasma disappearance of Factor VIII.     

Validation of a procedure for potency assessing of a high purity factor VIII concentrate--comparison of different factor VIII coagulant assays and effect of prediluent

The assessment of factor VIII coagulant activity (FVIII:C) in recently available highly purified and concentrated FVIII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVIII:C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVIII:C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVIII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVII-deficient plasma used as prediluents. In order to validate our "in vitro" data we performed 6 "in vivo" analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.     

In vivo recovery of factor VIII: a comparison of one-stage and two-stage assay methods.

The recovery and half-life of VIII:C in the plasma of severely haemophilic patients was measured by one-stage and two-stage assays after injection of two Factor VIII concentrates (Hemofil, Hyland and Fraction I-O, Kabi). Plasma volumes were measured with an Evans' Blue technique, and both concentrates and post-infusion samples were measured against the same plasma standard. There was a highly significant difference in recoveries estimated by the two assay methods. The one-stage assays gave the most consistent results, in that the average recovery was 100%, whereas the two-stage assays gave only about 80% of the value expected from in vitro assays. There was no differences in recoveries between the two concentrates. The two-stage assays gave a slightly shorter half-life than the one-stage assays, and the half-life of Hemofil was also shorter than that of Fraction I-O.     

Pharmacokinetics of recombinant factor VIII (recombinate) using one-stage clotting and chromogenic factor VIII assay

In a study designed to demonstrate the safety and pharmacokinetics of a recombinant factor VIII (Recombinate) manufactured in Andover, MA and Thousand Oaks, CA, two different methods of factor VIII assay (one-stage clotting and Chromogenic substrate) were compared in vivo. The study was performed in four centres in the UK: London, Oxford, Cardiff and Manchester. Two pharmacokinetic studies, at least one week apart, were performed in 30 patients with severe haemophilia A (VIII:C < 2 IU/dl). A dose of 50 IU/kg was administered with sampling pre-infusion, and +0.25, 0.5, 1, 3, 6, 9, 12 and 24 h post-infusion. The aggregate 60 pharmacokinetic study showed a half-life of 12.7 and 13.0 h (p = 0.28) and recovery of 127 and 161 IU/dl (p = 0.0001) using one-stage clotting or chromogenic substrate respectively. In a supplementary experiment, 20 post-infusion samples were re-assayed by 1-stage and chromogenic assay using two plasma (20th British plasma standard and an "in-house" pooled normal plasma) and two concentrate standards, derived from the same type, but different batch of infused concentrate (Recombinate) and pre-diluted in either individual pre-infusion sample or in pooled commercial haemophilic plasma. The use of the Recombinate concentrate standard overcame the significant difference in FVIII levels between 1-stage and chromogenic assay methods when a plasma standard was used (p <0.0001). It is concluded that where potency dosing designation is carried out by an assay system different to that used in the clinical situation, the use of the recombinant concentrate as a standard in post-infusion plasma samples is likely to give more reliable and reproducible results.     

A collaborative study to establish a Korean Standard for factor VIII:C concentrate

BACKGROUND AND OBJECTIVES: A collaborative study among five laboratories including three manufacturers and two national control laboratories was carried out to evaluate the suitability of a candidate to serve as a Korean Standard for factor VIII:C concentrate. MATERIALS AND METHODS: Two approaches were attempted to determine the potency of this candidate. The one is a one-stage clotting assay and the other is a chromogenic assay. To achieve acceptable precision and accuracy, the following recommendations by the International Society on Thrombosis and Haemostasis were adopted in the assays, e.g., pre-dilution of samples in factor VIII (FVIII)-deficient plasma, inclusion of 1% albumin in the dilution buffer and calibration against the sixth International Standard for the blood coagulation factor VIII:C concentrate, coded 97/616. RESULTS: The data collected within each laboratory and among laboratories for both assays employed here were in good agreements in the calibration of the candidate preparation against the International Standard. The overall potencies by the one-stage clotting assay and the chromogenic assay, however, showed recognizable differences between them. Each differed from each other in that the potency obtained from chromogenic assay was approximately 17% lower than that from one-stage clotting assay. The estimated geometric mean value obtained from the one-stage clotting assay was 8.4 international units (IU)/vial and that from the chromogenic assay was 6.7 IU/vial. CONCLUSIONS: Based on the results of this collaborative study, the candidate standard is judged to be suitable to serve as a Korean National Standard for factor VIII:C concentrate.     

Thrombin potentiation of factor VIII procoagulant activity: assessment by the two-stage assay

Factor VIII (FVIII) procoagulant activity is the function of a plasma glycoprotein that is missing or inactive in patients with classic hemophilia. Numerous studies have shown that trace thrombin causes rapid enhancement followed by gradual inactivation of FVIII procoagulant activity. Recent evidence suggests that thrombin activation of the FVIII/von Willebrand factor (vWF) protein is required for inactivation to occur. All of these studies have used the one-stage partial thromboplastin time to assay FVIII activity. Other investigators have used the two-stage assay of FVIII activity and have been unable to demonstrate thrombin-induced enhancement of FVIII activity, although inactivation has consistently occurred. We performed experiments designed to help resolve this disagreement, using the two-stage assay specifically modified to detect thrombin potentiation of FVIII activity. The length of the first-stage incubation time was found to be critical in demonstrating the initial effect of thrombin on FVIII activity. Taking advantage of this finding we were able to show a 4.1 +/- 0.5-fold enhancement of FVIII activity upon incubating purified FVIII/vWF with 0.04 NIH unit thrombin per ml. The apparent enhancement of FVIII activity declined with increasing thrombin concentration. Incubation with 0.08, 0.16, and 0.32 NIH unit thrombin per ml resulted in only 3.2 +/- 0.5, 2.6 +/- 0.5 and 1.5 +/- 0.3-fold enhancement, respectively, of FVIII activity. As with results from the one-stage assay, activation was followed by slow inactivation of FVIII/vWF. Using the two-stage assay we also showed 100% inactivation and 100% inhibition of FVIII activity by plasmin and human anti-FVIII IgG, respectively. Plasmin inactivation of FVIII activity showed a dose-response effect. Thrombin was unable to activate plasmin-degraded FVIII/vWF. Our results show that thrombin potentiation of FVIII activity is easily demonstrable in the two-stage assay. These findings support the contention that activation of FVIII activity by thrombin is prerequisite for inactivation and underscore the importance of thrombin activation of FVIII/vWF in the intrinsic clotting system.     

A collaborative study to establish the 6th International Standard for factor VIII concentrate.

A study was carried out to replace the 5th WHO International Standard (IS) for factor VIII concentrate, because of depletion of stocks. Two candidate concentrates (X and Y) were assayed as potential replacements against the 5th IS for FVIII concentrate, in a collaborative study involving 33 laboratories. Collaborators were asked to use the ISTH/SSC recommendations, including pre-dilution of concentrates in FVIII deficient plasma in their assays. Several laboratories performed more than one assay method and altogether there were 21 sets of assays with the one-stage method, 6 with the two-stage method and 26 with the chromogenic method. There was good agreement between laboratories using each method for the comparison of concentrates X and Y against the 5th IS, but the overall potencies by one-stage and chromogenic methods each differed by approximately 5% from the overall mean, with the chromogenic potency approximately 10% higher than the one-stage. Inter-laboratory agreement was slightly better for concentrate Y than X, and stability studies indicated that Y was more stable than X. After considering all the information, together with comments from participants and from the FVIII/FIX Subcommittee of the ISTH/SSC, candidate Y (NIBSC code [97/616]), was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 6th International Standard for Factor VIII Concentrate with an assigned potency of 8.5 IU/ampoule.     

Reproducibility of one-stage, two-stage and chromogenic assays of factor VIII activity: a multi-center study

This study was aimed at assessing the reproducibility of Factor VIII assays between different laboratories using the same reagents. A total of 176 post-dose plasma samples were obtained from 8 Italian subjects with hemophilia-A treated with a single dose of Factor VIII concentrates. Three laboratories (in FRG, Italy, and Sweden) participated in the study. Frozen aliquots of each sample were dispatched to each of the laboratories, where the aliquots were assayed using the same one-stage, two-stage and chromogenic methods. The one-stage and the chromogenic methods were well reproducible between the three centers: pairwise correlation analyses yielded r-values ranging from 0.88 to 0.91 for the one-stage method and from 0.91 to 0.96 for the chromogenic method. The agreement between these two assays was less evident in samples with activity below 200 IU/L in which the one-stage gave, on average, higher Factor VIII concentrations than those provided by the chromogenic method. The two-stage method was not well reproducible, and the pairwise r-values ranged from 0.48 to 0.73. Our study emphasises the need to develop multi-center quality control programs to verify the reproducibility of Factor VIII assays.     

Issues with the assay of factor VIII activity in plasma and factor VIII concentrates

A review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VII concentrates such as Hemofil M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements. it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor VIIIa during the assay process.     

Standardization of Factor VIII-IV. Establishment of the 3rd International Standard for Factor VIII: C concentrate

An international collaborative study was carried out to establish a replacement for the current (2nd) international standard for Factor VIII:C, concentrate. Twenty-six laboratories took part, of which 17 performed one-stage assays, three performed two-stage assays and six used both methods. The proposed new standard, an intermediate purity concentrate, was assayed against the current standard, against a high-purity concentrate and against an International Reference Plasma, coded 80/511, previously calibrated against fresh normal plasma. Assays of the proposed new standard against the current standard gave a mean potency of 3.89 iu/ampoule, with good agreement between laboratories and between one-stage and two-stage assays. There was also no difference between assay methods in the comparison of high-purity and intermediate purity concentrates. In the comparison of the proposed standard with the plasma reference preparation, the overall mean potency was 4.03 iu/ampoule, but there were substantial differences between laboratories, and the two-stage method gave significantly higher results than the one stage method. Of the technical variables in the one-stage method, only the activation time with one reagent appeared to have any influence on the results of this comparison of concentrate against plasma. Accelerated degradation studies showed that the proposed standard is very stable. With the agreement of the participants, the material, in ampoules coded 80/556, has been established by the World Health Organization as the 3rd International Standard for Factor VIII:C, Concentrate, with an assigned potency of 3.9 iu/ampoule.     

An effect of predilution on VIII:C determination by the one-stage assay

Factor VIII concentrates are usually prediluted in Owrens buffer, before use of the one-stage assay for VIII:C determinations. We found that predilution of high purity Factor VIII concentrates in fresh VIII:C deficient plasma gave VIII:C estimates about 3 times higher than predilution in Owrens buffer. Predilution in reconstituted lyophilized VIII:C deficient plasma gave VIII:C estimates about 1.6 times higher. Likewise the VIII:C in a plasma sample can be estimated to e.g. 0.03, 0.06 or 0.10 IU/ml depending on whether the reference curve is established by predilution of the reference plasma in fresh VIII:C deficient plasma, in reconstituted lyophilized VIII:C deficient plasma or in Owrens buffer, respectively. It is shown that the major part of the differences between the effects of the various prediluents on the VIII:C determinations, can be accounted for by differences in the content of Factor V, Fibrinogen, and vitamin K dependent coagulation factors in the assay mixtures.     

The assay of factor VIII: C in heparinized plasma: a polybrene neutralization method

Factor VIII coagulant activity (VIII:C) has been shown by several investigators to exhibit increased stability in vitro when physiological levels of plasma ionized calcium are maintained by anticoagulation with heparin rather than citrate. An increase in initial activity of VIII:C in heparin over that of VIII:C in citrate has been reported but this has not been confirmed. In order to assay VIII:C in heparinized plasma, the heparin anticoagulant effect must be excluded without interfering with the validity of the assay. A one-stage clotting assay for VIII:C has been developed where heparin is neutralized by Polybrene, a synthetic polymerized quaternary ammonium salt. VIII:C may be accurately measured by this method which satisfies the requirements for a valid assay of parallelism and linearity.     

Assay of AHF concentrates and standards: Failure to eliminate variability with a monographed assay

Previous calibration studies have shown a high interlaboratory variability in the potency of the proposed Office of Biologics (National Center for Drugs and Biologics, US-FDA) AHF standard relative to the 2nd International Standard for Factor VIII (Factor VIII:C) (WHO 73/552). This led to the formation of an Industry Collaborative Study group whose objective was to reduce the assay variability. The group, in collaboration with the Office of Biologics and the National Institute for Biological Standards and Control (UK), designed a study based on a monographed one-stage assay protocol, which specified all materials, assay methods, equipment, dilution technique, reagents, assay order, and calculation methodology. All participants received common reagents and samples, with the exception of substrate plasma. It was felt that substrate plasma could not be a common reagent in a monographed assay. However, each laboratory prepared substrate plasma according to the protocol. All data were analyzed by an independent statistical staff. Preparations assayed included two 10-donor plasma pools, the 2nd International Standard for Factor VIII (WHO 73/552), the proposed OoB Lot A internal standard, the participants' own house standards, and commercial AHF concentrate material. The results show a statistically insignificant reduction in the interlaboratory variability, but intralaboratory consistancy was generally maintained. The study shows that monographing an assay for Factor VIII standardization does not significantly reduce the interlaboratory variability. The most likely source of this variability was not identified in this study.     

In vivo recovery and survival of monoclonal-antibody-purified factor VIII concentrates.

In response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.     

Disparity between one- and two-stage factor VIII assays in measuring VIIIC in heparinized plasma

The level of Factor VIII procoagulant activity (VIIIC) was found to be more than 30% higher in heparinized plasma than in citrated plasma from the same donor when determined by a one-stage assay (1.34 U/ml versus 1.0 U/ml). However, after Al(OH)3 adsorption and two-stage assay, the VIIIC levels were not significantly different for the two types of plasma (0.90 +/- 0.25 U/ml versus 0.82 +/- 0.23 U/ml). If a one-stage assay was employed after adsorption, a similar low result was obtained (0.92 +/- 0.21 U/ml versus 0.91 +/- 0.12 U/ml). This indicated that the adsorption step itself was responsible for the difference in results. Parallel analyses of other VIII markers demonstrated that more VIIIC antigen (VIIICAg) was removed by adsorption of heparinized rather than citrated plasma (0.33 U/ml versus 0.12 U/ml) although there was no difference in the level of VIII-related antigen (VIIIRAg). The results show that adsorption of heparinized plasma prior to either one- or two-stage assay leads to a greater loss of both VIIIC and VIIICAg activity than observed with citrated plasma and indicate that, when applied to heparin plasma the two-stage assay for VIIIC underestimates the actual VIIIC content.     

Clinical application of a chromogenic substrate method for determination of factor VIII activity

A chromogenic substrate kit for the determination of factor VIII activity (COATEST Factor VIII) has been evaluated in five different laboratories, one of them using a semi-automated procedure. This chromogenic method was compared to one-stage clotting assays for factor VIII determination in plasmas from healthy subjects, carriers of hemophilia A, severe, mild and moderate hemophilia A as well as von Willebrand's patients. In all these cases, a high correlation between these two methods was obtained (r = 0.96-0.99, n = 385) with a good agreement of the assigned potencies at all levels of factor VIII. A good correlation (r = 0.94) was also obtained for the levels of factor VIII after infusion of concentrates in six severe hemophiliacs or after administration of DDAVP to von Willebrand's patients. The chromogenic method is insensitive to preactivation of factor VIII by thrombin, thus yielding valid potency assignments also in these situations. The precision was higher with the chromogenic method than with the one-stage clotting assays (C.V. = 2-5% vs 4-15%). Altogether, the new chromogenic substrate method has proven itself suitable for determination of factor VIII in plasma and concentrates.     

Standardisation of factor VIII and von Willebrand factor in plasma: calibration of the 4th International Standard (97/586)

The 4th International Standard (IS) Factor VIII/von Willebrand Factor (FVIII/VWF) plasma was calibrated in 25 laboratories by assay against the 3rd IS plasma and fresh normal plasma pools. Five parameters were measured, FVIII:coagulant activity (FVIII:C), FVIII:Antigen (FVIII:Ag), VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and a new parameter, VWF:collagen binding (VWF:CB). Mean potency estimates for the 4th IS, calculated relative to the 3rd IS, were significantly greater than the mean estimates calculated relative to the fresh normal pools by 15, 14 and 20% respectively for FVIII:C, VWF:Ag and VWF:RCof. These results indicate a drift in the International Unit away from the fresh plasma unit. Partial rectification of this drift was achieved by assigning the mean of the estimates calculated relative to the 3rd IS and the fresh plasma pools, i.e. FVIII:C 0.57 IU/ampoule, VWF:Ag 0.79 IU/ampoule and VWF:RCof 0.73 IU/ampoule. This represents a shift in the IU between the 3rd and 4th IS of 7.5% for FVIII:C, 7% for VWF:Ag and 10% for VWF:RCof. Mean estimates of FVIII:Ag relative to the 3rd IS and the fresh normal pools agreed to give an assigned value of 0.89 IU/ampoule. Excessive inter-laboratory variability and a low number of estimates (n = 6) precluded the assignment of a potency for VWF:CB. The 4th IS Factor VIII/VWF plasma (97/586) was established in October 1998.     

Human factor VIII inhibitor alloantibodies with a C2 epitope inhibit factor Xa-catalyzed factor VIII activation: a new anti-factor VIII inhibitory mechanism

Factor VIII (FVIII) inhibitor alloantibodies react with the A2, C2, or A3-CI domains of FVIII and inactivate FVIII activity. We recently demonstrated that an anti-C2 monoclonal antibody with a Val2248-Gly2285 epitope, inhibited factor Xa (FXa)-catalyzed FVIII activation, and that a FXa binding site for FVIII was located within residues Thr2253-Gln2270. In this study, we investigated whether anti-C2 alloantibodies inhibit FXa-catalyzed FVIII activation. Anti-C2 alloantibodies from four patients inhibited FVIII activation by FXa in one-stage clotting assay. Furthermore, analysis by SDS-PAGE showed that all alloantibodies inhibited FVIII proteolytic cleavage by FXa independently of phospholipid. To confirm direct inhibition of FVIII and FXa interaction, we examined the effect of alloantibodies on FVIII binding to anhydro-FXa, a catalytically inactive FXa, in ELISA. All alloantibodies and C2-affinity purified F(ab)'2 preparations inhibited FVIII binding to anhydro-FXa dose-dependently. Our results revealed a new inhibitory mechanism of FVIII, mediated by inhibition of FXa in the presence of anti-C2 alloantibodies.     

Stability of VIII:C in plasma: the dependence on protease activity and calcium

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48-72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.     

Factor VIII: C and factor VIII R: Ag in Argentine hemorrhagic fever

Factor VIII procoagulant activity (F VIII:C) and factor VIII related antigen (F VIII R: Ag) were investigated in 35 patients with Argentine hemorrhagic fever. Since the results obtained in the three clinical forms of the disease were not significantly different, they were tabulated altogether. F VIII:C was low in early stages of the disease but increased progressively in later days (days 5-6: 0.54 +/- 0.10 I. U/ml; days 13-14: 0.95 +/- 0.13 I.U./ml). In contrast, the levels of F VIII R: Ag were high all along the disease and they returned to normal values during the convalescence period (days 5-6; 2.58 +/- 0.54 I.U./ml; day 30: 1.30 +/- 0.14 I.U./ml). The levels of F VIII R: ag were similar in samples drawn before (11 cases) or after (10 cases) the treatment with immune plasma infusion. Plasma samples from 12 patients were studied by two-dimensional immunoelectrophoresis. The only abnormality found was increased height of the immune precipitation arc.