Stability of ADVATE, Antihemophilic Factor (Recombinant) Plasma/Albumin-Free Method, during simulated continuous infusion.

Continuous infusion of factor VIII (FVIII) concentrates during surgical procedures offers the potential for improved hemostatic control and reduced FVIII consumption, but requires stable FVIII concentrates. The stability of ADVATE, Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method (rAHF-PFM), was examined using various simulated conditions. Experiments performed with a multi-therapy 6060 pump showed FVIII recoveries of 95% or more after 48 h for multiple lots of high-potency and mid-potency rAHF-PFM, with or without heparin. Non-infused controls maintained at the same temperature showed similar FVIII recovery, demonstrating that the infusion system did not cause loss of FVIII activity. Supportive data generated using single lots of mid-potency or high-potency rAHF-PFM infused through a MEDEX or HARVARD syringe pump, or a CADD Pump-1, demonstrated FVIII recoveries of 83% or more at 24 or 48 h under all conditions tested. Additionally, rAHF-PFM was stable immediately after dilution in saline or saline/dextrose solutions, and after a 10-h exposure to ultraviolet and visible light. Taken together, these data demonstrate that rAHF-PFM is stable under conditions typically encountered during continuous infusion, and suggest that rAHF-PFM should be safe and effective when used for FVIII replacement by continuous infusion in patients with hemophilia A.

In vitro stability of recombinant human factor VIII (Recombinate).

Factor VIII (FVIII) is currently administered in diverse settings and by a range of methods, and it is important that the stability of specific FVIII preparations be documented for these varying uses. This study of Recombinate recombinant human FVIII (rhFVIII) evaluated: (i) thermostability; (ii) photostability; (iii) stability during simulated continuous infusion; and (iv) stability after dilution. This evaluation was conducted over a range of initial rhFVIII potencies and under differing conditions of temperature, light exposure, dilution and heparin usage. FVIII biological activity was measured by one-stage and chromogenic substrate assays. Microbiological assessment was also performed. Lyophilized rhFVIII was found to be highly thermostable, as evidenced by an energy of activation (Ea) of 16.2 kcal mol-1 and recovery of 99.3% of initial activity after incubation for 6 months at 40 degrees C and 93.8% at 60 degrees C for 2 months. No significant loss of activity could be detected after accelerated simulated natural daylight exposure of lyophilized rhFVIII, although partial activity loss was observed after similar exposure of reconstituted rhFVIII. Shielding in foil wrap effectively prevented such photodegradation of reconstituted rhFVIII. Based upon these results, exposure of lyophilized rhFVIII to sunlight is unlikely to affect stability adversely. Activity of reconstituted rhFVIII (22-106 IU mL-1) remained stable during simulated continuous infusion for 96 h at ambient (20-25 degrees C) and elevated (28-32 degrees C) temperature, and in the presence or absence of 1 U mL-1 heparin. After dilution of reconstituted rhFVIII, an immediate 14-42% loss of expected rhFVIII activity was observed depending upon diluent composition. Accordingly, potential partial loss of rhFVIII activity should be taken into account when dilution is being considered. rhFVIII remained sterile at least 96 h during simulated continuous infusion. rhFVIII is a robust preparation exhibiting biological stability under a wide array of clinically relevant conditions.

Virucidal short wavelength ultraviolet light treatment of plasma and factor VIII concentrate: protection of proteins by antioxidants.

The use of solvent/detergent mixtures and various forms of heat treatment to inactivate viruses has become widespread in the preparation of blood derivatives. Because viruses that lack lipid envelopes and/or are heat resistant, eg, hepatitis A virus (HAV) or parvovirus B19 may be present, the use of two methods of virus elimination that operate by different mechanisms has been advocated. We now report on short wavelength ultraviolet light (UVC) irradiation for virus inactivation and enhancement of its compatibility with proteins by quenchers of reactive oxygen species (ROS). Treatment of an antihemophilic factor (AHF) concentrate or whole plasma with 0.1 J/cm2 inactivated 10(5) to > or = 10(6) infectious doses (ID) of encephalomyocarditis virus (EMCV), HAV, bacteriophage M13, vesicular stomatitis virus (VSV), and porcine parvovirus. However, the recovery of factor VIII was 30% or lower on treatment of an AHF concentrate and 60% on treatment of plasma. Factor VIII recovery could be increased with little or no effect on virus kill by addition of rutin, a flavonoid known to quench both type I and type II ROS. On treatment of plasma in the presence of rutin, the recovery of several other coagulation factors was also enhanced by rutin addition and typically exceeded 75%. Electrophoretic analysis of treated AHF concentrate confirmed the advantage of rutin presence; UVC irradiation of plasma did not cause discernible changes in electrophoretic banding patterns, even in the absence of rutin. We conclude that addition of UVC treatment to existing processes used in the manufacture of blood derivatives will provide an added margin of safety, especially for nonenveloped or heat-stable viruses.

Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors.

The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

Inactivation of viruses in labile blood derivatives. II. Physical methods.

The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure to lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated.

The functional molecular weights of factor VIII activities in whole plasma as determined by electron irradiation.

Activities of the various components of the human factor VIII complex in citrated and heparinized human plasma have been determined following radiation inactivation of the plasma in a high energy electron beam at -135 degrees C in order to determine the molecular size of the functional units. In citrated and in heparinized plasma the functional size of VIII:C was 120,000 +/- 9,700 and 140,000 +/- 10,000, respectively. Taken together with previously published data, these results suggest that VIII:C exists in plasma as a dimer of noncovalently bonded functional subunits. The size of the functional unit of the ristocetin cofactor of the factor VIII complex was determined as being approximately 330,000 in both citrated and heparinized samples. Immunological assays for VIII:C (inhibitor neutralization assay), the VIII:C antigen, and the VIII:vWF-related antigen suggest that these may not be reliable under conditions favoring the activation and inactivation of factor VIII components by thrombin or other proteases.