Pharmacokinetic study of minipooled solvent/detergent-filtered cryoprecipitate factor VIII.

Eighteen cryoprecipitate minipools, each made of 30 units of low volume, concentrated cryoprecipitate, have been treated by solvent-detergent and filtration (S/D-F) in a single-use CE-marked bag system. The S/D-F cryoprecipitate contained a mean of 10.5 IU mL⁻¹ factor VIII (FVIII), 17 mg mL⁻¹ clottable fibrinogen, and >10 IU mL⁻¹ von Willebrand factor ristocetin co-factor, and anti-A and anti-B isoagglutinins were undetectable. The products have been infused in 11 severe (FVIII <1%) haemophilia A patients (mean age: 17.4 years; mean weight: 57.6 kg) at a dose close to 40 IU kg⁻¹. Patients were hospitalized for at least 36 h to determine FVIII recovery, half-life and clearance. They were also closely monitored for possible adverse events. None of the infused patients demonstrated reactions or adverse events even though they did not receive anti-allergic drugs or corticosteroids prior to infusion. The mean recovery of FVIII 10 min postinfusion was 69.7%. Mean FVIII half-life was 14.2 h and clearance was 2.6 mL h⁻¹ kg⁻¹. All patients had a bleeding-free interval of 8-10 days postS/D-F cryoprecipitate infusion. The data show that S/D-F cryoprecipitate FVIII presents a normal pharmacokinetics profile, and support that it could be safely used for the control of acute and chronic bleeding episodes in haemophilia A patients.

[Solvent-detergent viral inactivation of minipools of plasma for transfusion, cryoprecipitate and cryo-poor plasma in single-use bag systems]. / Inactivation virale par procédé solvant-détergent de minipools de plasma, de cryoprécipité et de surnageant de cryoprécipité dans des dispositifs à usage unique.

Non-virally inactivated plasma, cryoprecipitate and cryoprecipitate-poor plasma, prepared by blood establishments, are still used in many countries in the world, in both the developing world and industrialized countries, for the treatment of various hematological disorders. In the absence of viral inactivation treatment, these fractions may be involved, in spite of increasingly sensitive viral detection methods, into the transmission of plasma-borne viruses, most critically HIV and Hepatitis B (HBV) or C (HCV). We have adapted the well-established industrial solvent-detergent (SD) viral inactivation treatment to allow its application in a small scale using a single-use plastic bag system. The procedure can be used by blood establishments, without the need to build an industrial-scale manufacturing facility. Results show a good recovery of the functional activity of plasma proteins, including coagulation factors (such as factor VIII and coagulable fibrinogen) and/or protease inhibitors (such as alpha 2-antiplasmin). Viral validation studies revealed reduction factors greater than 4.17, greater than 4.73 and greater than 4.72 for HIV, BVDV and PRV, respectively, within a few minutes of treatment. A single-use SD treatment and SD-elimination system is currently under development to allow standardized use of the procedure by blood establishments or national or regional service centers.

Properties of a concentrated minipool solvent-detergent treated cryoprecipitate processed in single-use bag systems.

Cryoprecipitate is still used to treat factor VIII (FVIII), von Willebrand factor (VWF) and/or fibrinogen deficiency. Recently a solvent-detergent (S/D) process of minipools of cryoprecipitate performed in a closed bag system has been designed to improve its viral safety. Still, cryoprecipitate has other drawbacks, including low concentration in active proteins, and presence of haemolytic isoagglutinins. We report here the biochemical evaluation of S/D-treated minipools of cryoprecipitates depleted of cryo-poor plasma. Cryoprecipitates were solubilized by 8 mL of a sterile glucose/saline solution, pooled in batches of 40 donations and subjected to S/D treatment in a plastic bag system using either 2% TnBP or 1% TnBP-1%Triton X-45, followed by oil extractions (n = 10). Mean (+/-SD) FVIII and fibrinogen content was 8.86 (+/-1.29) IU mL(-1) and 16.02 (+/-1.98) mg mL(-1), and 8.92 (+/-1.05) IU mL(-1) in cryoprecipitate minipools treated with 2% TnBP, and 17.26 (+/-1.71) mg mL(-1), in those treated by TnBP-Triton X-45, respectively. The WWF antigen, ristocetin cofactor and collagen binding activities were close to 10, 7 and 8 IU mL(-1), respectively, and were not affected by either SD treatment. VWF multimeric pattern of SD-treated cryoprecipitates were similar to that of normal plasma, and the >15 mers and >10 mers content was identical to that of the starting cryoprecipitates. The anti-A and anti-B titre was 0-1 and 0-1/8, respectively. Therefore, it is possible to prepare virally inactivated cryoprecipitate minipools depleted of isoagglutinins and enriched in functional FVIII, VWF and clottable fibrinogen.

A minipool process for solvent-detergent treatment of cryoprecipitate at blood centres using a disposable bag system.

BACKGROUND AND OBJECTIVES: Single-donor or small-pool cryoprecipitates are produced by blood establishments, mostly in developing countries, for substitute therapy in haemophilia A, von Willebrand disease and fibrinogen deficiency, as well as for the manufacture of fibrin sealant. As cryoprecipitate may be contaminated with pathogenic plasma-borne viruses, there is an urgent need to develop a simple method for the viral inactivation of cryoprecipitate. MATERIALS AND METHODS: Cryoprecipitate was obtained according to standard procedures. Ten minipools of five or six donations of cryoprecipitate were prepared and subjected, in sterile closed bags, to a viral inactivation treatment using either 2% tri(n-)butyl phosphate (TnBP) for 4 h at 37 degrees C or the combination of 1% TnBP and 1% Triton X-45 for 4 h at 31 degrees C. The cryoprecipitates were subsequently extracted three times in their processing bags by mixing and decantation using 7.5% sterile ricinus oil. The TnBP-treated cryoprecipitates were further subjected to a clarifying centrifugation step at 3800 g for 30 min. The final products were dispensed into individual bags and frozen at -30 degrees C or lower. RESULTS: The cryoprecipitates treated with either 2% TnBP or 1% TnBP + 1% Triton X-45 showed excellent (> 93%) mean recovery of coagulant factor VIII (FVIII), ristocetin cofactor Von Willebrand factor (VWF:RCo), and clottable fibrinogen activity. Prothrombin time, international normalized ratio and activated partial thromboplastin time increased during solvent-detergent treatment but returned to initial values after oil extractions. The final content of TnBP and Triton X-45 was < 10 and 50 ppm, indicating excellent removal by the oil-extraction procedure. CONCLUSIONS: Viral inactivation treatment by TnBP, with or without Triton X-45, can be applied to minipools of cryoprecipitate, with good recovery of FVIII, VWF and fibrinogen. The viral inactivation and solvent-detergent removal process can be performed in a closed bag system and using simple blood establishment techniques and equipment. This technology could be considered for the improved viral safety of cryoprecipitate which is used to treat haemophilia A, von Willebrand disease or fibrinogen deficiency, or to prepare fibrin sealant.

Nanofiltration of single plasma donations: feasibility study.

BACKGROUND AND OBJECTIVES: Major technical developments have been made in recent years to improve the quality and safety of human plasma for transfusion and fractionation. The present study was performed to assess, for the first time, the feasibility of applying a nanofiltration process, using 75-nm and 35-nm mean pore size membranes (Planova) 75N and Planova 35N), to human plasma. MATERIALS AND METHODS: Ten apheresis plasma units were obtained from 10 plasma donors. Within 4 h of collection, plasma was subjected to leucoreduction and filtration (using 75-nm and 35-nm mean pore size membranes) at 35 degrees C, at less than 1 bar pressure. Aliquots of plasma were taken at all steps of the filtration procedure and numerous plasma quality parameters were measured. In addition, six hepatitis C virus (HCV)-positive plasma donations were experimentally subjected to the same filtration sequence and subsequently assessed by RNA polymerase chain reaction (PCR) and branched-chain DNA-quantification assays. RESULTS: Leucoreduced plasma can be reproducibly nanofiltered onto a sequence of 75-nm and 35-nm membranes, at a flow rate of 450 ml/h and a temperature of 35 +/- 0.5 degrees C. Some protein dilution, or loss, was found during filtration, but the plasma filtered through membranes with a mean pore size of 75 nm and 35 nm met in vitro specifications for use in transfusion or fractionation. There were no signs of activation of the coagulation system. HCV-positive plasma donations became negative, as judged by PCR and branched-chain DNA assay results, after filtration through the 35-nm membrane. CONCLUSIONS: It is possible to apply a 75 + 35-nm filtration process to leucoreduced human plasma. This technology may have important future benefits in improving the quality and safety of plasma, by removing blood cell debris and infectious agents.

Virucidal heat-treatment of single plasma units: a potential approach for developing countries.

Since HIV first burst onto the scene of transfusion medicine, the quest for viral inactivation processes for plasma and plasma products has not ceased. Sophisticated methods for improving viral safety are currently used in the industrial world. However, in developing countries, with no facilities for treating plasma, nonviral-inactivated fresh frozen plasma [FFP] continues to be used extensively, and as screening may not be optimal (or may even be absent), FFP still contributes to the spread of HIV and other infectious viruses. The feasibility of heat-treating FFP at the liquid state, in its collection bag, was explored by testing diverse conditions of temperature and duration, in the presence of biologically compatible stabilisers. Quality of the heat-treated plasma was evaluated by haematological, biochemical and animal assays. The efficiency of the method to inactivate viruses was validated using HIV and model viruses. The selected heating conditions are 50 degrees C for 3 h. The optimized combination of stabilizers is composed of 30 mM trisodium citrate, 10 g L-1 L-lysine, 12 mM calcium gluconate and 150 g L-1 sorbitol. Plasma coagulability is appropriately preserved as shown by the KCT ratio (1.4). Recovery of biological activity of most coagulation factors is higher than 70% (including fibrinogen & von Willebrand factor). Electrophoretic and immunoblotting studies did not evidence protein aggregation and/or degradation. Viral validation studies of this procedure have shown complete inactivation of HIV (> 6.6 log) in less than 1 h of treatment. A viral reduction of at least 4 log for various model viruses, including those of hepatitis A and C viruses, suggests a potential contribution of the method to diminish the risk from various blood-borne viruses. The selected formulation appears to preserve plasma protein integrity and properties. The procedure does not require sophisticated equipment but it is mandatory to monitor it carefully to ensure quality and reproducibility. If properly controlled and standardized, this approach offers an opportunity to reduce the risk of transmission of HIV and other viruses, particularly in poor countries with a high incidence of HIV.