The approach followed in the design of a large-scale pasteurization treatment (60 °C for 10 hours in the liquid state) of fresh frozen plasma is presented. Various aspects thought to influence the viral safety of such a product are discussed. They are based largely upon the fact that, although it is subjected to a specific viral inactivation treatment, this plasma does not benefit from any fractionation steps known to participate in the potential elimination of infectious agents during the manufacture of plasma derivatives. Consequently, the plasma is obtained from regular plasmapheresis donors, and the plasma donations used to make the pool must be negative for anti-HIV-1 and -2, anti-HCV, anti-HBc, anti-HTLV-l and -2, HBs antigen and parvovirus Bl9 antigen, and have a normal level of ALT. The batch size is limited to 100 plasma units to limit the potential infectious risk associated with very large batches, especially if an infectious agent, resistant to pasteurization, is present. Pasteurization has been chosen for this procedure, as applied to plasma derivatives, has been shown to inactivate a broad spectrum of viruses, both enveloped and non-enveloped. The process is relatively simple. The forzen plasma units are opened, and the plasmas are mixed and thawed at 30 °C to avoid the formation of cryoprecipitate. The liquid plasma is transferred to a sterilized container and stabilizers are added. The mixture is then transferred to the pasteurization unit to be heat-treated at 60 °C for at least 10 hours under gentle mixing. Following cooling, the mixture is ultrafiltrated to eliminate the stabilizers and to concentrate the plasma pool to its initial volume. The plasma is sterile-filtered, then dispensed into bottles and frozen. Virus validations of this pasteurization process, carried out by independant virology laboratories, have confirmed the ability of the process to inactivate more than 4 to 6 logs of non-enveloped or enveloped, DNA or RNA, viruses, including HIV-l and Sindbis virus, in less than 5 hours of heat-treatment. The biological characteristics of the pasteurized plasma include a good preservation (75 to 95%) of the activity of clotting factors, including FI, FV, FVIII, FXI, and FXIII, and protease inhibitors. The overall clottability of the plasma, as expressed by the APTT, is almost unchanged. In vitro electrophoretic, immunoelectrophoretic, and chromatographic analyses, as well as preclinical studies in animals (rat model, Wessler model in rabbit, toxicity tests in mouse, neoantigen detection in rabbit) did not reveal any alteration (such as protein aggregation) or any hypotensive or thrombogenic side-effects. The industrial pasteurization unit has been designed to fulfill the most stringent criteria of good manufacturing practices, in terms of traceability, automation and computerization. The designs of the plant and of the process take into consideration the necessity of avoiding the potential risks of introducing extraneous pathogenic agents (e.g. from buffers) as well as the risks of downstream contamination following the pasteurization step. In conclusion, our work demonstrates that pooled whole human plasma can be pasteurized under conditions that preserve its biological properties while inactivating significant doses of viruses. The absence of any fractionation step, in contrast to the manufacturing processes of plasma derivatives, and thus the impossibility of eliminating some additional infectious agents, has made additional precautions necessary in the selection of the donors, the type of viral screening tests performed on each donation, and the limitation of the batch size.
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