Abstract
The possibility of the transmission of HIV-1 by non-heated Factor-VIII and Factor-IX preparations has made it urgent to confirm the safety of our non-heated fibrinogen preparation with respect to the possibility of HIV-1 transmission. Fortunately, no cases of HIV-1 transmission by our former non-heated fibrinogen preparation have been reported. We studied potentially effective steps for HIV-1 inactivation or elimination in the manufacturing process for the former non-heated fibrinogen preparation. Using HIV-1 spiking, four steps were studied: cryoprecipitation, 8% ethanol fractionation, 6.5% ethanol washing and ultraviolet (UV) irradiation. MT-4 or human H9 cells were used for HIV-1 assay. Process samples (cryoprecipitate-rich plasma, cryoprecipitate-poor plasma, fibrinogen solution etc.) and 1/10 volume of HIV-1 solution were mixed and then processed under manufacturing conditions.
The cryoprecipitation step did not reduce HIV-1 infectivity. HIV-1 was inactivated by the 8% ethanol fractionation step. This inactivation was confirmed by two independent laboratories (>3.0log, experiment 1, and >5.0log, experiment 2). The two 6.5% ethanol washing steps each inactivated HIV-1 (2.6log). UV irradiation of the fibrinogen preparation at one Joule/ml inactivated HIV-1 (1.1-1.2log). The total reduction and clearance factors for all process steps combined were >6.7 and >12.3log, respectively.
Since these validation tests were performed under worst case conditions for both the ethanol fractionation steps (short incubation time, lower ethanol concentration) and UV irradiation (higher protein concentration), we expect greater reduction and clearance factors in actual production.
