VALIDATION OF THE HEAT-TREATMENT STEP USED IN THE PRODUCTION OF DIASPIRIN CROSS-LINKED HEMOGLOBIN (DCLHB(TM)) FOR VIRAL INACTIVATION - EFFECT OF CROSS-LINKING

Two experiments were performed to assess viral inactivation during the crosslinking and heat treatment steps of the DCLHb(TM) manufacturing process. Stroma free hemoglobin (SFHb) collected from a large scale ma nufacturing lot was tested in a 1:680 scaled down system in which the key parameters used in the manufacturing process were replicated. In t he first study Porcine Parvovirus (PPV), a non-enveloped virus, was us ed to assess inactivation, while in the second study Bovine Viral Diar rhea Virus (BVDV), an enveloped virus, was utilized. Tn both experimen ts, the SFHb solution was deoxygenated and an aliquot of virus suspens ion was added. To initiate the crosslinking reaction, a solution of bi s(3,5-dibromosalicyl) fumarate (DBBF) in HEPES buffer was added to the test solution. In both experiments the reaction times and the degree of crosslinking were normal. After crosslinking, the reaction mixtures were heated to 74 +/- 1 degrees C over 30 minutes, held at 74 +/- 1 d egrees C for 90 minutes, and cooled to less than 10 degrees C over 30 minutes. In each experiment the degree of crosslinking of final produc t was 100% and yield of hemoglobin recovery was normal. Samples were r emoved prior to crosslinking, after crosslinking and before, during an d after heat treatment for determination of virus titer and evaluation of key process parameters. The results from these experiments were co nsistent with those obtained from the full scale manufacturing process for the deoxygenation, crosslinking and the heat treatment step durin g the production of DCLHb(TM). The results of virus assays showed that crosslinking has no effect on viruses and their subsequent inactivati on by heat treatment.