No abstract

A series of experiments was performed to assess the ability of the heat treatment step used in the manufacture of diaspirin crosslinked hemoglobin (DCLHb) to inactivate viruses. In-process solutions (reaction mixtures after the crosslinking process) from six different manufacturing lots were used as test media in a 1:680 scaled down system in which the key process parameters used in the large scale production were duplicated. The inactivation of five different viruses (Bovine Viral Diarrhea Virus, Pseudorabies Virus, Human Immunodeficiency Virus 1, Porcine Parvovirus and Hepatitis A Virus) was evaluated. Each validation experiment consisted of spiking the solution at 37 degrees C with virus, heating to 74 +/- 1 degrees C over a period of 30 minutes, holding at 74 +/- 1 degrees C for 90 minutes and cooling from 74 +/- 1 degrees C to less than 10 degrees C over a period of 30 minutes. Duplicate experiments were performed with each of the viruses with the exception of Human Immunodeficiency Virus 1, for which three experiments were performed. In each experiment samples were removed before, during, and after heating for the purpose of determining virus titer and evaluating key process parameters. The results obtained from these experiments confirmed that the key process parameters in these experiments using the scaled down test system reproduced those of the large scale manufacturing process. The results of the virus assays showed at least a 7 log reduction was accomplished by the heat treatment for each of the viruses tested.

Two experiments were performed to assess viral inactivation during the crosslinking and heat treatment steps of the DCLHb manufacturing process. Stroma free hemoglobin (SFHb) collected from a large scale manufacturing lot was tested in a 1:680 scaled down system in which the key parameters used in the manufacturing process were replicated. In the first study Porcine Parvovirus (PPV), a non-enveloped virus, was used to assess inactivation, while in the second study Bovine Viral Diarrhea Virus (BVDV), an enveloped virus, was utilized. In both experiments, the SFHb solution was deoxygenated and an aliquot of virus suspension was added. To initiate the crosslinking reaction, a solution of bis (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 degrees C for 90 minutes, and cooled to less than 10 degrees C over 30 minutes. In each experiment the degree of crosslinking of final product was 100% and yield of hemoglobin recovery was normal. Samples were removed prior to crosslinking, after crosslinking and before, during and after heat treatment for determination of virus titer and evaluation of key process parameters. The results from these experiments were consistent with those obtained from the full scale manufacturing process for the deoxygenation, crosslinking and the heat treatment step during the production of DCLHb. The results of virus assays showed that crosslinking has no effect on viruses and their subsequent inactivation by heat treatment.