During 1990 and 1991 the capability for repetitive, consecutive production of DCLHb solution to meet a rigorous and complete set of product criteria was demonstrated. In addition, through periodic monitoring of product stored under controlled conditions, the stability of all lots of DCLHb solution during frozen storage was demonstrated for more than a year. In this way, assurance was provided that the DCLHb solution used in preclinical testing met all product criteria throughout the biological testing period.
The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88-97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5-6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situ from a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spin at 22 degrees C). Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
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