Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale |
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Authors: | Mathias R. Fibi Michael Bröker Rainer Schulz Roloff Johannsen Gerd Zettlmeissl |
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Affiliation: | (1) Department of Molecular Biology/Central Analytics, Research Laboratories of Behringwerke AG, Behringwerke AG, P.O. Box 1140, W-3550 Marburg, Federal Republic of Germany;(2) Department of Animal Cell Culture Technology, Behringwerke AG, P.O. Box 1140, W-3550 Marburg, Federal Republic of Germany |
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Abstract: | Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 103 per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.Offprint requests to: M. R. Fibi |
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