| Automated fed-batch fermentation with feed-back controls based on dissolved oxygen (DO) and pH for production of DNA vaccines. | |
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MedLine Citation:
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PMID: 9079287 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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A fermentation process in Escherichia coli for production of supercoiled plasmid DNA for use as a DNA vaccine was developed using an automated feed-back control nutrient feeding strategy based on dissolved oxygen (DO) and pH. The process was further automated through a computer-aided data processing system to regulate the cell growth rate by controlling interactively both the nutrient feed rate and agitation speed based on DO. The process increased the total yield of the plasmid DNA by approximately 10-fold as compared to a manual fed-batch culture. The final cell yield from the automated process reached 60 g L-1 of dry cell weight (OD600 = 120) within 24 h. A plasmid DNA yield of 100 mg L-1 (1.7 mg g-1 cell weight) was achieved by using an alkaline cell lysis method. Plasmid yield was confirmed using High Performance Liquid Chromatography (HPLC) analysis. Because cells had been grown under carbon-limiting conditions in the automated process, acetic acid production was minimal (below 0.01 g L-1) throughout the fed-batch stage. In contrast, in the manual process, an acid accumulation rate as high as 0.36 g L-1 was observed, presumably due to the high nutrient feed rates used to maintain a maximum growth rate. The manual fed-batch process produced a low cell density averaging 10-12 g L-1 (OD600 = 25-30) and plasmid yields of 5-8 mg L-1 (approximately 0.7 mg g-1 cells). The improved plasmid DNA yields in the DO- and pH-based feed-back controlled process were assumed to be a result of a combination of increased cell density, reduced growth rate (mu) from 0.69 h-1 to 0.13 h-1 and the carbon/nitrogen limitation in the fed-batch stage. The DO- and pH-based feed-back control, fed-batch process has proven itself to be advantageous in regulating cell growth rate to achieve both high cell density and plasmid yield without having to use pure oxygen. The process was reproducible in triplicate fermentations at both 7-L and 80-L scales. |
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Authors:
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W Chen; C Graham; R B Ciccarelli |
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Publication Detail:
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Type: Comparative Study; Journal Article |
Journal Detail:
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Title: Journal of industrial microbiology & biotechnology Volume: 18 ISSN: 1367-5435 ISO Abbreviation: J. Ind. Microbiol. Biotechnol. Publication Date: 1997 Jan |
Date Detail:
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Created Date: 1997-04-30 Completed Date: 1997-04-30 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 9705544 Medline TA: J Ind Microbiol Biotechnol Country: ENGLAND |
Other Details:
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Languages: eng Pagination: 43-8 Citation Subset: B |
Affiliation:
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Apollon, Inc, Malvern, PA 19355, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Acetic Acid
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metabolism Automatic Data Processing / methods* Bacteriological Techniques Biomass Chromatography, High Pressure Liquid Culture Media / metabolism* DNA, Bacterial / analysis, biosynthesis DNA, Superhelical / biosynthesis*, immunology* Electrophoresis, Agar Gel Escherichia coli / genetics*, growth & development, metabolism* Fermentation Glucose / metabolism Hydrogen-Ion Concentration Oxygen / metabolism* Plasmids / analysis, genetics Vaccines, DNA / biosynthesis* |
| Chemical | |
Reg. No./Substance:
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0/Culture Media; 0/DNA, Bacterial; 0/DNA, Superhelical; 0/Vaccines, DNA; 50-99-7/Glucose; 64-19-7/Acetic Acid; 7782-44-7/Oxygen |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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