Highly Improved Plasmid DNA Production by Optimizing the Plasmid Construct and Fermentation Conditions

Thursday, October 20, 2011: 10:40 AM
L100 I (Minneapolis Convention Center)
Ram N. Trivedi, Chemical and Petroleum Engineering; Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, Patrick R. Bartlow, Bioengineering, University of Pittsburgh, Pittsburgh, PA, Jonathan Meade, Chemical Engineering, Carnegie Mellon, Pittsburgh, PA, Saleem A. Khan, Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, Michael M. Domach, Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA and Mohammad M. Ataai, Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA

This presentation details our effort to develop a combined host-plasmid system that provides plasmid copy numbers that approach 104 copies/cell. The work is driven by DNA vaccines and gene delivery vectors which are becoming a growing segment of new clinical trials. Over 20% of ongoing trials rely on plasmid vectors spanning all disease categories and including nine trials currently in Phase III. Moreover, four DNA vaccines have been approved for veterinary use since 2005. 

Given the large number of potential vaccines and the size of the target animal and human populations, large amounts of plasmid DNA will be required. Our ultimate goal is to develop a production platform that has high volumetric productivity when inexpensive and simple minimal media is used. To this end, we employ plasmid pNTC8485 in DH5α that possess a temperature-responsive ColE1 origin of replication, encodes reporter EGFP, and has a major advantage of reduced metabolic load by encoding a small antisense RNA that mitigates chromosomally-endowed toxicity from sacB when sucrose is present in the media. Replication region contains two promoters which direct the synthesis of two RNA molecules (RNA I and RNA II) involved in control of plasmid copy number.  We have modified the origin of replication, yielding approximately 8000 plasmid copies/cell, a 6-fold increase in plasmid copy number relative to unaltered DH5α pNTC8485. Further increase in process productivity was achieved by improving the fermentation conditions that enables the selective agent (sucrose) to be metabolized once the catabolic resources are exhausted from the medium. After reaching stationary phase in media with 8% sucrose, treatment with invertase releases glucose that spurs added cell growth without noticeable plasmid dilution, nearly tripling DNA volumetric productivity. Overall, about a 20-fold increase in volumetric productivity was achieved by combining the productive mutations of origin of replication and improved fermentation conditions.

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