532. An Inducible Fed-Batch Fermentation Process for Scale-Up and Production of DNA Vaccines and Gene Medicines
Year: 2006
Type: article
Abstract: It is essential to devise industrial processes whereby plasmid DNA can be manufactured to meet the quality, economy, and scale requirements projected for future gene medicine and DNA vaccine production. To date, most plasmid fermentation media and processes result in low yields, imposing a cost and purity burden on commercialization of plasmid DNA production. Here we report the development of an inducible fed-batch fermentation process that dramatically increases volumetric yield and specific plasmid yield with respect to cell mass, while maintaining or enhancing plasmid integrity. Additionally, this process is well suited for producing unstable plasmids. This inducible process utilizes commercially available media that we designed specifically for plasmid production. The process consists of an initial biomass accumulation phase, followed by a plasmid accumulation phase. The plasmid is stably maintained at low levels during a period of nutrient restricted growth and reduced temperature (30°C), and then the temperature is increased (37-42°C) to induce plasmid amplification. Typically, the specific plasmid yield increases over a period of up to 8 hours following temperature up-shift. A set of ten replicate fermentations producing a DNA vaccine plasmid was performed. The total process lasted 38.5 hours, with a 7 hour induction time. The overall plasmid yield averaged 760 mg/L, and the average specific plasmid yield was 8.5 mg/L/OD600. A total DNA analysis by AGE of total cell lysates indicated that plasmid DNA accounted for the majority of the total DNA in the cells at harvest. A high degree of process robustness and reproducibility has been demonstrated with many different plasmids and with multiple batches of a single plasmid. Fed-batch fermentation productivity exceeding 1000 mg plasmid DNA/L fermentation media has been obtained with pUC origin containing plasmids (see figure). This five to ten fold increase in plasmid yield dramatically decreases plasmid manufacturing costs, and improves the effectiveness of downstream purification by reducing the fraction of impurities.
Source: Molecular Therapy
Authors: Aaron E. Carnes, James A. Williams, Clague P. Hodgson
Institution: Nature Technology Corporation (United States)
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2067718937