title: Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.
abstract: The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.
authors.affiliation: MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: Geisa A L
authors.initials: GA
authors.lastname: Gonçalves
authors.affiliation: MIT-Portugal Program, Portugal; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States.
authors.forename: Kristala L J
authors.initials: KL
authors.lastname: Prather
authors.affiliation: MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
authors.affiliation: Nature Technology Corporation, Lincoln, NE, United States.
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Carnes
authors.affiliation: MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address: miguelprazeres@ist.utl.pt.
authors.forename: Duarte M F
authors.initials: DM
authors.lastname: Prazeres
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005285:Fermentation; D055786:Gene Knockout Techniques / Q000379:methods*; D005947:Glucose; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019444:Vaccines, DNA
chemical_list: D029968:Escherichia coli Proteins; D019444:Vaccines, DNA; D005956:Glucose-6-Phosphate Isomerase; C554958:pgi protein, E coli; D005947:Glucose
keywords: DNA vaccine; Escherichia coli; Fermentation; Plasmid biopharmaceutical