Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: Fermentation strategies and impact on downstream processing

摘要

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, MG1655 Delta endA Delta recA, and the common laboratory strain, DH5 alpha, were used for comparison purposes and pVAX1GFP, a C0lE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (similar to 141 mg/L) than MG1655 Delta endA Delta ArecA (similar to 48 mg/L) and DH5 alpha (similar to 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 MG1655 Delta endA Delta recA (31%) and DH5 alpha. (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. (C) 2014 Elsevier B.V. All rights reserved.