Engineering of bacterial strains and vectors for the production of plasmid DNA

Abstract

The demand for plasmid DNA (pDNA) is anticipated to increase significantly as DNA vaccines and non-viral gene therapies enter Phase 3 clinical trials and are approved for use. This increased demand, along with renewed interest in pDNA as a therapeutic vector, has motivated research targeting the design of high-yield, cost-effective manufacturing processes. An important aspect of this research is engineering bacterial strains and plasmids that are specifically suited to the production of plasmid biopharmaceuticals. This review will survey recent innovations in strain and vector engineering that aim to improve plasmid stability, enhance product safety, increase yield, and facilitate downstream purification. While these innovations all seek to enhance pDNA production, they can vary in complexity from subtle alterations of the host genome or vector backbone to the investigation of non-traditional host strains for higher pDNA yields.2

Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters

“…Different E. coli strains present different heterologous gene expression capacities [138,139], but few studies have attempted to determine the effect of host strain on recombinant protein production using plasmids that contain λ elements. In one of such studies, different strains were transformed with plasmid λPL-DsbA (peptide signal of the periplasmic protein DsbA) containing the coding hGH gene [125].…”

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

“…As earlier reviewed, strain engineering can increase pDNA percentage per cell, reduce genomic DNA and RNA impurities, and decrease process stream or lysate viscosity (Bower and Prather, 2009). Moreover, a carefully designed strain may result in improved plasmid topology and segregational stability, and eventually contribute to increased transfection efficiency of the final plasmid product (Gonç alves et al, 2012).…”

DNA manipulation by means of insulator‐based dielectrophoresis employing direct current electric fields

“…DEP can be envisaged as a technique that could answer many of the requirements for purification of pharmaceutical-grade nucleic acids. Gene therapy and DNA vaccines will become a commercial reality in the following years [28][29][30], in fact, large-scale production and purification of nucleic acids are currently on demand for both laboratory and clinical trials [31,32]. Microdevices used in massively parallel systems could accomplish large-scale DNA purification, leading to faster separations while decreasing reagent consumption, making the purification processes more economically favorable.…”