Production Of Biopharmaceutical Plasmid DNA By Recombinant Escherichia Coli

With ongoing identification of functional genes modern medical treatments like genetic vaccination or gene therapy are beginning to evolve because with these novel therapies illness can be cured at the genetic level (genotype) and not by treating the symptoms (phenoptype). Production of nucleic acids as an active pharmaceutical ingredient (API) in gene therapy and genetic vaccination is gaining more and more importance. Non-viral delivery systems like plasmid DNA have become more and more desirable as they are the most attractive gene transfer systems for commercial products. Non-viral vectors own theoretically an unlimited insertion capacity. They are safety, stable, and in comparison with viruses relatively easier to produce. The number of approved gene therapy protocols using plasmid DNA-based delivery vectors has increased exponentially since 1995, representing approximately 25% of the ongoing gene therapy trials. Consequently, it is important to develop an economic, repeated, easy to be magnified plasmid production technology which could yield a great lot of plasmid DNA for clinic application.In this study the recombinant Escherichia coli DH5αcontaining pUK21CMVβ1.2 was employed to produce plasmid pUK21CMVβ1.2. The medium composition for plasmid production was optimized, and the fermentation strategy was systematically studied to improve the plasmid production. Culture time, transfer times and selectivity press were found less influence on plasmid production. Plasmid pUK21CMVβ1.2 was found to have both separate stability and structure stability, and the best plasmid harvest time was thirteen hours in LB medium under shake flask cultivation.Through one-factor-at-a-time experiments, sucrose and casein peptone were found to be the most suitable carbon and nitrogen sources, for plasmid production by recombinant E. coli pUK21CMVβ1.2, respectively. The statistical analyses of the effect of the carbon and nitrogen source as well as C: N ratios in the growth medium on plasmid production, by employing RSM and the Box-Behnken design indicated that the optimized medium should contain (g L^-1): sucrose 9.96, glycerol 9.8, casein peptone 10.42, yeast 5.0, (NH₄)₂SO₄ 6.4, and NaCl 10.0. By shake flask cultivation of the recombinant cells on the optimized medium, a plasmid concentration of 51.80 mg L^-1 and plasmid selectivity of 23.49 mg g^-1 were achieved after 24 h of incubation, which were approximately 3.5 and 1.7 times higher than those obtained in the LB medium at the same conditions, respectively.Batch and fed-batch cultivation was carried out in 2 L fermenter. Feeding strategies at exponential and stationary phase were designed according to the characteristics of cell growth. The results showed that feeding at exponential phase with glucose or the optimized medium couldn't significantly improve cell growth and plasmid production. Also feeding at stationary phase with optimized medium had the same results as feeding at exponential phase. However, feeding at stationary phase with glucose could significantly improve plasmid production with plasmid concentration being three times higher than that of batch fermentation, although it couldn't yield higher biomass. Fed-batch cultivation under pH-stat feeding control was found to be able to improve significantly cell growth and plasmid production when 2-fold concentration optimized medium was used in the batch phase. A plasmid concentration of 601.9 mg L^-1 and biomass of 16.78 g L^-1 were achieved, which were approximately 6.63 and 2.53 times higher than those obtained in batch fermentation, respectively.