Folarin, Olusegun; (2020) A New Approach to Plasmid Upstream Processing for Vaccine and Gene Therapy Applications. Doctoral thesis (Ph.D), UCL (University College London.

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Abstract

There has been a rise in the interest of plasmid DNA as therapeutics. The rise is evident in the number of ongoing clinical trials involving the use of plasmid DNA. To be useful as therapeutics, the DNA needs to be of high yield and high level of supercoiling. From the bioprocessing point of view, the level of supercoiling can potentially have an impact on the ease of downstream processing. We have approached meeting these requirements through plasmid engineering and developing an optimized fermentation strategy. Two different plasmids (small and large size) were developed. A 7.2kb plasmid was developed by insertion of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) to 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentation and analysis were carried out in triplicates cultures to screen for best-performing strains. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further, and an increase in supercoiling and plasmid yield was also observed. The extent of supercoiling was examined by superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter (Dh) measurement using the Nanoparticle Tracking Analysis (NTA), and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59 nm compared to pSVβ-Gal with Dh of 70-90 nm for both strains examined. In order to investigate this approach can be scaled on plasmid, a 27 kb plasmid pSTFλ398 was constructed with its respective control plasmid, 26.6 kb pSTFλ. There was an almost 2-fold increase in the plasmid yield for the SGS containing plasmid pSTFλ398 and a supercoiled content of 93%. In addition, SGS containing plasmid was maintained integrity when subjected to shear. An approach was also developed to increase the plasmid yield by developing a batch fermentation for high cell density which involves supplementation with minimal media with amino acids. The media was supplemented with histidine and glycine which have been reported to be implicated in increasing plasmid yield, and methionine for suppressing acetate inhibition. The supplementation allowed the use of initial glycerol concentration as high as 100 g/L with a volumetric yield and plasmid yield of twice as much as using 100 g/L glycerol in the media without supplementation. The report of this study has shown that plasmid modification with the Mu-phage SGS sequence and optimizing fermentation strategies have beneficial effects on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

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