Michael Danquah 1 , Jamil Chowdhury 2 , Gareth M. Forde 1

1. Michael Danquah1, Jamil Chowdhury2, Gareth M. Forde1 1: Department of Chemical Engineering, Monash University, Australia. 2: School of Chemistry, Monash University, Australia. Introduction > Plasmid DNA has gained tremendous interest in vaccination and gene therapy applications recent years. > Plasmid DNA for vaccination and gene therapy applications requires extremely high levels of quality and purity to conform with Food and Drug Administration (FDA) regulations. > Commercial adsorbents geared toward small molecule applications such as proteins (<10 nm) are unsuitable for the purification of the large plasmid DNA molecules (> 100 nm). Objective This research is directed toward developing a fully-scalable, commercially viable method for the production of plasmid DNA. In particular, the unit operations of fermentation (through medium selection and fed-batch optimisation) and chromatography (single-stage monolith purification) will be considered. Through process innovation, the time yield for the production of high purity therapeutic plasmid DNA will be improved. Bacterial Fermentation Optimisation Monolith Synthesis EDMA + GMA porogen Epoxy matrix DEAE Immobilization Epoxy matrix + DEAE I- EX resin Electrostatic binding system Conclusion The most favourable growth medium for pDNA production of those assessed is TB. Further scale-up and optimisation of the fermentation medium and process will be performed. Methacrylate monolith displays physical characteristics suited to the chromatographic purification of plasmid DNA – ease of functionalisation and large pore diameter leading to low mass transfer resistance whilst maintaining a suitable surface area. Methacrylate monolith will be used in further purification studies. Economics of plasmid DNA production for different media Monolith Characterization Results Pore size distribution and BET surface area analysis were carried out for the monolith as shown in figures A and B respectively. Results show a modal pore diameter of 350 nm which is suitable for use as a stationary phase adsorbent for plasmid DNA purification. The BET surface area of the monolith is 12 m2/g; the low value of the surface area shows the absence significant micro or mesoporosity Plasmid DNA production – A scaleable, whole-system approach to fermentation and downstream purification . The synthesis of plasmid DNA is greatly influenced by the medium of cultivation. The nutrients available in a particular medium effect the plasmid DNA volumetric yield and specific yield which in turn has ramifications for subsequent down stream purifications. A TB medium displayed economic advantages over the other medium on a lab scale. The dollar amount per μg of plasmid DNA produced from TB is 5.27 compared to the highest from LB which is 10.84 (with all other parameters kept constant). A) E.coli DH5α was used for the production of pUC19 (~2700bp). Plasmid DNA specific yield comparison between LB (1), TB (2) and SOC (3). Ethidium bromide agarose gel electrophoresis with 1% agarose in TAE buffer, 5 μg/ml EtBr at 66 V. Pictures showing bands of pDNA purified from LB, TB and SOC B A) Differential pore volume against pore diameter of monolith B) Nitrogen adsorption- desorption isotherm at 77 K A Acknowledgements: