(493d) A Membrane-Based Purification Platform for Plasmid-DNA Production

Bacterial plasmid DNAs (pDNAs) have attracted significant attention in biopharmaceutical industries as vectors for gene therapy and DNA-based vaccines. With the growing demand for pDNAs for various therapeutic applications, large scale production and purification process is emerging as the major bottleneck. This study aims to evaluate membrane-based purification platform for pDNA production as an alternative to traditional column chromatography- or precipitation-based platforms.

E. coli cells were transformed with a model plasmid followed by harvesting with centrifugation and alkaline lysis. The lysis step led to a mixture of the desired form of pDNA (supercoiled-SC) in the broth along with cell debris, host cell proteins (HCPs), RNA and unwanted forms of DNA (open circular DNA-OC and linear DNA-L) as the major impurities. Removal of cell debris was conducted using two sequential depth filtration steps. Depth filtration steps were able to remove the particulates without significant loss in pDNA. Then, anion exchange (AEX) membrane chromatography with Mustang^® Q membrane was conducted to purify pDNA. Effect of operating parameters, such as buffer composition and flow rate, was investigated. Performance of membrane chromatography was compared with the standard AEX bead-based chromatography. Quantification of the purified supercoiled form of pDNA (SC) was conducted using fluorometer and HPLC. Qualitative analysis of the purified product was performed using gel electrophoresis. Detailed description of the experimental methods and in-depth analysis of the experimental results will be conducted during the presentation. This study is supported by Pall Corporation and hosted at KGI as a part of the Pall-KGI collaboration on Gene Therapy Initiative.