Separation and purification of linear covalently closed deoxyribonucleic acid by Q-anion exchange membrane chromatography

Abstract

We have constructed an in vivo system for rapid, scalable production of linear covalently closed (LCC) DNA from precursor circular covalently closed (CCC) plasmid DNA (pDNA) that offers a stronger safety profile compared to conventional CCC pDNA vectors. In the processing of LCC DNA products from the precursor CCC pDNA, LCC minivector DNA is produced in addition to other precursor DNA species and isoforms. DNA purification by anion exchange chromatography (AEC) attains high vector purity, making it an efficient and valuable approach to purification processes for the production of clinical grade DNA. Membrane chromatography offers significant advantages over traditional column chromatography including large convective pores, higher binding capacities, high throughput, scalable purification processes, and disposability. A hydrogel-based strong Q-anion exchange membrane for anion exchange chromatography can bind DNA with high capacity and recovery upon purification. We exploited these membrane properties in the separation of DNA sizes and isoforms to purify LCC DNA. We employed a NaCl concentration gradient at varying flow rates to successfully achieve effective separation of parental supercoiled CCC pDNA from processed isogenic LCC derivatives generated by the LCC DNA vector production system. We propose that anion exchange membrane chromatography is well positioned to play an integral role in large scale LCC DNA vector purification, successfully separating vectors by DNA isoforms.