화학공학소재연구정보센터
Separation and Purification Technology, Vol.182, 94-100, 2017
A process for supercoiled plasmid DNA purification based on multimodal chromatography
Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and prevention of genetic and acquired diseases. The large-scale manufacturing of plasmid DNA (pDNA) vectors, and the downstream processing in particular, is one of the key aspects of process development required to move non-viral gene therapy to the clinic. To address the problematic isolation and purification of pDNA molecules, an alternative and cost effective process based on multimodal chromatography was developed. In particular, the possibility of using a cationic multimodal ligand (Capto(TM) adhere) to remove RNA impurities from E. coli pre-purified lysates and to isolate supercoiled (sc) pDNA isoforms from open circular (oc) pDNA was explored. The process involves E. coli culture, cell harvesting and alkaline lysis followed by isopropanol, ammonium acetate and PEG-8000 precipitation. Finally, sc pDNA is isolated by multimodal chromatography using a stepwise NaCl elution method that includes washing of unbound oc pDNA at 830 mM NaCl, sc pDNA elution at 920 mM and removal of RNA at 2 M. The method provided baseline separation of isoforms and yielded sc-rich fractions (> 90%) that are virtually free from RNA and have levels of gDNA (1.3 +/- 0.3% mu g gDNA/mu g sc pDNA) and protein (4.97 +/- 1.34 mu g/mL) impurities within specifications. Approximately 376 mu g of sc pDNA were obtained from 200 mL of bacterial cell culture (OD600nm approximate to 19). The process was reproducible and performed similarly with differently sized plasmids (2686 bp, 3696 bp and 10,410 bp). (C) 2017 Elsevier B.V. All rights reserved.