Developing an RNase-free bioprocess to produce pharmaceutical-grade plasmid DNA using selective precipitation and membrane chromatography

• Published in: Separation and purification technology 2011-11, Vol.83, p.121-129
• Main Authors: Zhong, Luyang, Srirangan, Kajan, Scharer, Jeno, Moo-Young, Murray, Fenner, Drew, Crossley, Lisa, Howie Honeyman, C., Suen, Shing-Yi, Perry Chou, C.
• Format: Article
• Language: English
• Subjects: anion exchange; Anion-exchange membrane chromatography; Applied sciences; bioprocessing; Calcium chloride; Chemical engineering; chromatography; Drinking water and swimming-pool water. Desalination; endotoxins; Exact sciences and technology; Ion exchange; Isopropanol; isopropyl alcohol; Membrane separation (reverse osmosis, dialysis...); Plasmid DNA; plasmids; Pollution; product quality; proteins; ribonucleases; RNA; RNase-free; Water treatment and pollution
• ISSN: 1383-5866; 1873-3794
• DOI: 10.1016/j.seppur.2011.09.024

► We developed a novel, RNase-free and scalable bioprocess for plasmid DNA production. ► Calcium chloride precipitation was optimized to pre-treat Escherichia coli lysates. ► Anion-exchange membrane chromatography was used as a major polishing step. ► Selective precipitation with isopropanol was implemented as a key coupling step. ► Complete removal of various major contaminants was successfully demonstrated. A novel, RNase-free, and potentially scalable bioprocess was developed for the production of pharmaceutical-grade plasmid DNA. High bioprocess recovery and product quality were primarily associated with the optimal integration of impurity removal by calcium chloride precipitation and anion-exchange membrane chromatography and the implementation of isopropanol precipitation as a coupling step between the two impurity-removing steps. Complete removal of total cellular RNA was demonstrated without the use of animal-derived RNase. High-molecular-weight (HMW) RNA and genomic DNA (gDNA) were removed by selective precipitation using calcium chloride. Complete removal of the remaining low-molecular-weight (LMW) RNA was achieved by membrane chromatography using a high-capacity hydrogel-based strong anion-exchange membrane. The simultaneous achievement of desalting, concentrating and buffer exchange by the coupling step of isopropanol precipitation and the high efficiency and resolution of DNA–RNA separation by anion-exchange membrane chromatography significantly reduced the operating complexity of the overall bioprocess, increased the overall recovery of plasmid DNA, and enhanced product quality by removing trace amounts of major impurities of concern for biomedical applications, such as gDNA, proteins, and endotoxin.