Developing an RNase-free bioprocess to produce pharmaceutical-grade plasmid DNA using selective precipitation and membrane chromatography
| Autor: | Shing-Yi Suen, C. Perry Chou, C. Howie Honeyman, Murray Moo-Young, Drew Fenner, Jeno M. Scharer, Kajan Srirangan, Luyang Zhong, Lisa Crossley |
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| Rok vydání: | 2011 |
| Předmět: |
0303 health sciences
Chromatography Resolution (mass spectrometry) RNase P Precipitation (chemistry) 010401 analytical chemistry chemistry.chemical_element RNA Filtration and Separation Calcium 01 natural sciences 0104 chemical sciences Analytical Chemistry 03 medical and health sciences genomic DNA Membrane chemistry Biochemistry Bioprocess 030304 developmental biology |
| Zdroj: | Separation and Purification Technology. 83:121-129 |
| ISSN: | 1383-5866 |
| DOI: | 10.1016/j.seppur.2011.09.024 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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