This title appears in the Scientific Report :
2005
Please use the identifier:
http://dx.doi.org/10.1042/BA20040107 in citations.
Scalable recovery of plasmid DNA based on aqueous two-phase separation
Scalable recovery of plasmid DNA based on aqueous two-phase separation
Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are req...
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Personal Name(s): | Frerix, A. |
---|---|
Müller, M. / Kula, M.-R. / Hubbuch, J. | |
Contributing Institute: |
Biotechnologie 2; IBT-2 |
Published in: | Biotechnology and applied biochemistry, 42 (2005) S. 57 - 66 |
Imprint: |
Hoboken, NJ [u.a.]
Wiley
2005
|
Physical Description: |
57 - 66 |
DOI: |
10.1042/BA20040107 |
PubMed ID: |
15612880 |
Document Type: |
Journal Article |
Research Program: |
Biotechnologie |
Series Title: |
Biotechnology and Applied Biochemistry
42 |
Subject (ZB): | |
Publikationsportal JuSER |
Future developments in gene therapy and DNA vaccination depend on cost-effective large-scale production of pharmaceutical-grade pDNA (plasmid DNA). Given the large amount of impurities present in the feedstock, purification processes that have high specificity and capacity at a moderate cost are required. In the present study, we describe a non-chromatographic procedure based on aqueous two-phase extraction allowing a fast and simply scalable capture step. PEG [poly(ethylene glycol)] in combination with potassium citrate or potassium phosphate was tested as phase component for extraction. By increasing either PEG or salt concentration, the partitioning of nucleic acids changed from bottom to top phase. Phase systems with a composition of 15% PEG 800 and 20% potassium phosphate at pH 7.0 showed a strong partitioning of pDNA to the bottom phase, linked to a clear decrease in open circular pDNA, while proteins, genomic DNA and RNA remain at the top or at the interphase. A great advantage of the current process is that the complete procedure of lysis, precipitation, clarification and extraction can be performed in a single vessel. The number of denatured and sheared genomic DNAs in a spiking experiment was found to be depleted by more than 99%. |