Dissertation / PhD Thesis/Book

PreJuSER-50226

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Entwicklung und Untersuchung eines Verfahrens zur integrierten Aufreinigung von Plasmid DNA mittels wäßriger Zweiphasenextraktion

Frerix, A. (Corresponding author)FZJ*

2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 3-89336-442-0

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Please use a persistent id in citations: http://hdl.handle.net/2128/2477

Abstract: Plasmids are double stranded circular DNA molecules, mainly known as vectors for transferring genes. Therefore, plasmids have a high importance for molecular biology and gene technology respectively. Presently, there is a high interest to utilize plasmids in gene therapy and DNA vaccination due to their ability to replace or supplement genes. Compared with viral based gene transfer, the use of plasmid for gene therapy is less risky, however, efficiency of transfection and duration of expression are relatively low. Thus, high doses per application are necessary, making effective processes for the manufacture of large quantities of plasmids in pharmaceutical quality essential. The manufacturing of plasmid DNA is performed using biotechnological processes by cultivation of optimized E. coli strains. Since current downstream processing is comparatively elaborate, there is a high demand for new approaches. This thesis deals with the development of an alternative and cost-effective purification process based on aqueous two phase extraction (ATPS). The developed three step process begins with an alkaline lysis of bacterial cells and separation of crude impurities and cell debris using two phase extraction. In this study, a systematic screening led to an optimal system comprising 15 % PEG 800 and 20 % potassium phosphate. This system resulted in a plasmid DNA recovery of about 90 % plasmid recovery in the bottom phase, while most of contaminants together with solids (cell precipitate) are separated into the top phase. It was found that conditions of the standard alkaline lysis can be adopted. Reducing the duration of neutralization by immediate extraction after lysis stabilizes plasmid DNA due to separation of nucleases to the opposite phase. By increasing the temperature of extraction, plasmid DNA yields could be optimized, however, to the expense of purity. Partitioning experiments of plasmid DNA and RNA, performed as a function of temperature, pH and PEG molecular weight, showed a close relation between system conditions and partitioning of plasmid DNA. In the investigated two phase system it was found that changing the temperature from 15 to 20 °C leads to a partitioning of plasmid DNA from top to bottom phase. This could be attributed to the plasmid solubility in the top phase, which is drastically affected by variation of PEG molecular weight and temperature [...]

Note: Record converted from VDB: 12.11.2012
Note: RWTH Aachen, Diss., 2006

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Contributing Institute(s):

1. Biotechnologie 2 (IBT-2)

Research Program(s):

1. Biotechnologie (PBT)

Appears in the scientific report 2006

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Database coverage:

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