Abstract:
: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%.
journal_name
Biotechnol Appl Biochemjournal_title
Biotechnology and applied biochemistryauthors
Frerix A,Müller M,Kula MR,Hubbuch Jdoi
10.1042/BA20040107subject
Has Abstractpub_date
2005-08-01 00:00:00pages
57-66issue
Pt 1eissn
0885-4513issn
1470-8744pii
BA20040107journal_volume
42pub_type
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