Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1‐CF...

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Bibliographic Details
Published in:Biotechnology and bioengineering 2000-06, Vol.68 (5), p.576-583
Main Authors: Diogo, M. M., Queiroz, J. A., Monteiro, G. A., Martins, S. A. M., Ferreira, G. N. M., Prazeres, D. M. F.
Format: Article
Language:English
Subjects:
Ammonium compounds
Chromatography - methods
Chromatography, High Pressure Liquid
cystic fibrosis
Cystic Fibrosis Transmembrane Conductance Regulator - genetics
Cystic Fibrosis Transmembrane Conductance Regulator - isolation & purification
DNA
Endotoxins - analysis
Escherichia coli - chemistry
Escherichia coli - genetics
gene therapy
Genetic Therapy - methods
Genetic Vectors - isolation & purification
Humans
Hydrophobic chromatography
hydrophobic interaction chromatography
nucleic acids
plasmid purification
Plasmids - genetics
Precipitation (chemical)
Purification
Quality Control
RNA
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Summary:The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1‐CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4‐butanediol‐diglycidylether. The use of HIC took advantage of the more hydrophobic character of single‐stranded nucleic acid impurities as compared with double‐stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14‐cm HIC column. Anion‐exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (
ISSN:0006-3592
1097-0290
DOI:10.1002/(SICI)1097-0290(20000605)68:5<576::AID-BIT13>3.0.CO;2-5