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Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

Wahlund, Per-Olof LU ; Gustavsson, Per-Erik LU ; Izumrudov, Vladimir LU ; Larsson, Per-Olof LU and Galaev, Igor LU (2004) In Biotechnology and Bioengineering 87(5). p.675-684
Abstract
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N,N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the... (More)
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N,N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production. (C) 2004 Wiley Periodicals, Inc. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biotechnology and Bioengineering
volume
87
issue
5
pages
675 - 684
publisher
John Wiley & Sons
external identifiers
  • pmid:15352066
  • wos:000223500700013
  • scopus:4644265849
ISSN
1097-0290
DOI
10.1002/bit.20180
language
English
LU publication?
yes
id
2e0e006f-4a94-427d-89a1-0763f3d24706 (old id 140593)
date added to LUP
2007-07-03 09:37:58
date last changed
2017-06-11 03:55:34
@article{2e0e006f-4a94-427d-89a1-0763f3d24706,
  abstract     = {The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N,N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production. (C) 2004 Wiley Periodicals, Inc.},
  author       = {Wahlund, Per-Olof and Gustavsson, Per-Erik and Izumrudov, Vladimir and Larsson, Per-Olof and Galaev, Igor},
  issn         = {1097-0290},
  language     = {eng},
  number       = {5},
  pages        = {675--684},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate},
  url          = {http://dx.doi.org/10.1002/bit.20180},
  volume       = {87},
  year         = {2004},
}