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Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

Kepka, Cecilia LU ; Lemmens, R; Vasi, J; Nyhammar, T and Gustavsson, Per-Erik LU (2004) In Journal of Chromatography A 1057(1-2). p.115-124
Abstract
An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the... (More)
An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%. (C) 2004 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Chromatography A
volume
1057
issue
1-2
pages
115 - 124
publisher
Elsevier
external identifiers
  • wos:000225143900014
  • pmid:15584230
  • scopus:7744222633
ISSN
0021-9673
DOI
10.1016/j.chroma.2004.09.060
language
English
LU publication?
yes
id
4532e3b3-f94d-4f33-a4fe-4d21a24108bb (old id 138655)
date added to LUP
2007-07-05 16:49:50
date last changed
2017-01-01 06:53:01
@article{4532e3b3-f94d-4f33-a4fe-4d21a24108bb,
  abstract     = {An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%. (C) 2004 Elsevier B.V. All rights reserved.},
  author       = {Kepka, Cecilia and Lemmens, R and Vasi, J and Nyhammar, T and Gustavsson, Per-Erik},
  issn         = {0021-9673},
  language     = {eng},
  number       = {1-2},
  pages        = {115--124},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography},
  url          = {http://dx.doi.org/10.1016/j.chroma.2004.09.060},
  volume       = {1057},
  year         = {2004},
}