Biomedical Engineering Reference
In-Depth Information
Source Microorganism
Fermentation
Cellular recovery and lysis
Removal of cell debris
Plasmid precipitation
Chromatographic purification
Concentration, if required
Formulation and packaging
Figure 14.11
Overview of the manufacturing process for the large-scale production of plasmid DNA. Refer
to the text for further details
SDS-protein complexes, which can subsequently be removed by centrifugation or fi ltration. The
plasmids can then themselves be precipitated from the resultant solution by the addition of ap-
propriate solvent (usually either isopropanol or ethanol). Upon resuspension, the plasmid prepara-
tion can then be subjected to chromatographic purifi cation. The major contaminants likely still
present include RNA, genomic DNA fragments, nicked or other plasmid variants, and endotoxins.
Gel-fi ltration chromatography can effectively remove contaminants that differ substantially in
shape/size from the desired plasmid. These can include most genomic DNA fragments, RNA and
(most) endotoxins. It can also achieve partial removal of plasmid variants, such as open circular
plasmids from the main (supercoiled) plasmid preparation. Ion exchange can remove many protein
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