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Precipitation of RNA impurities with high salt in a plasmid DNA purification process: Use of experimental design to determine reaction conditions
Author(s) -
EonDuval Alex,
Gumbs Karlene,
Ellett Christopher
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10704
Subject(s) - precipitation , rna , plasmid , dna , salt (chemistry) , plasmid preparation , chemistry , calcium , chromatography , impurity , chloride , recombinant dna , biochemistry , organic chemistry , gene , physics , meteorology , pbr322
The use of high salt solution to precipitate RNA in a pharmaceutical‐grade plasmid DNA purification process was investigated. Five antichaotropic salts were tested for their potential to precipitate RNA. Calcium chloride was by far the best precipitant with high RNA removal in a very short incubation time. Calcium chloride precipitation conditions were investigated at two stages of a plasmid purification process using experimental design techniques. The effect of up to five factors on RNA precipitation and plasmid recovery was assessed by statistical modeling. Optimized conditions for calcium chloride precipitation were then introduced to the plasmid purification process resulting in the efficient removal of most impurities (RNA, chromosomal DNA, proteins, and endotoxins). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 544–553, 2003.