Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography.
A hydrophobic interaction HPLC method was developed for the quantification of plasmid DNA and assessment of its purity in crude Escherichia coli lysates and other process streams. A Phenyl Sepharose Source (Amersham Biosciences) column was used to separate the double-stranded plasmid DNA molecules from the more hydrophobic impurities present in the process streams. The method is rapid (each analysis takes 7 min), reproducible, easy to perform and does not require previous digestion of RNA in samples with RNase or other pre-treatment. Furthermore, it is capable of handling heavily contaminated samples, with less than 5% of plasmid DNA thus constituting a good alternative to other less robust analytical techniques currently in use.
Diogo
M M
MM
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Queiroz
J A
JA
Prazeres
D M F
DM
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis*; D010957:Plasmids; D015203:Reproducibility of Results; D012996:Solutions / Q000737:chemistry*
D012996:Solutions; D004247:DNA