Plasmid Isoform Separation and Quantification by Anion-Exchange Chromatography (AEX)

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Hua Yang, Stephan M. Koza, Weibin Chen
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Anion-exchange chromatography has advantages of consuming small amounts of sample and being able to yield robust and reproducible results using a standard LC instrumentation. While this technique has been widely used for plasmid purification/preparation,1,2 it has great potentials, as an analytical technique, in separating and quantifying plasmid isoforms, and provides a simple means for plasmid purity testing. Indeed, while not specifically investigated in this work, the substantial binding strength of plasmid DNA to the quaternary ammonium ligands of the column used may allow for plasmid analysis in low purity lysate without an additional sample clean-up step or the use of DNA specific dyes. Here, we report an analytical method which separates supercoiled, open circular, and linear form of a 5.4 kbp plasmid, using a Waters Protein-Pak Hi Res Q strong anion exchange Column. The relative quantification of the three primary plasmid forms are consistent for total sample loads ranging from approximately 117 ng to 1875 ng.

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