A chromatographic method for determination of supercoiled plasmid DNA concentration in complex solutions.
2009 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 877, no 24, 2530-6 p.Article in journal (Refereed) Published
A method for determination of the plasmid DNA concentration with subsequent analysis of the ratio supercoiled to open circular form is presented. The method is suitable for samples from all steps of the manufacturing process, from fermentation to final product. The analysis consists of size exclusion chromatography, followed by analytical thiophilic aromatic chromatography. In the first step, the plasmid DNA concentration is determined by group separation, including removal of RNA and other impurities, within less than 2 min. The limit of detection and quantification was 0.28 and 0.83 microg/ml, respectively. The precision of the method is high, providing a coefficient of variation as low as below 2%. In the second step, the ratio of open circular to supercoiled plasmid DNA is determined following separation of the two plasmid DNA isoforms with a linear salt gradient. The precision of the second step was evaluated using serial injections of aliquots of a sample stock solution. In comparison with the two most commonly used methods, the developed analysis was found to be significantly more accurate than agarose gel electrophoresis and equivalent to capillary gel electrophoresis. The combined methods for quantification and control of homogeneity of plasmid DNA presented here enable reliable and precise analysis at all steps of the manufacturing process.
Place, publisher, year, edition, pages
2009. Vol. 877, no 24, 2530-6 p.
Concentration determination, Supercoiled plasmid DNA, Analytical group separation, Thiophilic aromatic chromatography
Research subject Chemistry with specialization in Surface Biotechnology
IdentifiersURN: urn:nbn:se:uu:diva-172792DOI: 10.1016/j.jchromb.2009.06.037PubMedID: 19616488OAI: oai:DiVA.org:uu-172792DiVA: diva2:515769