title: Application of a ultra performance liquid chromatography method in the determination of DNA quality and stability.
abstract: The development of plasmid DNA as a pharmaceutical requires that integrity (i.e., supercoil content) be monitored as part of quality control. The standard method of determining supercoil content is gel electrophoresis followed by staining and imaging, which is complicated by a variety of factors. Previously described chromatographic methods used to quantify supercoil content have had difficulty obtaining reliable separation of the different isoforms. Using ultra performance liquid chromatography, we have optimized buffer conditions, and utilized increased column temperatures in developing a method that allows accurate quantification of each isoform by ultraviolet detection. We found that increasing the column temperature to 55°C improved separation of the isoform peaks as well as increased the resolution of each peak. We demonstrate the utility of this method by quantifying supercoil content of samples subjected to sonication, acidification or lyophilization, and storage. Our results demonstrate that this method allows for a precise quantification of individual DNA isoforms within a heterogeneous sample.
authors: University of Colorado School of Pharmacy, Aurora, Colorado 80045, USA.
authors: Jamie
authors: J
authors: Betker
authors: Tyson
authors: T
authors: Smyth
authors: Wei
authors: W
authors: Wang
authors: Thomas J
authors: TJ
authors: Anchordoquy
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004270:DNA, Circular / Q000737:chemistry*; D005612:Freeze Drying; D010957:Plasmids / Q000737:chemistry*; D013010:Sonication; D013696:Temperature
chemical_list: D004270:DNA, Circular