Supercoiled plasmid quality assessment by analytical arginine-affinity chromatography.
Supercoiled plasmids are an important component of gene-based delivery vehicles, applied in new therapeutic strategies such as gene therapy or DNA vaccination. However, aiming at the general distribution of plasmid DNA (pDNA) therapeutics requires a procedure to easily and efficiently assess the purity and recovery yield of the supercoiled (sc) plasmid isoform. Based on affinity interactions between amino acids and nucleic acids, an arginine affinity methodology with UV detection was established to quantify and to control the quality of sc plasmid biopharmaceuticals. The fact that this new technique allows to distinguish between plasmid isoforms represents an advantage, since it allows the selective quantification of the biologically active pDNA topology, and a more accurate analysis of the quality of the isolated plasmid. The analytical experiments were performed in 12 min and the method was found to be accurate, precise, reproducible and linear for a sc plasmid concentration range between 2 and 150 μg/mL. In comparison with other established methods used in the quantification of native pDNA (oc+sc), the main advance introduced by this new method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, ensuring the purity of plasmid products to be therapeutically applied.
CICS-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, 6201-001 Covilhã, Portugal. fani.sousa@fcsaude.ubi.pt
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Queiroz
D001120:Arginine / Q000737:chemistry*; D002138:Calibration; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D016503:Drug Delivery Systems; D004587:Electrophoresis, Agar Gel; D015316:Genetic Therapy; D016014:Linear Models; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004278:DNA, Superhelical; D001120:Arginine