Analytical and biological characterization of supercoiled plasmids purified by various chromatographic techniques

Irina Tumanova, Jean Boyer, S. Fernando Ausar, Joseph Burzynski, Dana Rosencrance, Julie White, Jason Scheidel, Rose Parkinson, Henry Maguire, C. Russell Middaugh, David Weiner, Anthony P. Green

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


Supercoiled plasmids are an important component of gene-based delivery vehicles. A number of production methods for clinical applications have been developed, each resulting in very high-quality product with low levels of residual contaminants. There is, however, no consensus on the optimal methods to characterize plasmid quality, and further, to determine if these methods are predictive of either product stability or biological activity. We have produced two plasmids using four production purification methodologies based on PolyFlo® and hydrophobic interaction chromatography (HIC), either alone or in tandem processes. In each case, the product was analyzed using standard molecular biological methods. We also performed a number of biophysical analyses such as dynamic light scattering (DLS), circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Minimal differences were detected among the preparations based on the more standard molecular biological methods. Some small differences were detected, however, using biophysical techniques, particularly FTIR and DSC, which may reflect small variations in plasmid tertiary structure and thermal stability. Stability after heat exposure at 60°C, exposure to fetal bovine serum and long-term storage at 4°C varied between plasmids. One plasmid showed no difference in stability depending on the production process, but the other showed significant differences. Evaluation in vivo in models for gene immunization and gene therapy showed significant differences in the response depending on the method of purification. Preparations using a tandem process of PolyFlo used in two separation modes provided higher biological activity compared to a tandem HIC/PolyFlo process or either resin used alone in a single column process. These data indicate that the process by which supercoiled plasmids are made can influence plasmid stability and biological activity and emphasize the need for more rigorous methods to evaluate supercoiled plasmids as gene-delivery vehicles.

Original languageEnglish (US)
Pages (from-to)819-831
Number of pages13
JournalDNA and Cell Biology
Issue number12
StatePublished - Dec 2005
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology


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