Circumventing Plasmid DNA Shear-Induced Degradation

University Of Colorado At Denver, Aurora CO

Investigators

Abstract

The goal of this project is to correlate degradation rates to strain rates in a fashion that creates general guidelines by which bioprocessing systems can be modified to minimize plasmid DNA degradation and maintain biological activity. To this end, specific hypotheses regarding mechanisms to minimize the impact of shear, and how to scale behaviors based on molecular characteristics, will be tested. This knowledge will then be applied to the maintenance of supercoil structure and biologic activity. These hypotheses include: (1) altering molecular diffusion times by increasing solution viscosity to protect DNA from shear induced degradation, (2) reducing shear stresses arising from velocity gradients by reducing the lengths of DNA particles, (3) increasing the level of turbulence which intuitively should promote conformational alterations that enhance covalent bond breakage, and (4) using the knowledge gained after testing in 1-3 toward protecting plasmids during atomization. Different aerosolisation strategies (e.g., turbulence, temperature, and particle size) will be analyzed with the help of computational analysis to optimize the atomization process. The success of an atomization strategy will be judged based on the molecular stability and biological activity of the plasmid DNA after aerosolisation.