Browsing by Author "Dutra, Gregory Silva"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- Understanding the interaction between pDNA and different chromatographic supportsPublication . Dutra, Gregory Silva; Cabral, Ana Cristina Mendes DiasGene Therapy and DNA vaccines are recent therapies that take advantage of the potential of plasmid DNA as a therapeutic molecule for the treatment and cure of several diseases. In the last decade both techniques have received a great deal of attention by pharmaceutical companies due to their simplicity, versatility and safe profile. Hence, the usage of pDNA as biopharmaceutical molecule requires its production at the gram scale, with a high purity and homogeneity level as a vital parameter to ensure a good response and the patient safety. Anion-exchange chromatography and hydrophobic interaction chromatography have been successfully used in pDNA purification. Nevertheless, the mechanism of pDNA separation for both purification techniques is still not completely understood. A better understanding of the driving forces and mechanisms underlying both purification processes are of great interest to help optimize chromatographic systems. Flow Microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces, mechanisms and kinetics involved in the interaction process during biomolecules adsorption onto several chromatographic systems]. Thus, using Flow Microcalorimetry as a central technique, this study aims to understand and compare the interaction between different pDNA isoforms (pVAX1-LacZ) and the anion-exchange support (Q-sepharose Fast Flow) or the hydrophobic support (Phenyl Sepharose 6 Fast Flow), considering only the isotherm linear conditions, showing the role of nonspecific effects on the adsorptive process. The results obtained in the binding capacity studies revealed that the ln pDNA adsorption process follows a Langmuir isotherm up to a specific ln pDNA equilibrium concentration. In all cases, the binding capacity increases with the plasmid concentration in equilibrium until it reaches a level at which saturation of the chromatographic medium is achieved. The anion-exchange support was found to have a higher binding capacity for ln pDNA adsorption than the hydrophobic interaction support, as expected. FMC results reveled that for both processes the endothermic heat major contributor was suggested to be the desolvation and changes in the solvation shell processes while exothermic heats were related to the interaction (electrostatic attraction) between pDNA and support and also to the secondary adsorption of already adsorbed pDNA molecules. The enthalpies of adsorption showed that the overall adsorption process is mainly enthalpically driven for the adsorption of ln pDNA onto Phenyl Sepharose and entropically driven for the sc pDNA-Q-sepharose system.