title: Thermodynamic study of the interaction between linear plasmid DNA and an anion exchange support under linear and overloaded conditions.
abstract: Anion-exchange chromatography has been successfully used in plasmid DNA (pDNA) purification. However, pDNA adsorption mechanism using this method is still not completely understood, and the prediction of the separation behavior is generally unreliable. Flow microcalorimetry (FMC) has proven its ability to provide an improved understanding of the driving forces and mechanisms involved in the adsorption process of biomolecules onto several chromatographic systems. Thus, using FMC, this study aims to understand the adsorption mechanism of linear pDNA (pVAX1-LacZ) onto the anion-exchange support Fast Flow (FF) Q-Sepharose. Static binding capacity studies have shown that the mechanism of pDNA adsorption onto Q-Sepharose follows a Langmuir isotherm. FMC experiments resulted in thermograms that comprised endothermic and exothermic heats. Endothermic heat major contributor was suggested to be the desolvation process. Exothermic heats were related to the interaction between pDNA and Q-Sepharose primary and secondary adsorption. Furthermore, FMC revealed that the overall adsorption process is exothermic, as expected for an anion-exchange interaction. Nevertheless, there are evidences of the presence of nonspecific effects, such as reorientation and electrostatic repulsive forces.
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, 6200-001 Covilhã, Portugal.
authors.forename: P A
authors.initials: PA
authors.lastname: Aguilar
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal; Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Cracow, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Twarda
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, 6200-001 Covilhã, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Sousa
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, 6200-001 Covilhã, Portugal. Electronic address: ccabral@fcsaude.ubi.pt.
authors.forename: A C
authors.initials: AC
authors.lastname: Dias-Cabral
keywords: Adsorption isotherms; Fast flow Q-Sepharose; Flow microcalorimetry; Ion-exchange chromatography; Plasmid DNA