title: Effect of chromatographic conditions and plasmid DNA size on the dynamic binding capacity of a monolithic support.
abstract: DNA therapies are becoming recognized alternatives for the treatment and prevention of severe pathologies. Although most current trials have used plasmids <10 kbp, in the future larger plasmids would be required. The purpose of this work was to study the chromatographic behavior of nongrafted carbonyldiimidazole monolithic disks using plasmids with different sizes under hydrophobic conditions. Thereunto, the purification of several plasmids was performed. Higher size plasmids needed lower ammonium sulfate concentration, due to the greater number of interactions between the plasmids and monolith. The dynamic binding capacity experiments for the different plasmids revealed a lower capacity for bigger plasmids. It was also verified that the increase of salt concentration from 2.5 to 3 M of ammonium sulfate increased the capacity. At the highest salt concentration, a slight improvement in the capacity using lower flow rate was observed, possibly due to compaction of plasmid molecules and its better organization on the monolith channels. Finally, a low pH also had a positive effect on the capacity. So, this monolithic support proved to be appropriate to purify the supercoiled isoform of different plasmids with different sizes, providing a valuable instrument as a purification technique.
authors.affiliation: CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, Covilhã, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Bicho
authors.forename: Ângela
authors.initials: Â
authors.lastname: Sousa
authors.forename: Fani
authors.initials: F
authors.lastname: Sousa
authors.forename: João
authors.initials: J
authors.lastname: Queiroz
authors.forename: Cãndida
authors.initials: C
authors.lastname: Tomaz
mesh_terms: D000327:Adsorption; D000645:Ammonium Sulfate / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry* / Q000302:isolation & purification; D057927:Hydrophobic and Hydrophilic Interactions; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification; D012117:Resins, Synthetic / Q000737:chemistry
chemical_list: D004278:DNA, Superhelical; D012117:Resins, Synthetic; D000645:Ammonium Sulfate
keywords: Affinity chromatography; Dynamic binding capacity; Monoliths; Supercoiled plasmid DNA