Performance of a non-grafted monolithic support for purification of supercoiled plasmid DNA.
The use of therapeutics based on plasmid DNA (pDNA) relies on procedures that efficiently produce and purify the supercoiled (sc) plasmid isoform. Several chromatographic methods have been applied for the sc plasmid purification, but with most of them it is not possible to obtain the required purity degree and the majority of the supports used present low capacity to bind the plasmid molecules. However, the chromatographic monolithic supports are an interesting alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. The separation of pDNA isoforms, using short non-grafted monolithic column with CarbonylDiImidazole (CDI) functional groups, is described in the current work. The effect of different flow rates on plasmid isoforms separation was also verified. Several breakthrough experiments were designed to study the effect of different parameters such as pDNA topology and concentration as well as flow rate on the monolithic support binding capacity. One of the most striking results is related to the specific recognition of the sc isoform by this CDI monolith, without flow rate dependence. Additionally, the binding capacity has been found to be significantly higher for sc plasmid, probably because of its compact structure, being also improved when using feedstock with increased plasmid concentrations and decreased linear velocity. In fact, this new monolithic support arises as a powerful instrument on the sc pDNA purification for further clinical applications.
CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. fani.sousa@fcsaude.ubi.pt
A
A
Sousa
D
D
Bicho
C T
CT
Tomaz
F
F
Sousa
J A
JA
Queiroz
D000645:Ammonium Sulfate; D055598:Chemical Phenomena; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*
D004278:DNA, Superhelical; D000645:Ammonium Sulfate