Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography.
BACKGROUND
It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (sc) plasmid isoform. Plasmid DNA (pDNA) purification strategies that use amino acids as immobilized ligands have recently yielded interesting results.


METHODS
The present study describes a strategy that uses arginine-chromatography to specifically purify sc pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate.


RESULTS
Control analysis shows that the purity of the sc pDNA is 100% with a homogeneity higher than 97% of sc. Furthermore, no RNA was detectable, the protein content was lower than 10 microg/ml and a 117-fold reduction on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatographic process demonstrated an impressive performance on sc isoform recovery (79% yield). Furthermore, the sc transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control.


CONCLUSIONS
With the simplified sc pDNA purification process, a high yield was achieved, sc pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatography is thus an interesting option for use as a late stage plasmid purification step.
Sousa
Fani
F
CICS, Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal. fani.sousa@fcsaude.ubi.pt
Prazeres
Duarte M F
DM
Queiroz
João A
JA
D000818:Animals; D001120:Arginine / Q000737:chemistry*; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000302:isolation & purification* / Q000378:metabolism; D004731:Endotoxins / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000302:isolation & purification*; D014162:Transfection / Q000379:methods*
D004278:DNA, Superhelical; D004731:Endotoxins; D001120:Arginine