Fani Sousa1, Duarte M F Prazeres, João A Queiroz. 1. CICS, Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal. fani.sousa@fcsaude.ubi.pt
Abstract
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. (c) 2008 John Wiley & Sons, Ltd.
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. (c) 2008 John Wiley & Sons, Ltd.