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J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Jan 26;978-979:145-50. doi: 10.1016/j.jchromb.2014.12.004. Epub 2014 Dec 13.

Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

Author information

1
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
2
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. Electronic address: angela@fcsaude.ubi.pt.

Abstract

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

KEYWORDS:

Affinity chromatography; Arginine monolith; Composite Central Face design; Design of experiments; HPV-16 E6/E7 vaccine; Supercoiled plasmid DNA

PMID:
25544011
DOI:
10.1016/j.jchromb.2014.12.004
[Indexed for MEDLINE]

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