Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography

Abstract

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.

Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis

“…Hydrophobic interaction chromatography (HIC) has been shown to have significant utility for the separation of proteins from complex mixtures (Diogo et al, 2000(Diogo et al, , 2001Husi and Walkinshaw, 1999;Machold et al, 2003;O'Connor et al, 2000;Pomazal et al, 2002;Sunasara et al, 2003). HIC stationary phases are manufactured by attaching relatively weak hydrophobic functional groups to an agarose or polymer backbone .…”

Purification of plasmid (pVaxLacZ) by hydrophobic interaction chromatography

“…The best purification factor was obtained after HIC, using the precipitation with 2.5M ammonium sulphate, 51% yield. Studies accomplished by Diogo et al (2000) working in the purification of plasmid using hydrophobic interaction chromatography obtained similar results. Fig.…”

“…The use of the recombinant DNA with progress of the gene therapy or nucleic acid vaccination DNA in the cure of diseases such as cancer and AIDS, has increased the need to obtain pure plasmid of DNA (Diogo et al, 2000). In some research applications it is possible to use crude cell extracts of varying degrees of purity.…”

“…Large-scale purifications require scalable methods such as column chromatography (Prazeres et al, 1998). Hydrophobic interaction techniques have been used in the purification of therapeutic proteins, but so far there are no reports on the application of HIC for plasmid purification (Diogo et al, 2000). The use of HIC has be advantage of the different hydrophobic character of double-stranded plasmid DNA and other nucleic acids impurities with high content in single strands, such as RNA and denatured gDNA.…”

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

“…In addition, most recombinant proteins can be obtained at therapeutic grade of purity, by processes of the same structure . Then, HIC often forms part of processes to yield a purified macromolecule of biomedical interest, such as therapeutic proteins (Seely & Richey, 2001), DNA vaccines (Diogo et al, 2000), and enzymes (Teng et al, 2010), among others. Besides, the use of HIC to purify protein complexes (McCue et al, 2008), as well as to study protein folding from a thermodynamic point of view (Geng & Wang, 2007), have been reported.…”