Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography.
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.
Diogo
M M
MM
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1000 Lisboa, Portugal.
Queiroz
J A
JA
Monteiro
G A
GA
Martins
S A
SA
Ferreira
G N
GN
Prazeres
D M
DM
D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000302:isolation & purification*; D004731:Endotoxins / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011786:Quality Control
C505032:CFTR protein, human; D004731:Endotoxins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator