Stabilization of DNA utilizing divalent cations and alcohol.
A novel method for protection of DNA from high shear induced damage is presented. This method uses simple divalent cations and the lyophilizable alcohol, tert-butanol, to self-assemble DNA into condensed, shear-resistant forms. The DNA used in these studies was a 5600 BP plasmid DNA encoding a therapeutic gene. Various solvents and salts were used to identify optimal conditions to condense plasmid DNA. A stable formulation was identified with plasmid DNA condensed in a cosolvent solution containing 20% (v/v) tert-butanol and 1mM calcium chloride. The DNA was formulated at 100 microg/ml and condensed into rod and toroidal shapes that were approximately 50-300 nm in diameter. The rods were found to be kinetically stable for greater than 24h following their preparation. Condensation of the plasmid DNA in this manner results in nearly 100% of the plasmid DNA remaining intact after 1 min of high shear stress applied by a 50 W probe sonicator. Uncondensed control plasmid DNA is completely fragmented following 30s of identical sonication. It is believed that condensation of DNA in this manner will permit utilization of high shear-stress inducing processing techniques, such as lyophilization or spray-drying without resulting in damage to the DNA.
Knight
Jefferson D
JD
Department of Pharmaceutical Research and Development, Pfizer Global Research and Development, Groton Laboratories, MS 8156-26, Groton, CT 06340, USA.
Adami
Roger C
RC
D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D002415:Cats; D002626:Chemistry, Pharmaceutical; D004247:DNA / Q000737:chemistry*; D004355:Drug Stability; D004921:Erythropoietin / Q000235:genetics*; D007700:Kinetics; D008854:Microscopy, Electron; D010316:Particle Size; D012997:Solvents / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical; D020002:tert-Butyl Alcohol / Q000737:chemistry*
D002413:Cations, Divalent; D012997:Solvents; D004921:Erythropoietin; D004247:DNA; D020002:tert-Butyl Alcohol