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Stabilization of DNA utilizing divalent cations and alcohol.
Jefferson D Knight1, Roger C Adami
1Department of Pharmaceutical Research and Development, Pfizer Global Research and Development, Groton Laboratories, MS 8156-26, Groton, CT 06340, USA.
International Journal of Pharmaceutics
|September 16, 2003
Summary
We developed a new method to protect therapeutic plasmid DNA from damage using simple chemicals. This process condenses DNA into stable forms, preserving its integrity during high-shear stress for applications like lyophilization.
Area of Science:
- Biochemistry
- Molecular Biology
- Materials Science
Background:
- High shear stress can damage plasmid DNA, limiting its use in processing techniques.
- Protecting therapeutic DNA is crucial for its stability and efficacy in pharmaceutical applications.
Purpose of the Study:
- To present a novel method for protecting plasmid DNA from high shear-induced damage.
- To identify optimal conditions for DNA condensation using divalent cations and tert-butanol.
- To enable the use of high shear-stress processing techniques for DNA formulations.
Main Methods:
- Plasmid DNA (5600 BP) was condensed using various solvents and salts.
- Optimal condensation achieved in a cosolvent solution of 20% tert-butanol and 1mM calcium chloride.
- DNA condensation resulted in rod and toroidal shapes (50-300 nm diameter).
Main Results:
- Condensed plasmid DNA showed nearly 100% integrity after 1 min of high shear stress (50 W probe sonicator).
- Uncondensed control DNA fragmented completely within 30s under identical conditions.
- Condensed DNA rods demonstrated kinetic stability for over 24 hours.
Conclusions:
- This novel condensation method effectively protects plasmid DNA from high shear stress.
- The method allows for the application of shear-inducing processing techniques like lyophilization and spray-drying.
- This approach enhances the stability and utility of therapeutic DNA formulations.