Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies

The stability of highly purified supercoiled plasmid DNA formulated in simp le phosphate or Tris-buffered saline solutions has been characterized, to e stablish the overall degradation processes that occur during storage in aqu eous solution. Plasmid DNA stability was monitored during accelerated stabi lity studies (at 50 degrees C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8-hydroxyguanosine residues over time. The effects of formulation pH, demet alation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on t he supercoiled content of plasmid DNA during storage at 50 degrees C were a lso determined. The results indicate that free radical oxidation may be a m ajor degradative process for plasmid DNA in pharmaceutical formulations unl ess specific measures are taken to control it by the addition of free radic al scavengers, specific metal ion chelators, or both. The generation of hyd roxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability a nd to inhibit the hydroxylation of phenylalanine; both observations are con sistent with the known ability of ethanol to serve as a hydroxyl radical sc avenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDT A) and ethanol had a synergistic enhancing effect on DNA stability. However , the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as p otent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and S-elimination were then determined, allowing accurate predictions of DNA st orage stability as a function of formulation pH and temperature. The abilit y to predict plasmid DNA storage stability in the absence of free radical o xidation should prove to be a valuable tool for the design of stable pharma ceutical formulations of plasmid DNA. (C) 2000 Wiley-Liss, Inc. and the Ame rican Pharmaceutical Association.