Plasmid DNA processing for gene therapy and vaccination: Studies on the membrane sterilisation filtration step

Membrane filtration through 0.2 μm pores is typically the last operation in the production of pharmaceutical grade plasmid DNA. The membrane sterilisation of purified DNA solutions containing plasmids and bacterial artificial chromosomes (BAC) is investigated in this paper. A linear relationship between total DNA transmission and vector size was observed when filtering through 0.2 μm polyvinylidene difluoride (PVDF) membranes. The percentage of DNA transmission assessed spectrophotometrically varied from 98 to 13% for vector sizes ranging from 6 to 116 kb. There was no significant change in transmission during filtration when controlled flux was increased from 0.1 to 2.3 mL/min cm2 or DNA concentration changed from 25 to 100 μg/mL. For vectors ≥20 kb; (i) the level of backbone breakage increased with molecular weight, flux and number of filtration passes; (ii) consecutive filtration experiments indicated that greater DNA loss occurred during the first pass of filtration; and (iii) the use of polyethersulfone (PES) membranes with asymmetrical pores improved DNA transmission and decreased DNA damage. The addition of 150 mM NaCl in the formulation buffer improved filtration transmission by 47 and 11% for the 72 and 116 kb vectors, respectively. Complexation with polyethylenimine (PEI) and a lipid–integrin binding peptide (LI) complex did not improve product transmission.