Development of a phenyl membrane chromatography-based process yielding pharmaceutical grade plasmid deoxyribonucleic acid for mammalian cells transfection.

Production of plasmid DNA pharmaceuticals requires fast, robust and cost effective methodologies able to deliver high amounts of the target molecule in short periods of time. Membrane adsorbers can be tailored and operated to suit such criteria. This study focuses on the impact of pDNA samples produced by a membrane chromatography-based purification methodology on the transfection efficiency of CHO cells. Chromatographies were performed with 5mL of plasmid-containing clarified bacterial lysate each on a Sartorius® Phenyl 3mL spiral cartridge using a bind and elute mode to purify the GFP expressing pVAX1/GFP model plasmid. The developed methodology could deliver up to 285µg pDNA samples per run that were virtually RNA free (over 99% removal) and chromatographic step yields of 85%. The purified samples had a reduced content of OC pDNA (∼15% less in average). Additionally, robustness of the process was assessed up to nine chromatographic runs without noticing any relevant loss in chromatographic performance and transfection capabilities. The increase of productivity was also studied by increasing the flow rate 5 fold-a maximum productivity of 100µg pDNA/(hmL-BV) was achieved. The pDNA samples produced led to transfection efficiencies that were comparable among all experiments-72% and within 4% relative standard deviation when samples were produced using a lower throughput. Transfection efficiencies obtained by the membrane process were comparable to a combined HIC/SEC bead-based purification process, with values ranging within 74-113% of the values obtained from the latter.

Impact of plasmid size on the purification of model plasmid DNA vaccines by phenyl membrane adsorbers.

Plasmid DNA (pDNA) offers a versatile platform for the development of new pharmaceuticals. This versatility also adds in variability among plasmid products most of the times sharing only the same basic molecular structure. Membrane chromatography experiments performed with a Sartorius(®) Phenyl 3 mL spiral cartridge and differently sized plasmids (3.70 kbp, 6.05 kbp and 10.4 kbp) show that the strength of interaction of pDNA isoforms with HIC membrane adsorbers depends on size. These differences in relative binding strength were explored using a stepwise elution strategy of decreasing buffer conductivities in order to increase the purity of supercoiled (SC) pDNA isoforms. The open circular (OC) isoforms of all plasmids eluted earlier at a similar conductivity of 190 mS/cm, independently of the hydrodynamic diameter (Dh). A drop in conductivity of 16.0 mS/cm, 23 mS/cm and 19 mS/cm had to be imposed to elute the supercoiled (SC) counterparts of the 3.70 kbp, 6.05 kbp and 10.4 kbp, respectively. This corresponds to relative binding strengths of the SC over OC isoforms of 1.09, 1.14 and 1.11. Unlike the OC isoforms, the behavior of SC isoforms was dependent of the Dh. The purified and pooled plasmid fractions were assayed and demonstrated high degree of purity, compliant with regulatory agencies criteria: over 99% RNA removal, endotoxin levels below 0.001 EU/µg pDNA and undetectable protein content by BCA assay.