Purification of plasmid DNA using a novel two stage chromatography process

Abstract

The chromatography process of large-scale plasmid purification with high efficiency and low cost has always been a major challenge. We established a two-step plasmid chromatography purification process combining multimodal and thiophilic chromatography with an overall chromatography yield of nearly 70%. Capto Core 700, a multimodal core–shell particle, was firstly used to remove the impurities from the crude lysate. The effects of different experimental conditions on chromatography recovery and impurity removal were screened. Compared to conventional size exclusion chromatography, the sample load and flow rate of this step were enhanced by 40-fold and 5-fold, respectively, while maintaining a 90% yield. For the thiophilic chromatography (Capto PlasmidSelect), the method of Design of Experiments (DoEs) was used to study the influence of parameters on the results. The effects of ammonium sulfate concentration, sodium chloride concentration and flowrate in the elution phase were studied and optimized with a central composite design model consisting of 17 experiments. The versatility of this process was demonstrated by successfully purifying three different lentiviral packaging plasmids (pLP1, pLP2 and pLP/VSVG) and the target plasmid containing green fluorescent protein (GFP). Purified plasmids consistently achieved a supercoiled purity of at least 90% with endotoxin levels below 5 EU/mg. Lentiviral vectors packaged using these plasmids exhibited high infectious titers of 1 × 10⁷ TU/mL, thereby verifying the process applicability for diverse plasmid purification requirements.