Purification of plasmid DNA using a novel two stage chromatography process

Area of Science:

• Biotechnology
• Chromatography
• Molecular Biology

Background:

• Large-scale plasmid purification presents challenges in efficiency and cost.
• Current methods often struggle to meet the demands for high-purity plasmid DNA required for therapeutic applications.

Purpose of the Study:

• To develop and optimize a cost-effective, high-efficiency two-step chromatography process for large-scale plasmid purification.
• To demonstrate the versatility of the developed process for various plasmid types, including those used in lentiviral vector production.

Main Methods:

• A two-step chromatography approach combining multimodal (Capto Core 700) and thiophilic (Capto PlasmidSelect) chromatography.
• Screening of experimental conditions for multimodal chromatography to optimize impurity removal and recovery.
• Design of Experiments (DoEs) with a central composite design to optimize elution parameters for thiophilic chromatography.

Main Results:

• The multimodal step significantly enhanced sample load (40-fold) and flow rate (5-fold) compared to size exclusion chromatography, with a 90% yield.
• The optimized thiophilic chromatography step, guided by DoEs, yielded high-purity plasmids.
• Overall chromatography yield reached nearly 70% with supercoiled purity ≥90% and endotoxin levels <5 EU/mg.
• Successful purification of diverse plasmids, including lentiviral packaging plasmids and GFP-tagged plasmids.

Conclusions:

• The developed two-step chromatography process offers an efficient and scalable solution for large-scale plasmid purification.
• The purified plasmids are suitable for producing high-titer lentiviral vectors (1 × 10^7 TU/mL), confirming process applicability.
• This method provides a robust platform for meeting diverse plasmid purification needs in biotechnology and gene therapy.