Anion exchange purification of plasmid DNA using expanded bed adsorption

Area of Science:

• Biotechnology
• Bioprocessing
• Chromatography

Background:

• Gene therapy and DNA vaccination drive demand for pharmaceutical-grade plasmid DNA.
• High viscosity of plasmid process streams causes high back pressures in chromatography, limiting throughput.

Purpose of the Study:

• To evaluate expanded bed anion exchange chromatography as an alternative to fixed bed chromatography for plasmid DNA purification.
• To address challenges posed by high viscosity in plasmid purification processes.

Main Methods:

• Utilized a Streamline 25 column with Streamline QXL media.
• Equilibrated column with 0.5 M NaCl in TE buffer at an upward flow of 300 cmh-1.
• Injected E. coli lysates, washed unbound material, allowed media to sediment, and eluted plasmid with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1.

Main Results:

• Achieved purification factors of 36-fold, 26% purity, and near 100% yield with <1 column volume of feed.
• Processing larger volumes (250 ml) resulted in decreased recovery (35%) and purity (5%), with observed gel clogging and expansion collapse.
• Isopropanol precipitation prior to chromatography enabled processing of larger volumes, enhancing the purification step.

Conclusions:

• Expanded bed anion exchange chromatography is effective for plasmid DNA purification at smaller scales.
• Viscosity-related issues limit scalability; pre-treatment steps like isopropanol precipitation are necessary for processing larger volumes.
• Optimized chromatography methods are crucial for efficient, large-scale plasmid DNA production for therapeutic applications.