High-speed chromatographic purification of plasmid DNA with a customized biporous hydrophobic adsorbent

Interest in producing large quantities of plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Hydrophobic chromatography is a popular technique in the downstream processing of plasmid DNA. However, the low capacity and the high mass transfer resistance of most commercially available packings for bio-macromolecules limit their application in a large-scale process. In this work, a hydrophobic absorbent with wide pores was synthesized by the solid porogenic method. Analyses by scanning electron microscopy and mercury intrusion porosimetry revealed that the matrix contained two families of pores, i.e., micropores smaller than 100 nm and superpores of 500-7300 nm. The superpores provided not only convective flow channels for the mobile phase, but also a large surface for biomolecules binding. So the chromatographic process can be operated at high flow rate with high column efficiency and low backpressure as identified on a 2-mL column (5 mm i.d., 2 cm length). With a loading up to 2.6 mg of 5.4 kb plasmid (pcDNA3) in 8 mL feedstock and operated at a flow rate as high as 20 cm/min, nearly 100% of plasmid was recovered with a purity of 100%. The results indicate that the hydrophobic medium is promising for high-speed purification of plasmid DNA.