Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix.

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.