Purification Of Supercoiled Plasmid DNA By Arginine-affinity Chromatography

Compared with viral vectors, simple plasmid DNA injection appears moreattractive because of its low toxicity and immunogenicity, improved safety profiles,and ease of manufacture that has largely been used in gene therapy and DNAvaccination. Amino acid-affinity chromatography based on specific interactionbetween the supercoiled plasmid and immobilized amino acid ligands which presentshigh efficiency and low cost. The concept of using less-selective ligands has also beenapplied to pDNA purification, mainly by using amino acids as immobilized ligands.Studies have shown that arginine-affinity chromatography can selectively purifysupercoiled plasmid DNA, the purification condition is mild and ease to manufacture.In this study, a series of novel arginine ligand-attached agarose-based beads with3-and10-carbon spacer arms as well as different ligand densities were prepared toinvestigate their chromatographic performance in the purification of pDNA.Firstly, a series of arginine-modified agarose beads of different ligand densitieswith3-or10-carbon spacer arms were fabricated with Sepharose CL-6B gel. Prior tothe preparation of the arginine beads, epichlorohydrin and1,4-Butanediol diglycidylether were applied to activate the agarose-based Sepharose CL-6B gel so that spacerarms with different lengths were introduced. The results indicated that the arginineligand density of the affinity beads with3-or10-carbon spacer arms was21.8mmol/L gel and21.2mmol/L gel, respectively. Based on the1,4-Butanedioldiglycidyl ether activated Sepharose CL-6B beads, three arginine beads with liganddensities of9.6,28.5and47.4mmol/L gel were prepared.Secondly, the frontal analysis chromatography was applied to investigate theeffects of ligand density and spacer arm length on the dynamic binding capacity forsupercoiled plasmid DNA. The results show that a longer spacer arm improves thepDNA DBC of arginine-affinity columns significantly. It is also obvious that the DBCincrease with increasing ligand density, and the maximum DBC for pDNA was6.32mg/mL gel at the ligand density of47.4mmol/L gel.Thirdly, using arginine-affinity chromatography, supercoiled pDNA wassuccessfully purified from E. coli clarified cell lysate by a two-step elution procedure.The effects of loading volume and ligand density on the chromatographic performance of the arginine-affinity columns in the purification of plasmid DNA wereexplored. The results show that the purity and recovery yield decreased withincreasing loading volume, but the recovery yield increased with increasing liganddensity. In this study, the R47-L-Sep column exhibited much higher productivity, andabout0.35mg of sc pDNA could be obtained during the chromatographic processfrom3mL of clarified cell lysate. It is approximately27times higher than theprevious results reported by Sousa et al in the purification of pDNA byarginine-affinity chromatography (12.8μg pDNA/mL gel).