The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates
a corresponding need for simple, scalable, and cost-effective purification processes. Due to the
pDNA large size and complex shape, the use of commercial chromatographic beads often
results in poor yields and low binding capacities when operated in a positive mode. An
alternative to overcome this limitation is the design of chromatographic ligand-resin systems
able to efficiently operate in negative mode, where host impurities (especially low molecular
weight RNA) are efficiently captured and separated from the target pDNA. In this work,
arginine amino acid and di-arginine peptide (arginine-arginine) were immobil... More
The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates
a corresponding need for simple, scalable, and cost-effective purification processes. Due to the
pDNA large size and complex shape, the use of commercial chromatographic beads often
results in poor yields and low binding capacities when operated in a positive mode. An
alternative to overcome this limitation is the design of chromatographic ligand-resin systems
able to efficiently operate in negative mode, where host impurities (especially low molecular
weight RNA) are efficiently captured and separated from the target pDNA. In this work,
arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose
resins and evaluated for negative chromatographic purification of pDNA from bacterial cell
lysates. The results showed that RNA was preferentially bound to the ligands, interfering with
the binding of pDNA. The amount of plasmid processed per column volume by arginine and diarginine,
under negative mode, was substantially larger comparing with the conventional
positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host
impurities. This study shows that negative mode chromatography using arginine-based ligands
poses as an interesting alternative for intermediate and polishing pDNA purification
operations, with considerable economic and environmental advantages.