The increasing development and future application of molecular therapies such as gene
therapy and DNA vaccination is expected to have a great impact in health care. However
for their wide application large amounts of plasmid DNA (pDNA) are required with a
stringent clearance of impurities. This prompted the development of new, efficient and
cost-effective large-scale processes for the production and purification of pDNA. Most of
the purification processes described are based on chromatography but dispite their high
resolution, frequently they are difficult to scale-up, have low capacity and present low
yields. In order to overcome these disadvantages other methodologies are also being
developed.
Aqueous two-phase systems (ATPS) are one of the most promising approaches for pDNA
purification given their several advantages like easy scale-up, high capacity and the
possibility of continuous operation. Despite their great potential ATPS have low selectivity,
which limits the purification outcome. The addition of certain molecules with affinity for
the target molecules (pDNA in this case) may increase their selectivity.
In this work it was studied the possibility of using amino ligands for the affinity purification
of pDNA from bacterial alkaline lysates. Two free amino acids, lysine and arginine, their
respective Polyethylene glycol (PEG) conjugates, PEG-lysine and PEGarginine, and PEGamine
were tested. The system used was composed of 16,2% (w/w) PEG 600 and 17,4%
(w/w) dextran 100 (DEX) and it was evaluate the ability of each ligand to steer the pDNA
to the phase where less impurities are accumulated (PEG rich phase). The results show
that free amino acids did not have any effect on pDNA partitioning but the PEG conjugates
were able to steer the pDNA to the PEG phase, at low concentrations. With the addition of
0,2% of PEG-lysine, or 0,5% of PEG-arginine or 4% of PEG-amine in relation to the total
PEG, all the pDNA is recovered in the PEG phase. However it presents some RNA
contamination, that could be removed by re-extracting with a new phase containing 30%
of ammonium sulphate (NH4)2S04. The purified pDNA is obtained in the bottom phase of
this new system with no measurable presence of RNA or proteins
