Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography - A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography. - Bio

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Plasmid DNA Recovery Using Size-Exclusion and Perfusion Chromatography
A new plasmid DNA purification method uses a nonenzymatic approach to RNA removal based on size-exclusion chromatography.


BioPharm International


RESULTS

Lysis and Plasmid Recovery


Figure 2
In the alkaline environment, the chDNA is denatured, while within a pH range at 12–12.3 pIDKE2 plasmid remains double stranded. During neutralization, the pH of the solution is reduced to a value close to 5.5 by adding potassium acetate. The change in physicochemical conditions of the solution causes the renaturation and flocculation of the chDNA, as well as the precipitation of protein-SDS complexes and cell-wall debris. The insoluble material can be separated from the liquid containing the pIDKE2 plasmid by centrifugation. Once the pellet is separated, the clarified alkaline lysate containing the pIDKE2 plasmid is concentrated 5-fold using a TFF system. This step, however, is not enough to remove all the RNA (Figure 1, Lane 4). On the other hand, the 260 nm/280 nm absorbance ratio was 1.8, which is in the range of the purity parameter regulated for a drug substance containing a purified pDNA.10 RNA removal can be performed enzymatically using RNase or by selective precipitation using Ca2+; NH4+, or Mg2+ ions or polyethylene glycol.9,11 Depending on the qualitative and quantitative composition of the sample, this polymer involves the risk of co-precipitation of DNA and needs to be removed before subsequent ion-exchange steps. RNA removal also can be achieved by size-exclusion chromatography using Sepharose 6 Fast Flow, which recovers 92% of the pDNA. In this process, RNA was removed from the concentrated cell lysate during the first chromatographic step on Sepharose CL-4B recovering 91% of the pDNA by group separation in the presence of buffer containing 1.5 M (NH4)2SO4.12 The use of high salt concentration is beneficial because of the different hydrodynamic size of doubled-stranded pDNA compared with small single-strand content such as RNA and other nucleic acids impurities. Therefore, pDNA eluted in the void volume can be clearly separated from RNA (Figure 1, Lane 5). The elution profile, obtained during the pDNA elution from Sepharose CL-4B in the presence of (NH4)2SO4, is shown in Figure 2.


Figure 3
The second step was designed as a concentration step, and therefore, a support with high capacity for large molecules was used: a reverse phase POROS R1 50 matrix with a dynamic binding capacity between 5 and 1.5 mg pDNA/mL using a high flow rate (500 cm/h). The high flow rate is the main advantage of POROS. It does not affect the resolution or capacity during pDNA purification because of the intraparticle, convective, solute transport, which is a fundamental new approach to reducing mass-transfer limitations in chromatography. This new approach will dramatically decrease separation time and increase throughput and productivity for pDNA recovery.13


Table 1. The purified plasmid DNA had 95% purity. Contaminants such as host-cell RNA and DNA were undetectable by agarose gel electro-phoresis assay. Plasmid identity was confirmed by restriction enzyme digestion and activity was confirmed by an in vivo assay, in which the results show that the purified pIDKE2 plasmid induced a positive and long-term antibody response against HCV core protein and enveloped proteins.
Another size-exclusion chromatography method was chosen as the last purification step. Agarose gel electrophoresis of column fractions showed that purity of the pDNA increased from the first fraction, which had <60%, to the last, in which purity of >95% was obtained (Figure 3). The pDNA fractions with a purity >89% were pooled and further concentrated by a 10% w/v PEG-8000 precipitation, which concentrated the plasmid and reduced levels of E. coli host proteins and undesirable DNA contamination.9 The pellet was resuspended in the formulation buffer at 2 mg/mL and was finally filtered (0.22 μm) with a yield of about 100% before further analyses were performed. Analytical methods were followed according to the criteria recommended by the FDA. A summary of the analytical specifications and final results for pIDKE2 is shown in Table 1.


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