RESULTS AND DISCUSSION
Lysis and plasmid recovery
The most common method for isolating pDNA is based on alkaline treatment and detergent-mediated solubilization of the bacterial
cell membranes. In the second stage, the pH of the solution is adjusted to a value close to 5.5 by adding potassium acetate.12 The change in the 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.12 The insoluble material can then be separated from the liquor containing the pDNA by filtration or centrifugation; once separated,
the liquor containing pDNA is subjected to downstream processing to recover and purify the product.2,13,14 In our process, the insoluble material is separated from the liquor containing the pIDKE2 plasmid by centrifugation. Loading the crude lysate
containing large amounts of impurities such as chDNA, RNA, proteins, and endotoxins directly to a chromatography matrix is
not recommended; primary purification is essential.13 The clarified lysate containing the plasmid is concentrated five times using a TFF system; however, this step is not sufficient
to remove all the RNA (Figure 1, lane 2). RNase is commonly added to degrade the RNA but this procedure is not recommended
by regulatory agencies; consequently the RNA content in the clarified lysate is very high—about 25 times the amount of the
pDNA in weight.3 In the present process, 100% of the remaining RNA is removed from the concentrated cell lysate during the first chromatographic
step on Sepharose CL-4B, with 91% of the pDNA recovered.4 The analysis on the agarose gel shows that pDNA eluted in the void volume can be efficiently separated from RNA (Figure
1, lane 4).
pIDKE2 purification on a Sartobind D membrane
Large biomolecules such as pDNA bind only to the surface of traditional chromatography beads; hence, the capacity for pDNA
is much lower than it is for small biomolecules which are able to access the full volume of the beads.15
Figure 2 shows the chromatographic profile for the separation of pIDKE2 under the selected conditions. During loading, no
significant amount of pDNA was detected in the flow-through at the high flow rate of 150 mL/min. We calculated the average
dynamic binding capacity for 10 batches at 3.3 ± 0.8 mg pDNA per mL. This means that the capture of pDNA from E. coli lysate is more efficient and rapid using the Sartobind D membrane, which has a dynamic binding capacity between 4.1 and 2.5
mg pDNA per mL support using a flow rate of 4.3 column volumes per minute. This is a more desirable result than what can be
achieved by conventional anion-exchange resins, which bind about 1 mg of pDNA per mL of resin at flow rates that typically
are lower than 0.5 column volume per minute.16 Seventy percent of the pDNA was eluted with 90% purity as determined by agarose gel, and no RNA is found (Figure 1 lane
6, Table 1). In addition, the assays for proteins and endotoxins indicate that there was a reduction of these contaminants.
Table 1: Testing results of released purified pIDKE2 compared to specification limits.
The loading, washing, elution, and membrane regeneration procedures took place in 1.5 h. Thus, this approach significantly
reduced the separation time and increased the throughput and productivity for pDNA recovery. The Sartobind D membrane was
repeatedly used for 10 batches. After each run, the membrane was regenerated with 0.5 M sodium hydroxide, because of its high
stability in alkaline solutions. During the process, no change in backpressure was observed, demonstrating the stability and
reproducibility of the membrane.17
The final purification step was similar to that reported previously4: pDNA fractions were pooled, concentrated to 2 mg/mL by tangential flow filtration, and finally filtered (0.22 μm). The purification
steps render a final high purity pIDKE2 plasmid with an average yield of 50%. The yield is influenced also by the homogeneity
of pDNA in the cells. A summary of the analytical specifications according to FDA criteria is shown in Table 2. The purified
plasmid pIDKE2 had 95% purity.
Table 2: Plasmid recovery, yield, protein, and endotoxin reduction for the Sartobind D capture step.
Plasmid identity was confirmed by restriction enzyme digestion and its activity confirmed by an in vivo assay. The purified pIDKE2 plasmid induced a positive response against HCV core and enveloped proteins, with 87% seroconversion
against Co.120 and 60% against E2.680 proteins, respectively.