The use of validated and pharmacopoeia-compliant quality control assays is essential to demonstrate the effectiveness of the
process to purify pDNA away from process-related impurities.4 The platform should be validated according to the cGMP guidelines and achieve a high quality of product to satisfy the current
An outline of the design of the cGMP process for manufacturing pDNA vaccines is shown in Figure 1. Each step should be attributed
a specific function.
Plasmid DNA is transfected into E.coli by heat-shock, and clones containing the pDNA are identified under a selective pressure
present on the vector (such as an antibiotic marker or by regulation of an essential E. coli protein). In an industrial setting,
a master cell bank (MCB) is laid down from a clone, which is then cultured in large-scale fermenters by fed-batch, high-cell
density culture.6 We have demonstrated that it is possible to omit the clone selection or MCB steps and culture a randomly selected clone
in low-tech disposable shaker flasks without compromising high pDNA yields (>3 mg pDNA per gram of wet weight E. coli). We
are able to routinely produce up to 200 mg of pDNA in a culture volume of less than 4 L in 24 hours. High specific yields
are favored over high volumetric yields (mg pDNA per liter of ferment) because they reduce the relative starting load of E.
coli-sourced contaminants. The ferment is harvested by centrifugation into single-use plastic centrifuge bottles at late log
for maximal pDNA yields. Because of cost, we decided not to use disposable fermenter systems or crossflow applications for
Standard alkaline lysis methods must be optimized to ensure the efficient lysis of cells and subsequent removal of major contaminants
such as genomic DNA and proteins.7 The lysis is performed in a plastic container containing a low-level outlet attached to prefiltration and 0.22 μM filtration
devices. The whole assembly and container are autoclaved together before use. Addition of a secondary salt such as ammonium
acetate or calcium chloride precipitates and removes RNA (up to 45% reduction in contaminating nucleic acid load observed).8 The secondary salt partitions the flocculent (precipitated material) in an upper layer away from the lysate, which enables
the subsequent clarification of the lysate by dead-end filtration (Figure 2). The clarified lysate can be stored in a bioprocess
container or can be pumped directly into the crossflow filtration container for the start of the purification step.
Figure 3 shows the complex nature of the lysate, which is known to contain large amounts of RNA, endotoxin, and trace amounts
of contaminating genomic DNA, protein, and other non-supercoiled pDNA related isoforms. The pDNA constitutes less than 5%
of the total mixture, which presents a challenge to purify the desired monomeric supercoiled pDNA to homogeneity from contaminants
that have similar physiochemical properties. Supercoiled pDNA is tightly coiled and is widely accepted as being the therapeutically
efficacious form, mainly due to its relatively small size compared to other isoforms.1