The immunogenicity of pIDKE2 plasmid obtained by the method described in the current work was compared with another method
previously described.9 All animals immunized with pIDKE2+Co.120 developed anti-HCV antibodies. Figure 4 shows the antibody response against HCV
structural antigens at week 17. Mean antibody titers above 1:800 were elicited against HCV structural antigens in animals
immunized with the mixture of pIDKE2 and Co.120. In fact, antibody titers against Co.120 and E2.680 reached values above 1:2,000.
No significant differences were observed in antibody titers generated by immunization with the plas-mid obtained by different
procedures. Mice immunized with normal saline did not induce any detectable antibody response.
Historically, highly purified pDNA recovery has been accomplished through the use of cesium chloride/ethidium bromide (CsCL/EtBr)
buoyant density gradient separation.14 This method allows the separation of pDNA by buoyant density into purified bands of different forms: supercoiled (sc), open
circular (oc), linear (l) and multimeric (m) plasmid. Although it yields a highly purified plasmid, this approach is not scalable
because of personnel safety issues and the hazardous waste considerations associated with the use of cesium chloride and ethidium
bromide. Using these process solutions at large scales requires safety measures such as designing explosion-proof facilities
or using appropriate protection. In addition, the use of ultracentrifugation is also a major impediment to the scale-up of
this technology. In contrast, simple unit operations and the avoidance of critical reagents such as animal-derived compounds
(e.g., enzymes), detergents, and organic solvents significantly reduce the need for validation efforts and precautions to
ensure patient and operator safety.
Our pDNA purification process is based on alkaline lysis, TFF, and size-exclusion chromatography as primary downstream steps
for extensive removal of RNA. Reverse phase interaction chromatography is then used to purify the pDNA from the remaining
impurities, particularly because of its ability to reduce the endotoxin burden to levels below the specifications. Volume
reduction of the resulting stream is achieved by precipitation of the plasmid with PEG instead of 2-propanol or ethanol. Finally,
size-exclusion chromatography is used as a polishing step and to exchange the buffer for an adequate formulation. The proposed
process does not use or generate significant amounts of hazardous materials and no special safety requirements are envisaged.
Thus, environmental or safety-associated costs are minimized. The reagents used do not pose any special regulatory concern
because they are nontoxic, nonmutagenic, and nonflammable.
It is also strongly recommended to spend sufficient time and efforts developing large-scale GMP processes. This may result
in a different approach when compared with 'kit' protocols, in which convenience and simple robustness play the most important
role. Depending on the final application as a therapeutic (high-dose single injection, or long-term, low-dose treatment) or
for diagnostics, specific demands may require individual solutions. Given the complexity of the starting material, certainly
single purification step will not be enough to meet the regulatory requirements. Nevertheless, the aim is to establish a robust
and preferably generic protocol that is applicable to a variety of plasmids of different sizes (regardless of individual precautions
related to stability or sensitivity to shear forces). When developing a multistep large-scale pDNA purification process, the
design will aim to begin with fast volume reduction. This can be achieved by ultrafiltration or any (chromatographic) capture
step, in which recovery (>90%) is more important than maximal capacity.15
Currently published processes for pDNA purification include precipitation and extraction of pDNA by organic solvents, ultrafiltration,
and predominantly liquid chromatographic techniques. Most of the available processes for pDNA purification are time-consuming
and not scalable. Furthermore, because these processes use materials that are not certified for application in humans and
also enzymes of avian or bovine origin, these processes do not meet the appropriate regulatory guidelines.
Chromatography is considered the highest resolution method, and therefore, it is essential for producing pDNA suited for therapeutic
applications. The most commonly used techniques for initial purification are anion-exchange and hydrophobic interaction.2,16 It has to be considered that the large pDNA molecules adsorb only at the outer surface of particulate supports.12 Consequently, capacities are usually on the order of hundreds of micrograms of plasmid per milliliter of chromatographic
support. In our process, we used a POROS R1 50 reverse-phase matrix, which has a dynamic binding capacity between 5 and 1.5
mg pDNA per mL support. Finally, as a polishing step, size-exclusion chromatography is the most suitable for removing undesired
pDNA isoforms and host cell proteins and to achieve buffer exchange. The processes recover 95% of pDNA similar to the process
described by Horn, et al.9 The results demonstrate that this process meets all regulatory requirements and delivers pharmaceutical grade pDNA. The final
chDNA content is <5 μg per dose, RNA is not detectable by agarose gel electrophoresis, protein content is lower than 5 μg
per dose, and the endotoxin content is 0.6 EU per kg of body weight.