Plasdmid DNA Purification Scale-Up
Tony Hitchcock, head of manufacturing technologies at RecipharmCobra Biologics, provided another approach to plasmid purification. The company's product line includes bacteria, animal cells, viruses, novel proteins, and antibodies. The overall process that Hitchcock sought to optimize was quite similar to that followed by Eurogentec—high density fermentation, followed by alkaline cell lysis, chromatographic purification, and final formulation.
“The plasmid purification flow consists of anion exchange to remove bulk contaminants, including RNA, next ion-pair chromatography for removal of endotoxins, SEC chromatography to remove low level RNA fragments, and finally formulation and 0.2 µm filtration,” Hitchcock explained.
Hitchcock considered potential solutions including modifications of upstream processing and alternative chromatography approaches. His group adopted the use of the GE PlasmidSelect Xtra™, designed for purifying supercoiled plasmid DNA, in order to deal with high levels of plasmid open forms and host DNA in the preparations.
In order to simplify and expedite the process, Hitchcock asked a number of questions: “Can we increase the loading on the PlasmidSelect™? Can we swap PlasmidSelect for ion-pair chromatography? Can we eliminate the SEC step? Does it work with challenging plasmids?”
To answer these questions, Hitchcock and his colleagues developed a two-step process in which anion exchange is followed by ion-pair chromatography, SEC chromatography, and, finally, formulation and 0.2 µm filtration. These modifications improved the process, with robust removal of host-cell proteins and endotoxin and high-quality purification of problem plasmids without the necessity of individual optimization per plasmid.
Rick Hancock, president of Althea Technologies, moved the discussion away from the molecular biology of the various systems in order to confront problems of formulation, stability, container selection, and cold-chain management.
“In the early phases of process development we want to look for scalable approaches with no obvious bottlenecks. This is not the time to incorporate mutagenic agents or animal-derived materials into the process. In addition, we need to minimize the use of flammable solutions. Finally, it's essential to have a firm grasp of intellectual property issues.”
According to Hancock, as one progresses through the various phases of clinical evaluation, manufacturing conditions are selected based on the increasing volume requirements and more stringent regulatory requirements. By Phase II, a robust formulation is required in order to meet the requirements for long-term stability, while in Phase III, large-scale demands of commercial distribution must be considered.
Ideally, one wants to have stability of the plasmid preparations at room temperature such that degradation of DNA, as measured by the loss of the super-coiled form, is minimized. Extensive evaluation of formulation buffers gave optimal results with a TRIS EDTA/EtOH buffer, in which plasmids were stable at 48 months.
“We observed that a combination of EDTA and ethanol had a synergistic enhancing effect on DNA stability.” This formulation is compatible with electroporation, which is an increasingly important consideration.
“Successful cold-chain management for the developing world will be an ongoing challenge. In an environment in which one cannot rely on refrigeration, these logistical issues will drive the search for successful lyophilization or liquid ambient temperature storage for all vaccine preparations, including DNA vaccines.”
Cleaning validation is critical for the successful cGMP manufacture of DNA vaccines, as Henry Hebel, COO of VGXI, explained. “We evaluated our cleaning procedures for removal of microorganisms and determined that our current protocols are quite effective, based on colony counts of suspected laboratory surfaces.”
A far more sensitive approach, however, is based on the use of PCR, as Hebel showed. Employing the fluorescently labeled Taqman probes provides a specific method of quantitation down as low as one part per trillion or less. The high sensitivity of the assay combined with its convenience make it especially desirable for the pharma industry.
Using a specific segment in the kanamycin-resistance gene as a PCR target, it is possible to run the samples over a cycle period of one hour and 45 minutes.
According to Hebel, sampling in the plant after completion of the cleaning process is a crucial step. The qPCR assay can be performed in conjunction with typical test methods such as TOC, pH, and conductivity. The assay fits in well with these requirements since it can detect even a few plasmid copies in a rinse sample. With such a sensitive procedure, false positives are always a concern, and for this reason control samples are essential at every step. Additionally, the design of the facility and the operation of the PCR laboratory must be carefully managed.
Although DNA vaccines have been around for many years, success has been quite limited. With a myriad of new approaches being brought into play, the future looks brighter.