September 1, 2011 (Vol. 31, No. 15)

K. John John Morrow Jr. Ph.D. President Newport Biotech

Plasmid Purification Issues Do Not Get the Same Attention that Delivery Obstacles Do, but They Remain Problematic Nonetheless

Delivery, delivery, delivery is the major focus of DNA vaccine research, according to David Weiner, Ph.D., University of Pennsylvania professor and also chair of the recent conference on “DNA Vaccines: Building on Clinical Progress and Exploring New Targets.”

In his keynote address, Dr. Weiner reviewed the history of the field, detailing how trials of a DNA HIV vaccine dealt a huge blow to the field when it was observed that plasmids carrying five different HIV proteins failed to induce immunity when injected into patients. In the years since, the technology has advanced dramatically, and therapies currently under evaluation are demonstrating the superb potential of plasmid-based vaccines.

DNA vaccines represent a simple, elegant, and straightforward approach. They do not require the handling of live, unstable, and dangerous pathogens; they can be produced and ramped up rapidly to confront epidemics; and because they are composed of DNA they are stable at room temperature, a claim that cannot be made for many proteins. Since no actual viruses are used in the vaccines there is no possibility of a debilitated form mutating back to an aggressive wild type. Finally, DNA is not perceived by the host to be a foreign material and so no immune response would be expected to be mounted against it.

Dr. Weiner emphasized at the meeting, which was sponsored by the International Society of DNA Vaccines and organized by BioConferences International, that in the intervening years, a coterie of new vaccine methods have been developed including new strategies for getting the plasmids into cells, increasing protein production once they are inside, and modifications of the vaccine proteins that increase their recognition and response by the immune system.

Some of the new heavy hitters include transdermal, needle-free patches; devices that blast the plasmids into the skin with air pressure; and electroporation, in which electrical pulses are used to temporarily open the cell membrane, allowing the plasmids easier access to the interior of cells.

Codon modification can also improve the rates of gene transcription, speeding protein manufacture. In addition, adding a so-called leader sequence can improve the stability of the final protein molecule.

Today, dozens of DNA vaccines are being evaluated. Targets include H1N5 avian flu, HIV, and cancer. Dr. Weiner cautioned that such efforts may appear to be simple and straightforward, yet present years of setbacks and frustrations. “The next two years of clinical testing of new and more complex DNA vaccines will be pivotal for either generating a true clinical success based on immune potency, or for telling us that we still have much further to go.”

GMP Plasmid Purification

According to Philippe Ledent, Ph.D., process transfer and development manager at Eurogentec Biologics, his company has faced major challenges upscaling output of both protein and nucleic acid products. “In plasmid production protocols we use fed-batch for better growth control of our cultures.” In a two-step process that was based on biomass expansion followed by plasmid DNA production, it was possible to increase fermentation yields 10-fold.

Dr. Ledent and his team have optimized plasmid extraction, which is based on alkaline lysis and neutralization, followed by clarification and filtration through 0.2 µm units. This is recognized as the most critical step of the entire process. They also worked to reduce shearing and bring about more rapid homogenization of NaOH levels, an action that is difficult to achieve when one is dealing with increasingly large volumes.

“Overall, through the application of our improvement strategies, we were able to increase yields 21-fold while decreasing the cost of goods to one-quarter of the original figure,” Dr. Ledent stated.

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.

Scientists at Althea Technologies are working to ensure that plasmid preparations are stable at room temperature. The firm has found that a combination of EDTA and ethanol has a synergistic enhancing effect on DNA stability.

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