(341e) Plasmid DNA Purification and Formulation for Vaccine Applications

According to a WHO ?Epidemic Alert and Response' report from November 2004, the present capacity to produce a pandemic vaccine is woefully inadequate. As the report states, this leads to a demand for forward-looking innovations to address production issues. DNA vaccines offers a solution to the current world vaccine crisis. An integrated, working solution for the production of DNA vaccines will be presented from development and fermentation to down-stream purification and formulation.

The Institute of Medicine (2004) has stated that the world is teetering on the verge of a massive influenza outbreak due to inadequate production systems. The bulk of the world's vaccine production comes from a very small number of companies compared to the number of companies producing drugs. How has this occurred? Because vaccines have long processing times, and are difficult, hence expensive, to produce. Technological issues, stemming from production difficulties, are crippling the vaccine industry and leading to vaccine shortages and financial woes for companies. For example, on the 5th October 2004, the FDA was caught off guard by a shortage of flu vaccine, caused by a contamination in a UK plant of one of only two suppliers of U.S. flu vaccines. In the case of an influenza pandemic, vaccination will be one of the key interventions. A crucial stage in the control of a pandemic will be to provide as many vaccine doses to the new viral strain in the shortest possible time, however the demand for a pandemic vaccine will significantly surpass the available supply from existing manufacturing capacities.

Solving the Production Crisis: What does DNA offer?

The current time scale for the vaccine development process from raw ingredient to the vaccine actually being in the surgery fridge can take almost 20 months (according to the UK Vaccine Industry Group). This includes 4-9 months for production, 3 months for quality control, 1-2 months for product finishing, up to 2 months for control agency testing and up to three months for distribution. It has been shown that a DNA vaccine could reduce the initial production period to 1 month, reducing the total process time by up to 8 months, or 40 %. How many lives could this save in the first 8 months of a pandemic?

Current vaccine production methods are based on 50 year old technology, which can confront such production issues such as requiring 100 % inactivation or attenuation of the vaccine and waiting for the delivery of hen's eggs. The often overlooked issue of vaccine production will be solved by the introduction of smarter, faster response purification procedures.

DNA has the following advantages over current vaccines: - An excellent safety profile, free from specific safety concerns associated with viruses. - Generally simpler to develop - Easier to manufacture than an inactivated pathogen, subcellular fraction or recombinant protein vaccine. - Different DNA encoding different antigens can be prepared in identical ways, which enables technology transfer and reduced production times; and - DNA is very stable and resistant to extremes of temperature; thus facilitating the storage, transportation and distribution of DNA-based vaccines to remote areas.

Developments in genomics and structural biology are enabling scientists and engineers to develop new and better purification processes for DNA such as affinity chromatography. Affinity chromatography enables the capture and purification of vastly different types of DNA in one chromatographic step without the co-purification of contaminants such as gDNA, RNA, protein and endotoxins, thus potentially eliminating the need for further purification or polishing stages.

There are relatively few companies producing vaccines compared with the number of pharmaceutical companies. This is a result of the size of the vaccine market (less than 2 percent of the pharmaceutical market) and the barriers of entry into the vaccine production market. These barriers include: - the huge fixed costs of research and development, quality control and assurance, distribution, and the construction and maintenance of production facilities. These reach up to 90 % of the total costs. - a need for economies of scale and productivity gains, both of which are obtained over time, in order to off-set the high fixed costs; and - barriers to technology transfer due to the low numbers of vaccine producers and competitive nature of the industry.

A simpler to develop vaccine based on DNA that harness identical purification procedures could assist to overcome the traditional barriers of entry to vaccine production by empowering countries with low technology platforms to produce their own vaccines rather than relying on a handful of large bio-manufacturers.

Research Topics and Results

Work currently being completed to address the following areas of DNA vaccine production will be presented.

Purification: The contaminants that pose a particular problem in the production of purified pDNA are anionic polymers of a similar structure, charge and physical behaviour to pDNA. These contaminating anionic polymers include genomic DNA, RNA and lipopolysaccharides. Affinity chromatography is a highly specific chromatographic technique that utilises ligands based on biological systems to obtain highly purified plasmid DNA in one unit operation. By the use of affinity ligands and solid phase adsorbent design, the stages of capture, concentration, and purification can be performed in a single unit operation. Approaches to the development of affinity ligands will be presented and results from scaleable unit operations employing affinity ligands will be shown.

Overcoming Low efficiency of cell transformation by pDNA: Current approaches to improving cell transformation address issues of the development of particles that mimic viruses. This can lead to a whole suite of unique safety issues. Small particles (sub-micron) are permitted to diffuse freely, hence increase transfection. Work is being completed to harness scaleable production systems (surface acoustic wave atomization and ultrasonic atomization) to reproducibly make biodegradable particles loaded with biomolecules. Protein and DNA has been encapsulated using this process. Comparisons between bovine serum albumin (protein biomolecule) and herring DNA (DNA biomolecule) are made with the ultimate aim being to improve cell transformation efficiencies.

Process Optimization - Reducing Cost per Dose: 50-80 % of the total costs of vaccine production are incurred during downstream purification. It is highly desirable to reduce the number of unit operations and hence the processing time and cost of purification. The development of an integrated purification process that considers all stages from development through to formulation will assist to reduce to coat per dose of DNA vaccines. The effect of plasmid DNA form and size, fermentation, lysis, as well as the afore mentioned stages of purification and formulation are considered in an integrated approach to DNA vaccine production.

The Future of Plasmid DNA Vaccines

It is likely that the most immediate use for pDNA will be in specialist, high value vaccine applications as was seen for the condors and for the immunization of domestic pets. Over time, the body of evidence supporting the use of pDNA as a vaccine will most likely lead to the acceptance of pDNA for time critical applications such a developing an influenza vaccine for high risk groups.

Wider use of DNA as a pharmaceutical in other applications such as gene therapy and research will lead to processing advances and technology transfer which will bring the cost per dose of a DNA vaccine down to levels that are economically competitive. Such advances may include the development of a process to create much shorter fragments of DNA that elicit the same response as the much larger pDNA, leading to high transformation efficiencies and better time yields from processing equipment. At this point in time, the barriers of entry into the vaccine development market will hopefully be broken down and a wider range of countries will be able to produce their own vaccines, rather than the world needing to rely on a small number of producers. For this to happen, it becomes painfully clear that research centers, public health institutions, governments and vaccine manufacturers throughout the world and particularly in countries that have strengths in vaccine production, will need to collaborate in taking action towards solving the current vaccine production problem in order to confront the looming pandemic. The countries and areas named by WHO in January of this year as having manufacturing capacity for influenza vaccines were Australia, Europe, Japan and North America. It is up to the countries and areas, including Australia, with strengths in vaccine production to lead the way in developing forward-looking innovations to address woefully inadequate production capacities.

Conclusion

Plasmid DNA offers the promise of a new generation of pharmaceuticals that will address the often overlooked issue of vaccine production by offering a simple and reproducible method for producing a vaccine. Through reverse engineering, production could be reduced from up to 9 months to as little as 1 month. Simplified development and faster turn-around times means that DNA offers a solution to the vaccine crisis and will help to contain future viral outbreaks as a vaccine against new viral strains will be able to be prepared in the shortest possible time. Groups with strengths in vaccine production need to play an active role in the development of new technologies, such as pDNA, to safeguard global human health against current and future viral pandemics.