The DNA vaccines field is definitely regaining the momentum that it had a few years ago, according to David Weiner, Ph.D., professor of pathology and laboratory medicine at the University of Pennsylvania and chair of the “DNA Vaccines” meeting held last month in New Orleans. “There are a lot of exciting breakthroughs, and the clinical data is much more encouraging.”
“DNA Vaccines,” sponsored by BioConferences International, a Mary Ann Liebert company, covered a range of noteworthy topics including: new approaches to electroporation, vaccine formulation, vector design, and recent clinical trials including a large-scale trial of an HIV/AIDS vaccine in Thailand.
In the HIV/AIDS trial described by Colonel Nelson Michael, M.D., Ph.D., of the Walter Reed Army Research Institute, the vaccine showed a modest (30%) but statistically significant protective effect over unvaccinated controls. “While a suitable HIV vaccine is quite a few years away, we’re in positive territory,” Dr. Weiner explained. “Getting from 30 to 60 percent effectiveness is a lot easier than getting from 0 to 30 percent. It is a big challenge, but several efficacy trials for HIV are quite encouraging.”
Matjaz Peterka, Ph.D., manager at BIA Separations, discussed the challenges of purifying plasmids,whose large size, negative charge, and varied levels of coiling and supercoiling present special challenges.
Plasmids represent less than 0.5% of the wet biomass, and endotoxins and other impurities must be completely removed. Because of their size, plasmids diffuse slowly through the solvent, and they may not be able to easily penetrate chromatographic beads if the pore size is too small. Since most chromatographic media is optimized for protein applications, viral particles and plasmids will tend to adhere to the outside, greatly reducing the absorptive capacity of the beads.
Monolithic columns, on the other hand, are better suited for plasmid separation, according to Dr. Peterka. With large flow-through channels (1.5 µm) and high surface accessibility they are useful for “mega-molecular” purification. The result is higher dynamic binding capacity, a flow-independent performance with lowered shear forces. “In our strategic planning, we focused on selective precipitation, anion exchange chromatography, and hydrophobic interaction chromatography,” he stated.
BIA Separations’ selective purification component employs CaCl2, and the separation of nucleic acids is accomplished through the use of a convection interaction media (CIM) DEAE-tube monolithic column, a weak anion exchanger. This column is used for extremely fast, highly efficient separations of large molecules such as proteins or DNA, as well as smaller molecules such as peptides.
It is followed by efficient separation of supercoiled and open circular pDNA on the CIM® C4 HLD-1 tube monolithic column. Following optimization, scale up of the process can continue using an 8,000 mL column, which will purify up to 48 g of plasmid DNA.
“The CIM DNA purification process removes over 99 percent of all major contaminants, and is fast and scalable,” said Dr. Peterka. “The economics are highly favorable, with high binding capacity and low buffer consumption, resulting in high productivity.”