SBIR-STTR Award

Plasmid DNA has become the basis for new approaches to gene therapy and polynucleotide-based vaccines. The use of plasmid DNA as a pharmaceutical has presented challenges for its manufacture and characterization. Current technology for the large-scale production of plasmid DNA using CsCl gradients or first-generation column chromatography for purification is often inefficient, resulting in low purity and yields. The ability to remove contaminants such as plasmid isoforms, chromosomal DNA, RNA and host endotoxins is also compromised. A new process is proposed, based on a novel polymer resin PolyFlo[R]. In this Phase I proposal, we propose to evaluate the entire manufacturing process, fermentation through formulation to achieve efficient, cost-effective large- scale production. In addition, we will evaluate the effects of residual contaminants on transfection efficiency. The results will be used to support a Phase II application to evaluate the correlation between purity, in vivo biological activity and safety. Results of this research will have immediate impact for those engaged in the use of plasmid DNA for gene delivery and have commercialization implications for PolyFlo. Additionally, the ability to manufacture plasmids at large-scale will eliminate a significant obstacle to the development of the gene therapy/DNA vaccine market. PROPOSED COMMERCIAL APPLICATIONS: The PolyFlo[R] resin is manufactured by Puresyn, Inc. and is to be used as part of the purification process in the development of gene therapy and nucleic acid-based products. The results of the proposed research will offer a process to a new technology area to Academic and Industrial Healthcare Markets. High purity and yield as well as a reduction in process time will lower production expenses from analytical through large-scale development

Puresyn, Inc. has developed purification processes for supercoiled plasmid DNA vaccine and gone therapy vectors utilizing its proprietary chromatographic resin, PolyFIo(r). In Phase I, a tandem chromatography processes was developed: hydrophobic interaction and PolyFIo. The process resulted in product of high purity and recovery that exceeds other chromatographic processes. We demonstrated this process removes significant amounts of host contaminants as well as other plasmid isoforms. The resulting product has improved biological activity as assessed in in vitro transfection assays. We are seeking Phase II funding to concentrate on supercoiled plasmid production to include large-scale purification up 5 grams per run without compromising purity, yield or biological activity. We will apply newly developed analytical methodologies to investigate the factors that may influence biological activity. Finally, we will conduct an in vivo mouse study to determine the correlation between purity and biological activity through the evaluation of immune and gone therapeutic responses to several vectors. Results of this research will have immediate impact for those engaged in the use of supercoiled vectors for DNA vaccines and gone delivery because it will allow the large-scale development of plasmid vectors. It will have positive commercialization implications for PolyFIo and Puresyn, Inc.

Thesaurus Terms:
analytical method, chromatography, nucleic acid purification, plasmid, technology /technique development drug design /synthesis /production, fermentation, transfection, vector vaccine biotechnology, laboratory mouse