The significance of plasmid DNA preparations contaminated with bacterial genomic DNA on inflammatory responses following delivery of lipoplexes to the murine lung.

Non-viral gene transfer using plasmid DNA (pDNA) is generally acknowledged as safe and non-immunogenic compared with the use of viral vectors. However, pre-clinical and clinical studies have shown that non-viral (lipoplex) gene transfer to the lung can provoke a mild, acute inflammatory response, which is thought to be, partly, due to unmethylated CG dinucleotides (CpGs) present in the pDNA sequence. Using a murine model of lung gene transfer, bronchoalveolar lavage fluid was collected following plasmid delivery and a range of inflammatory markers was analysed.

A novel mixing device for the reproducible generation of nonviral gene therapy formulations.

Nonviral gene therapy utilizing plasmid DNA (pDNA) complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has demonstrated considerable potential for the treatment of a variety of diseases. However, progress toward clinical application is often delayed by the lack of reliable and scalable mixing of components sufficient to guarantee consistent performance in vivo. Attempts to improve and standardize mixing have been limited by the sensitivity of pDNA to shear-related degradation.

Production of pharmaceutical-grade plasmids at high concentration and high supercoiled percentage.

The increased use of plasmid-based vaccines to replace their more challenging viral counterparts has increased the demand for high purity and high concentration plasmids. Here we report the production of plasmids encoding different transgenes for DNA vaccine candidates at gram scale with an integrated process consisting of batch fermentation and limited steps of purification. Plasmid products encoding for eight smallpox antigens that were combined into a bioterrorism DNA vaccine exhibited high purity with undetectable RNA, protein and endotoxin, concentration of up to 13.

DNA vaccine manufacture: scale and quality.

The demand for plasmid DNA in large quantities at high purity and concentration is expected to escalate as more DNA vaccines are entering clinical trial status and becoming closer to market approval. This review outlines different methods for DNA vaccine manufacture and discusses the challenges that hinder large-scale production. Current technologies are summarized, focusing on novel approaches that have the potential to address downstream bottlenecks and adaptability for large-scale application.

Successful parallel development and integration of a plasmid-based biologic, container/closure system and electrokinetic delivery device.

We have developed three major technologies that allow plasmid-based products to be used for large-scale vaccination or therapeutic protein applications. Our team has integrated these components into one complete, cost-effective, easy-to-use system capable of rapid implementation under field conditions. The proprietary manufacturing process uses a lysis method and membrane-based chromatography to rapidly produce large-scale batches of plasmid.