Conference Dates

June 6-11, 2010

Abstract

To commercialize DNA medicines, industrial plasmid DNA manufacturing processes are needed which meet the quality, economy, and scale requirements projected for future products. NTC has developed an inducible fed-batch fermentation process that incorporates novel cell bank and fermentation process innovations that reduce plasmid mediated metabolic burden. This process also incorporates a scalable plasmid induction profile that, in combination with vector backbone modifications that double fermentation productivity compared to existing high copy vectors such as pVAX1 and gWIZ, form a generic plasmid DNA production platform driving high plasmid yields up to 2.6 g/L, with specific yields of 5% of total dry cell weight.

We have investigated the effect of various epigenetic modifiers on plasmid performance in this process. For example, the dcm gene encodes a DNA methylase that methylates the internal cytosine residues in the recognition sequence 5‘-CC*AGG-3’ or 5‘-CC*TGG-3’. This creates 5-methyl-cytosine (5mC), a common mammalian pattern, although dcm methylated cytosine is in a different sequence context in bacteria compared to mammals (CG sequence). While plasmid production yields and quality are similar between dcm+ and dcm- host strains, CMV promoter expression is reduced by dcm methylation. Surprisingly, despite improved expression, dcm- plasmid DNA is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e. production strain) early in product development and suggest that dcm+ strains may be superior for vaccine applications.

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