June 6-11, 2010
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.
Aaron E. Carnes, Jeremy Luke, Justin Vincent, Clague Hodgson, and James Williams, "ESCHERICHIA COLI PLASMID DNA FERMENTATION: STRAIN AND PROCESS-SPECIFIC EFFECTS ON VECTOR YIELD, QUALITY, AND TRANSGENE EXPRESSION" in "Vaccine Technology III", John G. Auniņš,Merck, USA; Barry C. Buckland, BiologicB, USA; Kathrin U. Jansen, Pfizer, USA; Paula Marques Alves, IBET, Portugal Eds, ECI Symposium Series, (2010). http://dc.engconfintl.org/vaccine_iii/25