Generic plasmid DNA production platform incorporating low metabolic burden seed‐stock and fed‐batch fermentation processes

Abstract

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is 'plugged' into a validated vector, and rapidly produced in a validated, fermentation -purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably 'toxic' or otherwise low yielding in standard fermentation processes. We report cell bank and fermentation process unit operation innovations that reduce plasmid-mediated metabolic burden, enabling successful production of previously known toxic influenza hemagglutinin antigen genes. These processes, combined with vector backbone modifications, doubled fermentation productivity compared to existing high copy vectors, such as pVAX1 and gWIZ, resulting in high plasmid yields (up to 2220 mg/L, 5% of total dry cell weight) even with previously identified toxic or poor producing inserts.

Plasmid DNA fermentation strain and process‐specific effects on vector yield, quality, and transgene expression

“…A few high yield fed-batch plasmid fermentation processes (500-2,200 mg/L) have been described (Carnes et al, 2006;Listner et al, 2006;Mairhofer et al, 2010;Phue et al, 2008;Singer et al, 2009;Williams et al, 2009c). These processes all couple reduced growth rate (which generally increases copy number) with high copy replication origins (reviewed in Carnes and Williams, 2007).…”

“…This process in combination with vector backbone modifications (to incorporate a PAS-BH-SV40 high copy origin) doubled fermentation productivity compared to existing high copy vectors such as pVAX1 and gWIZ, with plasmid yields up to 2,200 mg/L (Williams et al, 2009c).…”

Antibiotic-free production of a herpes simplex virus 2 DNA vaccine in a high yield cGMP process

“…Fermentations were performed in semi-defined media according to the HyperGRO™ fed-batch fermentation process as described previously. 4,5,11 Inocula were prepared from RSBs or MCBs and grown at 30°C in seed medium containing 6% (w/v) sucrose. Batch phase fermentation media contained 5 g/L sucrose for plasmid selection.…”

“…US Patent 7 943 377). 4,5 VGXI, Inc., a provider of DNA plasmid cGMP manufacturing and development services, has licensed NTC's HyperGRO™ technology and successfully scaled-up cGMP fermentation using this process to manufacture Coridon's HSV-2 DNA vaccines for a Phase I clinical trial. VGXI achieved reproducible plasmid yields of up to 1.8 g/L at the 320 L scale without the use of antibiotic selection, the largest fermentation scale yet using HyperGRO™ technology.…”

Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza Virus DNA Vaccine

“…Vectors were made by standard restriction digestion-mediated transfer of fragments between vectors as described previously (68). The insertions, deletions, and substitutions in vectors were made by inverse PCR, using amplification primers containing terminal AarI type IIS restriction enzyme sites for seamless site-directed mutagenesis (J.…”