Segregated growth kinetics of Escherichia coli DH5α‐NH36 in exponential‐fed perfusion culture for pDNA vaccine production

Abstract

The clinical demand of plasmid DNA (pDNA) has been increasing constantly. An exponential-fed perfusion (EFP) culture is a new mode for plasmid production for clinical trials and commercialization. However, the culture conditions may lead to cell filamentation and growth cessation. In this study, the variation of the physiological state and the plasmid contents of Escherichia coli DH5α hosting pVAX1-NH36 in an EFP culture for application as a Leishmaniasis vaccine was investigated. The culture performance was monitored using flow cytometry (FC) and real-time quantitative PCR. The FC studies showed a high viability of cell population and a constant distribution of complexity and size. A high homogeneity of pDNA (>95 % of supercoiled) was obtained, which might be attributed to a better culture environment. The obtained plasmid specific and volumetric yields of 1.8 mg/g dcw and 36.5 mg/L represent typical values for laboratory-scale plasmid production in a defined medium. A segregated kinetic model of the perfusion system was developed and fitted to the experimental data (R(2) > 0.96). A practical conclusion of this work is that a space-time yield analysis of a bioprocess requires a viability evaluation. This new strategy of culture operation might help in the efficient production of pDNA for therapeutic use.

Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

“…A 100 mL sample of exponential phase culture was used to provide a 10% inoculum volume for the fermentations. Fermentation studies were conducted as described previously using a 3.0 L stirred tank bioreactor (Applikon® Biotechnology, Delft, The Netherlands). During fermentation, pH was controlled at 6.8 ± 0.1 by the automatic addition of 12.5% ammonia solution.…”

“…Plasmid DNA (pDNA) vaccine is a promising technology that allows foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Beside simplicity in manipulation and rapid manufacturing process, pDNA‐based vaccines have inherent features that make them promising vaccine candidates in a variety of diseases . The clinical application of pDNA vaccines is progressing and it is essential to devise industrial processes whereby pDNA can be economically manufactured .…”

“…These changes are generally associated with transition in growth conditions at the end of batch and fed‐batch cultures . It has been reported that EFP cultures produce a highly pure supercoiled isoform, perhaps due a healthier and more constant environment.…”

“…A perfusion culture for plasmid production may be an alternative mode to conduct HCDC to avoid the limitations of conventional cultures. It has been determined by electrophoresis and cell cytometry studies that perfusion cultures help to produce pure supercoiled plasmids and to minimize cell damage …”

“…Recently, a new culture strategy for plasmid production has been developed by combining an exponential feeding strategy with perfusion cultivation . In this mode of operation exponential cell growth and a constant specific growth rate can be achieved.…”