Fed‐batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

Abstract

The rising costs of bioprocess research and development emphasize the need for high‐throughput, low‐cost alternatives to bench‐scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed‐batch or continuous. We have developed a 1‐mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1‐GFP) in Escherichia coli via a fed‐batch temperature‐inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench‐scale bioreactor. The predictive abilities of the micro‐scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Biotechnol. Bioeng. 2012; 109:1976–1986. © 2012 Wiley Periodicals, Inc.

Development of new plasmid DNA vaccine vectors with R1-based replicons

“…Plasmid copy number was determined from 100 μL of culture using the quantitative PCR (qPCR) method described by Bower et al [29] In short, SYBR Green was used to detect amplification of plasmid-based and chromosomal gene targets from total DNA samples diluted 5-fold (if necessary) to be within the linear range of the assay. The ΔΔC T method [30] was used to calculate plasmid copy number.…”

“…Plasmid DNA was quantified from crude lysates prepared from OD 600 = 10 cell pellets using the method described previously [29]. Briefly, crude lysates were run on a Gen-Pak FAX anion-exchange column (Waters Corporation; Milford, MA) and pDNA was eluted using a NaCl gradient.…”

Consistent development of bioprocesses from microliter cultures to the industrial scale

“…Recently by Bower et al [34], a microbioreactor was presented that enables fed-batch operations in addition to the monitoring and control of temperature, pH and DOT, and even of the cell density, at 1 mL working volume. The growth chamber is connected to a sampling port and to three liquid reservoirs.…”

Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

“…Scale‐down bioprocesses form the backbone of rapid bioprocess design, allowing for cost‐effective and rapid development of biopharmaceutical production processes. Specifically, small‐scale fermentation process design combined with multivariate data analysis techniques and subsequent process upscaling has proven to be effective for characterizing and optimizing industrial‐scale operations …”

“…Specifically, small-scale fermentation process design combined with multivariate data analysis techniques and subsequent process upscaling has proven to be effective for characterizing and optimizing industrial-scale operations. [1][2][3][4] Pichia pastoris, recently reassigned to the genus Komagataella, is a methanol assimilating yeast that has been receiving increased industrial attention over the past two decades due to its potential to produce a wide array of complex biopharmaceuticals. [5][6][7][8][9][10][11][12] P. pastoris offers the potential of heterologous protein secretion, however depending on physical and chemical properties of the protein of interest, this is not always a viable option.…”