Jochen Büchs

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality.

Cell line generation of mammalian cells is a time‐consuming and labor‐intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource‐intensive and time‐consuming due to necessary sampling and offline analysis effort. This study, thus, explores the potential of online monitoring the oxygen transfer rate (OTR), as a non‐invasive and efficient alternative for kill curve experiments. The OTR is monitored using the Transfer‐rate Online Measurement (TOM) system and the micro(μ)‐scale Transfer‐rate Online Measurement (μTOM) device, which was used for mammalian cells first. It could be shown that the OTR curves for both devices align perfectly, affirming consistent cultivation conditions. The μTOM device proves effective in performing kill curve experiments in 96‐deep‐well plates without the need for sampling and offline analysis. The streamlined approach reduces medium consumption by 95%, offering a cost‐effective and time‐efficient solution for kill curve experiments. The study validates the generalizability of the method by applying it to two different CHO cell lines (CHO‐K1 and sciCHO) with two antibiotics (puromycin and hygromycin B) each. In conclusion, the broad application of OTR online monitoring for CHO cell cultures in 96‐deep‐well plates is highlighted. The μTOM device proves as a valuable tool for high‐throughput experiments, paving the way for diverse applications, such as media and clone screening, cytotoxicity tests, and scale‐up experiments.