Demand for high-quality plasmid DNA (pDNA) has greatly increased in recent years due to its myriad applications across therapeutic modalities – including gene therapies, vaccines, and mRNA. This ...increased demand poses a challenge to pDNA manufacturers, which must continue to increase yield and productivity without compromising on rigorous standards for purity, and ultimately, safety. Productivity in pDNA manufacturing is heavily influenced by host strain, plasmid type and size, growth medium and growth conditions.
A widely used culture medium, TB (Terrific Broth) offers high cell density cultures but lacks the capacity to generate large quantities of pDNA (>60 ng/µl). In understanding the significance and effect of peptones in TB a proprietary chemically defined media was designed to increase pDNA titers. This abstract captures efforts to create a high yielding and scalable fermentation process, involving the evaluation of various strategies in Escherichia coli DH5α transformed cells. The development of the process was driven by a three-level single-factor experimental design focused on media composition, temperature, concentration of peptone and carbon source. Improvements in small- and large-scale pDNA yield during fermentation, resulting from media selection and optimization are summarized. The results revealed that a chemically defined media used in conjunction with a nutrient-dense feed supported high cell density cultures and increased pDNA yield and specific productivity when compared to conventional exponential fed batch cultures in TB media. The feed approach developed was optimal for the three plasmids tested and constitute a robust pDNA yield optimization platform established at Akron enabling the production of cost effective pDNA for advanced therapies. This feed-based optimization platform is being tested with various E. Coli Stains and plasmid sizes for fitness and possible strain specific optimizations.
The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through ...the cell cycle causes metabolic regulations with variable productivities in antibody‐producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle‐derived population dynamics. In this study, process‐induced cell cycle synchronization was assessed in repeated‐batch and fed‐batch cultures. An automated flow cytometry set‐up was developed to measure the cell cycle distribution online, using antibody‐producing CHO DP‐12 cells transduced with the cell cycle‐specific fluorescent ubiquitination‐based cell cycle indicator (FUCCI) system. On the basis of the population‐resolved model, feeding‐induced partial self‐synchronization was predicted and the results were evaluated experimentally. In the repeated‐batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed‐batch process with up to 25×106 cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high‐frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle‐dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.
The authors report on their recent accomplishments in understanding and controlling the influence of process parameters on the formation of cell cycle‐enriched subpopulations and their dynamics in antibody‐producing CHO cells. They modified CHO cells genetically with the FUCCI (fluorescent ubiquitination‐based cell cycle indicator) system and developed an automated flow cytometer set‐up for high frequently measurement. Furthermore, feeding‐induced partial self‐synchronization was predicted based on a population‐resolved model and the simulation study was evaluated experimentally in repeated‐batch and fed‐batch cultivations.
To overcome catabolite repression, industrial fermentation processes are usually operated in substrate‐limited fed‐batch mode. Therefore, the implementation of such an operating mode at small scale ...is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well‐known producer of proteases, was cultivated with carbon (glucose)‐ and nitrogen (ammonium)‐limited fed‐batch conditions using the previously introduced membrane‐based fed‐batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5‐ and 2.1‐fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed‐batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (YP/Glu). In addition to YP/Glu, protease yields were determined based on the metabolized amount of oxygen (YP/O2). The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane‐based fed‐batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate‐limited fed‐batch conditions.
Small scale shaken bioreactors operated in fed‐batch mode have become important tools for screening and initial process development. In this study, Habicher and coworkers used a membrane‐based fed‐batch shake flask to realize continuous substrate release with parallel online monitoring of the oxygen transfer rate (OTR). It was demonstrated that defined substrate‐limited conditions allow overcoming catabolite repression in a protease producing Bacillus licheniformis strain.
•Optimizations in winery wastewater and sewage sludge treatment are tackled.•Recent metaheuristics namely CMAES, BSA and DE are found to give competent results.•Improved DE metaheuristic, BSA gives ...best overall performance for all problems.
Fed-batch fermentation has gained attention in recent years due to its beneficial impact in the economy and productivity of bioprocesses. However, the complexity of these processes requires an expert system that involves swarm intelligence-based metaheuristics such as Artificial Algae Algorithm (AAA), Artificial Bee Colony (ABC), Covariance Matrix Adaptation Evolution Strategy (CMAES) and Differential Evolution (DE) for simulation and optimization of the feeding trajectories. DE traditionally performs better than other evolutionary algorithms and swarm intelligence techniques in optimization of fed-batch fermentation. In this work, an improved version of DE namely Backtracking Search Algorithm (BSA) has edged DE and other recent metaheuristics to emerge as superior optimization method. This is shown by the results obtained by comparing the performance of BSA, DE, CMAES, AAA and ABC in solving six fed batch fermentation case studies. BSA gave the best overall performance by showing improved solutions and more robust convergence in comparison with various metaheuristics used in this work. Also, there is a gap in the study of fed-batch application of wastewater and sewage sludge treatment. Thus, the fed batch fermentation problems in winery wastewater treatment and biogas generation from sewage sludge are investigated and reformulated for optimization.
Despite wide applications of l‐tyrosine in the market, microbial overproduction of l‐tyrosine has been a great challenge due to the complex gene regulations involved in its biosynthetic pathway. To ...this end, effects of knocking out tyrR on the l‐tyrosine production were further explored during the strain development. Also, blocking cellular uptake of l‐tyrosine by knocking out tyrosine transporters was examined with respect to l‐tyrosine production. Using feedback‐resistant aroG and tyrA genes (aroGfbr and tyrAfbr hereafter) as initial overexpression targets, which encode 3‐deoxy‐7‐phosphoheptulonate synthase and chorismate mutase or prephenate dehydrogenase, respectively, various combinations of genes were subsequently overexpressed in the Escherichia coli wild‐type and tyrR knockout strain, and their effects on the l‐tyrosine production were examined. Co‐overexpression of aroGfbr, aroL and tyrC, a gene from Zymomonas mobilis functionally similar to tyrAfbr, but insensitive to l‐tyrosine, led to the greatest l‐tyrosine production regardless of the strains and plasmid constructs examined in this study. The strain BTY2.13 overexpressing the abovementioned three genes together with the removal of the l‐tyrosine‐specific transporter (tyrP) produced 43.14 g/L of l‐tyrosine by fed‐batch fermentation using the exponential feeding followed by DO‐stat feeding method. This outcome suggested that the tyrR gene knockout was not mandatory for the l‐tyrosine overproduction, but the production performance of strains having tyrR appeared to be highly affected by vector systems and feeding methods. With an optimal vector system and a feeding method, tyrP knockout appeared to be more effective in enhancing the l‐tyrosine than tyrR knockout.
Escherichia coli overexpressing aroGfbr, aroL and tyrC, with its l‐tyrosine‐specific transporter (tyrP) removed (strain name BTY2.13), produced up to 43.1 g/L of l‐tyrosine from its fed‐batch fermentation using the exponential‐to‐DO‐stat feeding method. The tyrC is a gene from Zymomonas mobilis functionally similar to tyrAfbr, but insensitive to l‐tyrosine.
•The concentration and yield of 1,3-PDO were 62.72 g/l and 0.73 mol/mol, respectively.•The 1,3-PDO productivity was 1.74 g/l/h by a continuous feeding strategy within 36 h.•By-products generation was ...reduced by a culture strategy of switching pH control.
In this study, crude glycerol was demonstrated as feedstock to use in the fed-batch fermentation of 1,3-propanediol production by Klebsiella pneumoniae ATCC 8724. To increase the yield and productivity of 1,3-propanediol, several factors for fed-batch fermentation were investigated, which included: initial substrate conditions, feeding strategy and fermentation conditions. Both performances of 1,3-propandiol and by-products were observed and studied for improved fed-batch fermentation. Increasing of initial working volume, continuous feeding, and culture pH switch strategy showed positive performance for this fed-batch process. The maximum 1,3-propandiol concentration, yield, and productivity were achieved 62.72 g/l, 0.73 mol/mol and 1.74 g/l/h, respectively. With the ignorable inhibitory effect, a large quantity of crude glycerol from industrial biodiesel process would be an attractive feedstock for value-added chemical, 1,3-propandiol, manufacture. The improvement of fed-batch fermentation could also be achieved by strategy optimization in further applications.
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