(160u) Development of a high-density perfusion bioreactor production unit using scale-down models and marine bacterium Rhodovulum sulfidophilum for the production of therapeutic oligonucleotides
AIChE Annual Meeting
Monday, November 8, 2021 - 3:30pm to 5:00pm
Furthermore, bioengineered ONs can decrease immunogenic responses when compared to its chemical synthetized counterparts . This justifies the current interest in producing ONs via bacterial fermentation. In previous work, the marine bacterium Rhodovulum sulfidophilum has successfully been used for the production of ONs , but a process applicable at the manufacturing level still needs to be defined. In this work, we determined the design space for a high cell density continuous perfusion bioreactor, which was already demonstrated amenable for high productivities in the integrated continuous biomanufacturing of other biopharmaceuticals . Towards this aim, we have used 50 mL spin-tubes as scale-down models of the perfusion bioreactor . The use of this model allows for the parallelization of experiments and cost reduction.
Firstly, a media optimization step was conducted leading to an increase of 44% in cell density, accompanied by a decrease in cost and complexity of the medium. Secondly, multiple experiments were run, screening perfusion rates from 0.05 to 1 RV/day. These experiments consisted of investigating the maximum achievable cell density before the collapse of the culture for each perfusion rate. This allowed the discovery of the minimum cell-specific perfusion rate (CSPRmin) and the maximum viable cell density (VCDmax). Figure 1 shows the possible operation area of the perfusion bioreactor. Specifically, this area is characterized by the already known limitation of the CSPRmin  and by a superior limitation that has not been described so far in literature: CSPRmax. The latter limitation is most likely caused by nutrient inhibition due to high level of media replenishment.
Concluding, this work shows the first steps in developing a high-density continuous fermentation unit for the production of oligonucleotides. Further improvements will consist validating the results at the laboratory scale in order to ensure culture stability.
- Catani, M., De Luca, J. Medeiros Garcia AlcÃ¢ntara, et al., âOligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification,â Biotechnology Journal, 15 (8), p. 1900226 (2020).
- Karst, D.J., F. Steinebach, and M. Morbidelli, âContinuous integrated manufacturing of therapeutic proteins,â Current Opinion in Biotechnology, 53, pp. 76â84 (2018).
- Pereira, P., Q. Pedro, J. TomÃ¡s, et al., âAdvances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum,â Applied Microbiology and Biotechnology, 100 (8), pp. 3723â3734 (2016).
- Wolf, M., -M. Bielser, and M. Morbidelli, âPerfusion Cell Culture Processes for Biopharmaceuticals,â Cambridge University Press, (2020).
- Wolf, M.K.F., V. Lorenz, D.J. Karst, J. Souquet, H. Broly, and M. Morbidelli, âDevelopment of a shake-tube-based scale-down model for perfusion cultures,â Biotechnology and Bioengineering, (2018).
- Yu, A.-M., C. Jian, A.H. Yu, and M.-J. Tu, âRNA therapy: Are we using the right molecules?,âPharmacology & Therapeutics, 196, pp. 91â104 (2019).