(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

The number of approvals of oligonucleotide (ON) based therapeutics has been steadily increasing throughout the last decade [1], testifying the increasing importance of this class of biopharmaceuticals. Currently, most of the production is done via solid-state synthesis, utilizing phosphoramidite chemistry. However, the yields of this technique are progressively lower as the length of the ON increases [1].

Furthermore, bioengineered ONs can decrease immunogenic responses when compared to its chemical synthetized counterparts [6]. 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 [3], 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 [2]. Towards this aim, we have used 50 mL spin-tubes as scale-down models of the perfusion bioreactor [5]. 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 [4] 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.

References

1. Catani, M., De Luca, J. Medeiros Garcia Alcântara, et al., “Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification,” Biotechnology Journal, 15 (8), p. 1900226 (2020).
2. Karst, D.J., F. Steinebach, and M. Morbidelli, “Continuous integrated manufacturing of therapeutic proteins,” Current Opinion in Biotechnology, 53, pp. 76–84 (2018).
3. Pereira, P., Q. Pedro, J. Tomás, et al., “Advances in time course extracellular production of human pre-miR-29b from Rhodovulum sulfidophilum,” Applied Microbiology and Biotechnology, 100 (8), pp. 3723–3734 (2016).
4. Wolf, M., -M. Bielser, and M. Morbidelli, “Perfusion Cell Culture Processes for Biopharmaceuticals,” Cambridge University Press, (2020).

5. Wolf, M.K.F., V. Lorenz, D.J. Karst, J. Souquet, H. Broly, and M. Morbidelli, “Development of a shake-tube-based scale-down model for perfusion cultures,” Biotechnology and Bioengineering, (2018).
6. Yu, A.-M., C. Jian, A.H. Yu, and M.-J. Tu, “RNA therapy: Are we using the right molecules?,”Pharmacology & Therapeutics, 196, pp. 91–104 (2019).

Figure 1 - Perfusion bioreactor operating region. OD600 – optical density at 600nm. RV – reactor volume