Poster De Conférence Année : 2024

Scalable production of small extracellular vesicles derived from a human beta cell line cultured in stirred tank bioreactor with high yield and purity

Résumé

FSEV2400067 Scalable purification of small extracellular vesicles derived from a human cell line cultured in stirred tank bioreactor with high yield and purity T. Dauphin1, F. Binet1,2, Q. Pruvost1, J. Makula1, C. Claire1,2, V. Lalanne1,2, J. Herve1,2, J.M. Bach1, S. Bosch1, B. Lieubeau1,2, L. De Beaurepaire1,2, M. Mosser1 1ONIRIS, INRAE, IECM - NANTES (France), 2 ONIRIS, B-FHIT - NANTES (France) Background. Therapeutic applications of small extracellular vesicles (sEV) are gaining significant attention, but challenges remain in ensuring standardization and large-scale production. sEV are a heterogeneous population of particles smaller than 200 nm, with their composition and purity varying according to the cell culture conditions and the methods of separation. Commonly, sEV are produced from adherent cells cultured in static T-flask, and isolated by ultracentrifugation based-methods (UC). The use of stirred-tank bioreactors is crucial for process scalability but requires fine-tuning of hydrodynamic parameters across scales. Furthermore, UC methods have drawbacks, such as EV aggregation and manual workload. Therefore, it is essential to establish a downstream processes that integrated scalable technologies such as in-depth filtration, tangential flow filtration and low-pressure chromatography. In this study, we present the development of a scalable process for sEV production and purification. Material & Methods. The human beta cell line 1.4E7 was used as a model of anchorage-dependent cells. A process was established in which the cell culture in T-flask was replaced by spheroid culture in stirred tank systems. It was evaluated whether the best scale-up strategy for standardizing spheroid formation was to keep either the volumetric power input (P/V) or the tip speed constant in spinner flask. The impact of this up-scale process was assessed by analysis of spheroid morphology and viability. The process was then transferred to a fully controlled bioreactor. For EV isolation, the feasibility of replacing differential centrifugation with depth filtration and gravity size exclusion column with an automated chromatography using columns based on size and charge were assessed. EV size and concentration was determined through nanoparticle tracking analysis, and purity was assessed by the ratio of particles or tetraspanin-positive particles to all protein. EV protein expression was analyzed by western blotting. Results. Culturing 1.4E7 cells in suspension reduced their specific growth rate, but promoted the formation of highly viable spheroids and increased sEV yields two-fold. Maintaining a constant power input proved to be the best strategy to scale-up spheroid formation. By defining the maximal volume that can be processed per filter area and flow rate, substituting differential centrifugation with scalable in-depth filtration maintained both sEV yield and purity. Automate multimodal chromatography columns isolated sEV of comparable yield and purity to manual gravity size exclusion chromatography. Preliminary results also indicated that a strong anion exchange column allowed the selection of different EVs population according to their charge, with acceptable yields and purities. Conclusion. This study underscores the main challenges in designing a scalable production and purification process for therapeutic sEVs, ensuring that the final products meet desired quality and quantity attributes.

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hal-05052858 , version 1 (30-04-2025)

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Thibaud Dauphin, Floriane Binet, Quentin Pruvost, Julien Makula, Clémentine Claire, et al.. Scalable production of small extracellular vesicles derived from a human beta cell line cultured in stirred tank bioreactor with high yield and purity. 7th Congress of the FSEV, Nov 2024, Strasbourg, France. ⟨hal-05052858⟩
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