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Exploring nutrient supplementation and bioprocess optimization to improve the production of lentiviral vectors in serum-free medium suspension cultures
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Orientador(es)
Resumo(s)
The use of lentiviral vectors (LV) in gene therapy has been growing in recent years. To meet the increasing clinical demand, LV production platforms will benefit from improved productivity and scalability to enable cost-effective manufacture of LV-based therapies. Here we report the adaptation of 293T cells to serum-free suspension cultures and the improvement of LV yields through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters. Cells were sequentially adapted to different serum-free culture media, transfection parameters were optimized and the two best-performing conditions were selected to explore process intensification by increasing cell density at the time of transfection. LV production at higher cell densities increased volumetric titers up to 12-fold and lipid supplementation was the most efficient metabolic optimization strategy further enhancing LV productivity by 3-fold. Furthermore, cell concentration was identified and validated as an important source of transfection variability impairing cellular uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers down to 6-fold. This work contributes to improving LV-based gene therapy by establishing new scalable manufacturing platforms and providing key metabolic insights, unveiling important bioreaction parameters to improve vector yields.
Descrição
Funding Information: T.A.V. acknowledges FCT for the award of the individual grant SFRH/BD/145270/2019. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.
Palavras-chave
bioprocess engineering gene therapy lentiviral vectors serum-free medium suspension culture transient transfection Applied Microbiology and Biotechnology Molecular Medicine