Publicação
Development of chemically defined medium for biopharmaceuticals production using mammalian cell lines guided by metabolic modelling tools and metabolomics measurements
| Resumo: | Systems biology and metabolic engineering tools hold a tremendous promise in improving biomanufacturing attributes. The emergence of omics tools and computational modeling potentiated the development of new approaches to optimize several expression platforms, in particular mammalian cell lines of which Chinese hamster ovary (CHO) cells, the most used platform for recombinant proteins production. This optimization envisions not only growth parameters of CHO, but also the final product titers. In this context, a CHO genome scale metabolic model (iCHO1766) and flux balance analysis (FBA) were used to study metabolic mechanisms in response to variations in environmental constraints (e.g., amino acids levels) aiming at optimizing cell culture medium formulations. Hence, iCHO1766, combined with an in-house developed algorithm (OptiModels) was first used to determine the minimal medium formulation able to sustain growth of both naïve and recombinant CHO cells lines. Subsequently, based on the prediction results, α-ketoglutarate (AKG) was determined as a potential media supplement and its effect on culture was investigated experimentally. Further, spent media analyses were performed to understand the influence of AKG on CHO metabolism and media formulation was optimized based on balancing the levels of non-essential amino acids together with supplementing AKG and ammonium. As a result of adding AKG to the media, growth parameters were improved, and ammonia accumulation during the process was reduced. In addition, recombinant protein titers were increased by 1.9-fold. Following, specific productivities were improved when rebalancing nutrient levels in the media, together with supplementing AKG, leading to more efficient metabolic features of CHO. In conclusion, in silico-based approaches for medium optimization are powerful tools for predicting the metabolic interconnexions within a cell and hold great potential in improving media design and bioprocess optimization. |
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| Autores principais: | Hamdi, Anis |
| Assunto: | CHO cells GSMM Media optimization α-ketoglutarate (AKG) Células CHO Otimização do meio de cultura α-cetoglutarato (AKG) Engenharia e Tecnologia::Biotecnologia Industrial |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Systems biology and metabolic engineering tools hold a tremendous promise in improving biomanufacturing attributes. The emergence of omics tools and computational modeling potentiated the development of new approaches to optimize several expression platforms, in particular mammalian cell lines of which Chinese hamster ovary (CHO) cells, the most used platform for recombinant proteins production. This optimization envisions not only growth parameters of CHO, but also the final product titers. In this context, a CHO genome scale metabolic model (iCHO1766) and flux balance analysis (FBA) were used to study metabolic mechanisms in response to variations in environmental constraints (e.g., amino acids levels) aiming at optimizing cell culture medium formulations. Hence, iCHO1766, combined with an in-house developed algorithm (OptiModels) was first used to determine the minimal medium formulation able to sustain growth of both naïve and recombinant CHO cells lines. Subsequently, based on the prediction results, α-ketoglutarate (AKG) was determined as a potential media supplement and its effect on culture was investigated experimentally. Further, spent media analyses were performed to understand the influence of AKG on CHO metabolism and media formulation was optimized based on balancing the levels of non-essential amino acids together with supplementing AKG and ammonium. As a result of adding AKG to the media, growth parameters were improved, and ammonia accumulation during the process was reduced. In addition, recombinant protein titers were increased by 1.9-fold. Following, specific productivities were improved when rebalancing nutrient levels in the media, together with supplementing AKG, leading to more efficient metabolic features of CHO. In conclusion, in silico-based approaches for medium optimization are powerful tools for predicting the metabolic interconnexions within a cell and hold great potential in improving media design and bioprocess optimization. |
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