Project description:Biotechnology-derived therapeutics manufacturing is highly regulated to assure product quality, safety and efficacy. Process conditions are closely monitored as they can influence product characteristics. Culture of mammalian cells at different scales is a vital part of biomanufacturing. Currently, scale up of bioreactors is largely done based on engineering parameters such as oxygen transfer and mixing characteristics. There is a lack of genomic translational studies in mammalian cell culture scale-up that can help delineate measurable cellular attributes for improved process understanding. These could be used for quantifying physiological response of cells due to changes in bioreactor environment. In this study, we identified 18 process-sensitive sentinel genes using microarray statistical analysis, which we further verified by qRT-PCR. These were differentially expressed transcripts between a typical 5 liter bench-scale bubble-aerated and impeller-agitated bioreactor and novel 35 mL high-throughput minibioreactors. Using expression changes and biochemical-pathway guidance of these sentinel genes, we were able to tune engineering parameters such that the improved scale-down resulted in both process parameter and sentinel gene profile convergence between the two systems, further solidifying the functionality evidence of these sentinel genes. This study serves as a starting point for developing qRT-PCR assays as comparability metrics that could be performed near-at-line during the cell culture process itself leading to enhanced process control. Additional broad applications of sentinel genes could be to validate different manufacturing facilities for the same product, and allow better process definition by fingerprinting the manufacturing process for more rapid biogenerics and vaccine approvals. We ran consecutive bioreactor (5L and HTCB) runs, each with an independent vial thaw, to achieve multiple biological replicates per time-point. Bioreactors were sampled approximately every 12 hours for RNA extraction. For the 5L bioreactors, microarray samples were run for day 1 (n=2), day 2 (n=2), day 3 (n=3), and day 3.5 (n=3). Here 2 or 3 of the three biological replicates run for each time-point were included in the analysis, based on >70% genes found. For the High-Throughput Controlled Minibioreactors (HTCB), microarray samples were run for day 1 (n=3), day 2 (n=3), day 3 (n=3), day 4 (n=2), and day 4.5 (n=2). Here 2 to 4 of the 4 biological replicate runs were included in the analysis, based on >70% genes found. We define early exponential as day 1, peak exponential as day 2 and day 3 and late stationary as day 3.5.

Project description:(iMTD22IC) This model is described in these articles: Casini, I., McCubbin, T., Esquivel-Elizondo, S., Luque, G.G., Evseeva, D., Fink, C., Beblawy, S., Youngblut, N.D., Aristilde, L., Huson, D., Drager, A., Ley, R., Marcellin, E., Angenent, L.T., Molitor, B. 2023. An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter. Iscience, 26(10). Casini, I., McCubbin, T., Esquivel-Elizondo, S., Luque, G.G., Evseeva, D., Fink, C., Beblawy, S., Youngblut, N.D., Aristilde, L., Huson, D., Drager, A., Ley, R., Marcellin, E., Angenent, L.T., Molitor, B. 2022. An integrated systems-biology platform for power-to-gas technology. bioRxiv, 2022.12.30.522236. Methanogenesis allows methanogenic archaea (methanogens) to generate cellular energy for their growth while producing methane. Hydrogenotrophic methanogens thrive on carbon dioxide and molecular hydrogen as sole carbon and energy sources. Thermophilic and hydrogenotrophic Methanothermobacter spp. have been recognized as robust biocatalysts for a circular carbon economy and are now applied in power-to-gas technology. Here, we generated the first manually curated genome-scale metabolic reconstruction for three Methanothermobacter spp.. We investigated differences in growth performance of three wild-type strains and one genetically engineered strain in two independent chemostat bioreactor experiments, first, with molecular hydrogen and carbon dioxide, and second, with sodium formate. In the first experiment, we found the highest methane production rate for Methanothermobacter thermautotrophicus ΔH, while Methanothermobacter marburgensis Marburg reached the highest biomass growth rate. Transcriptomics and proteomics data sets from these steady-state bioreactors, in combination with the implementation of a pan-model that contains combined reactions from all three microbes, allowed us to perform an interspecies comparison. While the observed differences in the growth behavior cannot be fully explained, the comparison enabled us to identify crucial differences in growth-related pathways such as formate anabolism. In the second experiment, we found stable growth with a M. thermautotrophicus ΔH plasmid-carrying strain on formate with similar performance parameters compared to wild-type Methanothermobacter thermautotrophicus Z-245. The results of the two studies demonstrate the advantages of an integrative approach combining fermentation and omics data with genome-scale modeling to reveal knowledge gaps of archaeal metabolisms and the biotechnological potential of Methanothermobacter spp. as production platform hosts.

Project description:This model is described in these articles: Casini, I., McCubbin, T., Esquivel-Elizondo, S., Luque, G.G., Evseeva, D., Fink, C., Beblawy, S., Youngblut, N.D., Aristilde, L., Huson, D., Drager, A., Ley, R., Marcellin, E., Angenent, L.T., Molitor, B. 2023. An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter. Iscience, 26(10). Casini, I., McCubbin, T., Esquivel-Elizondo, S., Luque, G.G., Evseeva, D., Fink, C., Beblawy, S., Youngblut, N.D., Aristilde, L., Huson, D., Drager, A., Ley, R., Marcellin, E., Angenent, L.T., Molitor, B. 2022. An integrated systems-biology platform for power-to-gas technology. bioRxiv, 2022.12.30.522236. Methanogenesis allows methanogenic archaea (methanogens) to generate cellular energy for their growth while producing methane. Hydrogenotrophic methanogens thrive on carbon dioxide and molecular hydrogen as sole carbon and energy sources. Thermophilic and hydrogenotrophic Methanothermobacter spp. have been recognized as robust biocatalysts for a circular carbon economy and are now applied in power-to-gas technology. Here, we generated the first manually curated genome-scale metabolic reconstruction for three Methanothermobacter spp.. We investigated differences in growth performance of three wild-type strains and one genetically engineered strain in two independent chemostat bioreactor experiments, first, with molecular hydrogen and carbon dioxide, and second, with sodium formate. In the first experiment, we found the highest methane production rate for Methanothermobacter thermautotrophicus ΔH, while Methanothermobacter marburgensis Marburg reached the highest biomass growth rate. Transcriptomics and proteomics data sets from these steady-state bioreactors, in combination with the implementation of a pan-model that contains combined reactions from all three microbes, allowed us to perform an interspecies comparison. While the observed differences in the growth behavior cannot be fully explained, the comparison enabled us to identify crucial differences in growth-related pathways such as formate anabolism. In the second experiment, we found stable growth with a M. thermautotrophicus ΔH plasmid-carrying strain on formate with similar performance parameters compared to wild-type Methanothermobacter thermautotrophicus Z-245. The results of the two studies demonstrate the advantages of an integrative approach combining fermentation and omics data with genome-scale modeling to reveal knowledge gaps of archaeal metabolisms and the biotechnological potential of Methanothermobacter spp. as production platform hosts.

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Differential gene expression in CHO cells cultivated in shake flasks and bioreactors

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