Presentation Information
[1AFOB-20]CHO Cell Spent Culture Media as Alternative Platform for Escherichia coli Recombinant Protein Production: Growth, Stability, and Proteome-Level Perspectives
○Judee Nogodula1, Takeyuki Mogi2, Tetsushi Namatame2, Pijar Religia3, Noriko Yamano-Adachi1,4, Kohsuke Honda3, Takeshi Omasa1,4 (1. Department of Biotechnology, Graduate School of Engineering, The University of Osaka (Japan), 2. Yokogawa Electric Corporation (Japan), 3. International Center for Biotechnology, Graduate School of Engineering, The University of Osaka (Japan), 4. Institute for Open and Transdisciplinary Research Initiatives (Japan))
Keywords:
CHO spent culture media,sustainable bioprocessing,recombinant protein production
Chinese hamster ovary (CHO) cells from the backbone of global monoclonal antibody manufacturing, yet their cultivation generates vast volumes of nutrient-rich spent medium that are routinely discarded. Transforming CHO spent medium into a usable feedstock for microbial systems offers a powerful route toward more sustainable and cost-effective biomanufacturing. Despite this potential, its suitability for supporting high-level recombinant protein production and maintaining microbial genetic stability has not been fully established.
Here, CHO spent media were assessed as a substrate for recombinant protein expression in GFP-expressing Escherichia coli. Multiple spent-medium formulations were benchmarked against Luria-Bertani (LB) broth. Cultures initiated at normalized starting cell densities and monitored over a 24-hour period. Growth and production were assessed through optical density and GFP fluorescence measurements, while plasmid stability was evaluated via antibiotic resistance profiling and plasmid copy number was quantified using qPCR. These data were used to calculate specific growth rates, and specific productivity.
Several CHO-spent media formulations supported growth kinetics that matched or exceeded those observed in LB, highlighting the presence of bioavailable residual nutrients and host-cell derived metabolites. These conditions sustained strong biomass accumulation, demonstrating that E.coli efficiently exploit recycled media. In the highest-performing formulation, biomass-normalized GFP yield and specific productivity were comparable to or greater than those achieved in LB throughout the primary growth and production phase.
Notably, cultures propagated in spent media consistently maintained a dominant population of antibiotic-resistant cells from the mid-logarithmic phase through to the late-log phase of growth. This observation indicates that selective pressure remained effective despite the use of a recycled and compositionally altered medium. The persistence of antibiotic resistance suggests that the plasmid-bearing population was not overtaken by plasmid-free cells, a common concern in suboptimal or stressful growth conditions where metabolic burden can favor plasmid loss.
Ongoing label-free quantitative proteomic analyses are investigating how E.coli adapts to growth in CHO spent media. Preliminary insights suggest substantial reprogramming of central carbon metabolism, activation of stress-response pathways, and modulation of translational machinery, collectively enabling efficient growth and protein production.
Overall, these results establish CHO spent medium as a robust and scalable substrate for microbial bioprocessing. This work advances a circular bioeconomy framework by bridging mammalian and microbial production platforms, reducing waste streams, and enhancing resource utilization. The integration of CHO and E.coli systems demonstrated a practical and innovative strategy for biomanufacturing.
Here, CHO spent media were assessed as a substrate for recombinant protein expression in GFP-expressing Escherichia coli. Multiple spent-medium formulations were benchmarked against Luria-Bertani (LB) broth. Cultures initiated at normalized starting cell densities and monitored over a 24-hour period. Growth and production were assessed through optical density and GFP fluorescence measurements, while plasmid stability was evaluated via antibiotic resistance profiling and plasmid copy number was quantified using qPCR. These data were used to calculate specific growth rates, and specific productivity.
Several CHO-spent media formulations supported growth kinetics that matched or exceeded those observed in LB, highlighting the presence of bioavailable residual nutrients and host-cell derived metabolites. These conditions sustained strong biomass accumulation, demonstrating that E.coli efficiently exploit recycled media. In the highest-performing formulation, biomass-normalized GFP yield and specific productivity were comparable to or greater than those achieved in LB throughout the primary growth and production phase.
Notably, cultures propagated in spent media consistently maintained a dominant population of antibiotic-resistant cells from the mid-logarithmic phase through to the late-log phase of growth. This observation indicates that selective pressure remained effective despite the use of a recycled and compositionally altered medium. The persistence of antibiotic resistance suggests that the plasmid-bearing population was not overtaken by plasmid-free cells, a common concern in suboptimal or stressful growth conditions where metabolic burden can favor plasmid loss.
Ongoing label-free quantitative proteomic analyses are investigating how E.coli adapts to growth in CHO spent media. Preliminary insights suggest substantial reprogramming of central carbon metabolism, activation of stress-response pathways, and modulation of translational machinery, collectively enabling efficient growth and protein production.
Overall, these results establish CHO spent medium as a robust and scalable substrate for microbial bioprocessing. This work advances a circular bioeconomy framework by bridging mammalian and microbial production platforms, reducing waste streams, and enhancing resource utilization. The integration of CHO and E.coli systems demonstrated a practical and innovative strategy for biomanufacturing.
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