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Exploring CHO cell stability during prolonged passaging via eXplainable AI driven flux balance analysis.

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Long-term Chinese hamster ovary (CHO) cell culture for protein production faces stability challenges. This study reveals late-passage CHO cells shift metabolism from growth to managing oxidative stress, impacting therapeutic protein yields.

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Metabolic Engineering

Background:

  • Production stability in Chinese hamster ovary (CHO) cell lines is crucial for therapeutic protein manufacturing but is often compromised during extended cell passaging.
  • The underlying mechanisms driving instability and reduced productivity in late-passage CHO cultures remain poorly understood.

Purpose of the Study:

  • To mechanistically characterize the phenotypic and metabolic differences between early-passage (EP) and late-passage (LP) CHO cultures.
  • To identify key metabolic pathways responsible for the divergence in cell behavior and productivity over extended passaging.

Main Methods:

  • Multivariate data analysis (MVDA) of temporal exometabolite profiles.
  • Flux balance analysis (FBA) integrated with explainable artificial intelligence (xAI) to interrogate metabolic rewiring.
  • Comparison of cell growth, therapeutic protein (IgG) titers, and key metabolite concentrations between EP and LP cultures.

Main Results:

  • Late-passage (LP) CHO cultures showed comparable peak viable cell densities but a significant reduction (~35%) in peak IgG titers compared to early-passage (EP) cultures.
  • LP cultures exhibited increased accumulation of lactate and ammonia, indicating altered metabolic function.
  • MVDA identified the exponential growth phase as the critical window for metabolic divergence between EP and LP cultures.

Conclusions:

  • Early-passage (EP) CHO cells prioritize acetyl-CoA for fatty acid biosynthesis to support proliferation.
  • Late-passage (LP) CHO cells shift metabolic focus towards oxidative stress mitigation via the trans-sulfuration pathway (cysteine and glutathione synthesis) and heightened TCA cycle activity for energy homeostasis.
  • The cysteine-glutathione axis represents a critical metabolic target for improving the long-term stability and productivity of CHO cell cultures.