Effect of cultivation conditions on growth, productivity and metabolism of CHO cellsBeltram, Ajda (Avtor) Župunski, Vera (Mentor) proizvodnja rekombinantnega proteinacelice CHObioreaktormetabolizem celic CHOBiopharmaceuticals, especially recombinant proteins produced in mammalian cells, are a cornerstone of modern medicine, with Chinese Hamster Ovary (CHO) cells being the most widely used host system due to their ability to perform human-like post-translational modifications. CHO cells were adapted to grow in suspension culture, which enabled the use of stirred tank reactors (STRs) and therefore production of proteins in larger quantities compared to adherent cultures. To be able to support process development, optimization, and troubleshooting efficiently and cost-effectively, each process at manufacturing scale must have a replicate at a smaller laboratory scale, known as scale-down model (SDM). SDMs must maintain equivalence in critical quality attributes and respond similarly to changes in process parameters. To investigate the suitability of various laboratory-scale vessels for developing a reliable SDM of a commercial recombinant protein production process using CHO cells, we performed the process using different lab-scale vessels (shake flasks, spinner flasks, 5 L STR) and assessed their ability to replicate key scale-dependent and scale-independent parameters observed at manufacturing scale as well as productivity and cell metabolism. The results indicated that neither spinner flasks nor shake flasks are suitable for SDM. Spinner flasks fail to provide adequate gas exchange and oxygen availability, leading to suboptimal growth, while shake flasks, despite having closer resemblance to STRs, still exhibit notable differences in metabolic activity and oxygen saturation. Furthermore, the investigation of CHO cells’ response to suboptimal environmental conditions, such as altered pH, low oxygen (hypoxia), and the absence of CO$_2$, showed that low pH and hypoxia significantly impaired cell growth and productivity, and the absence of CO$_2$ disrupted pH balance and metabolic stability. Although CHO cells attempted to adapt by shifting metabolic pathways (e.g., increasing glycolysis and lactate production), these adaptations were insufficient to maintain optimal performance. 20252025-09-24 14:15:01Magistrsko delo/naloga173873VisID: 24263COBISS_ID: 258829315sl