(24f) Hydrodynamic Stress Adaptation of CHO Cell Lines By Acoustic Resonance Mixing

Mammalian cells cultivated in bioreactors are pivotal to produce biopharmaceuticals. However, scalability often encounters obstacles due to hydrodynamic stress, which encompasses mechanical forces arising from fluid flow in bioreactors. Although higher agitation rates enhance mixing and oxygen transfer rates, they also increase shear stress, leading to adverse effects on cell viability and product quality. Within the realm of hydrodynamic forces present in cell culture, the mechanical force associated with the gas-liquid interface is the primary factor affecting cellular performance and product quality.

Several scaling-down approaches have been explored to investigate hydrodynamic stress. Nonetheless, a comprehensive model or specific parameter that encompasses all aspects of cellular response remains to be established. Particularly in the context of acoustic resonance mixing (RAM), increasing oxygen transfer rates in bacterial shake flask cultures has shown potential for improving productivity. Hence, reducing hydrodynamic stress with low-frequency acoustic energy, coupled with potential cell adaptation to agitation conditions, is anticipated to have a beneficial effect on CHO cells.

Here, we evaluated the impact of RAM on the production of a monoclonal antibody (mAb) alongside the adaptation of industrially relevant recombinant CHO DG44 cell lines. Cells were adapted to growth under RAM in 250 mL Corning polycarbonate Erlenmeyer flasks with 50 mL of culture media. The CHO cell's lethal and sub-lethal effects were assessed through specific growth rate analysis, cell viability, specific mAb productivity, and lactate dehydrogenase release. Additionally, glucose consumption and ammonia and lactic acid release were monitored throughout the cultures and compared with those with lower hydrodynamic stress (i.e. orbitally agitated T25 flasks at 60 rpm).

Cells grown using RAM at 3 x g seem to experience a significant increase in stress, resulting in a specific growth rate of 0.023 h^-1 compared to the same cell line evaluated under lower stress conditions of 0.029 h^-1 for the T25 flask system. No significant differences in mAb production were found (around 90 ng/mL).

As a novel strategy, allowing minor stages of viability recovery under lower (or none) agitation following stress exposure in RAM at 3 x g did not lead to observable adaptation, as the required time for this process was significantly increased. Direct adaptation, involving a continuous change in culture medium, maintained cell viability and mAb production under RAM for almost 500 hours. Obtaining five cell lines at different exposure times of the culture revealed an increase in specific growth rate of 10-15% when cells were grown without stress in T25 flasks.

From these results, RAM proved to be a robust method for cell adaptation to hydrodynamic stress. It is hypothesized that this adaptation is attributed to changes in differentially expressed protein patterns associated with cytoskeletal restructuring and regulation of factors related to apoptosis avoidance. The differences in cell growth rate and maximum viable cell density may be related to similar mechanisms, probably influenced by the cell's better capability to go beyond cell death.

A more comprehensive understanding of the cell response to hydrodynamic stress is essential for optimizing cell culture processes and ensuring cell viability, productivity, and product quality in biopharmaceutical production. , we present a study on the acquisition of a hydrodynamic stress-adapted CHO cell line, representing a significant advancement in understanding hydrodynamic stress compression in mammalian cell culture.