Overcoming CHO Cell Culture Challenges: Dissolved Oxygen Balance and Mass Transfer Optimization

Oxygen is essential for cellular respiration and plays a critical role in cell function.

In cells, oxygen serves as the terminal electron acceptor in the electron transport chain, where it combines with hydrogen ions to form water, releasing a large amount of energy for cellular use. During this process, oxidative phosphorylation enables the synthesis of adenosine triphosphate (ATP), which provides the energy required for various cellular activities and metabolic processes. Moreover, oxygen availability influences cell proliferation—under optimal oxygen concentrations, cells undergo mitosis and proliferate normally. Insufficient oxygen supply, however, may slow down or even halt cell proliferation. Therefore, precise control of oxygen concentration is essential in in vitro cell culture experiments to ensure an optimal environment for cell growth and proliferation.

In CHO cell culture, dissolved oxygen (DO) levels are typically maintained between 10% and 80% of air saturation. Excessive oxygen concentrations can lead to the accumulation of reactive oxygen species (ROS), which disrupt the mitochondrial respiratory chain and intracellular redox balance, ultimately inhibiting cell growth and reducing productivity. Conversely, hypoxic conditions are also known to induce ROS accumulation, which is associated with reduced oxygen consumption and increased lactate production.

At large scales (e.g., 2000 L), ROS accumulation has been linked to a significant decline in productivity, whereas this issue is not observed at smaller scales (e.g., 20 L). Thus, careful selection of DO setpoints is critical during scale-up, particularly due to hydrostatic pressure effects. At similar DO levels (expressed as a percentage of air saturation), the actual oxygen concentration in large-scale bioreactors is significantly higher compared to benchtop bioreactors.

Although surface aeration reduces shear stress caused by bubble rupture, its overall contribution to gas transfer becomes negligible at larger scales. To mitigate shear stress induced by bubble breakage at the gas-liquid interface, surfactants such as Pluronic F-68 (or Kolliphor P188) are commonly added to cell culture media. A Pluronic concentration of 1 g/L (up to a maximum of 5 g/L) is typically used to provide shear protection.

The oxygen transfer coefficient (kLa) is used to evaluate a system’s oxygen transfer capability. For a given bioreactor configuration, kLa primarily depends on volumetric power input (P/V) and superficial gas velocity (νs), as described by the following relationship: where K is a constant, and the exponents α and β are approximately 0.4 and 0.5, respectively.

Process parameters such as bubble size, mixing speed, gas flow rate, volume, and bioreactor geometry all influence kLa. Various medium components, including salts, antifoam emulsions, and surfactants, have also been shown to affect the mass transfer coefficient. In particular, antifoam emulsions significantly reduce kLa, while Pluronic F-68 also decreases kLa, though to a lesser extent. In contrast, the addition of salts increases the oxygen transfer coefficient.

Another factor influencing kLa is the number of impellers. In coalescence-inhibiting media (such as most culture media), when volumetric power input and gas flow rate are kept constant, using a single impeller results in a higher kLa compared to a three-impeller configuration. This suggests that concentrating power input at the bubble formation site enhances overall mass transfer. Generally, scaling up bioreactors with similar geometries leads to an increase in kLa, a trend observed in both traditional stirred-tank and single-use bioreactors. Additionally, increased liquid height extends gas residence time, thereby improving gas transfer efficiency.

Gas Diffusion in CytoLinX® BR Single-use Bioreactors

The structure and design of a bioreactor are closely linked to its functionality. An optimized reactor shape facilitates efficient gas transfer and partial pressure control, enhancing the regulation of culture conditions in microbial and cell cultivation. This, in turn, improves process control and simplifies parameter adjustments.

Bio-Link’s bioreactors are designed with robust structures and comprehensive functionality, offering a range of capacities including 10L, 50L, 200L, 500L, 1000L, and 2000L to accommodate various scale-up needs. These bioreactors are suitable for industrial-scale mammalian and insect cell cultures, as well as other applications requiring low shear conditions. Advanced pH and DO control systems enable optimal cell growth, while flexible customization services cater to specific customer requirements, fostering mutual success.

CytoLinX® BR Single-use Bioreactors

• Featuring an optimized tank design and comprehensive functionality, a single control cabinet can manage multiple bioreactor units via a plug-and-play system, significantly reducing costs. Different bioreactor volumes are available to support process scale-up based on specific operational needs.

• The system is built on a stable PCS7 platform, compliant with the ISA 88 control system, enabling full facility control.

• Its software design meets 21 CFR Part 11 requirements, with a user-friendly interface.

• The configuration is highly flexible, allowing partial customization to meet user specifications.

• All equipment components are sourced from premium international brands and undergo factory testing to ensure reliable performance.

• For single-use applications, the system supports bioreactor bags with bottom sparging options, including large bubbles, medium bubbles, and microbubbles, accommodating various process requirements.

References：

Lucas Lemire, Phuong Lan Pham, Yves Durocher, and Olivier Henry 《Practical Considerations for the Scale-Up of Chinese Hamster Ovary (CHO) Cell Cultures》under exclusive license to Springer Nature Switzerland AG 2021 R. Pörtner (ed.), Cell Culture Engineering and Technology, Cell Engineering 10, https://doi.org/10.1007/978-3-030-79871-01_2