The Art of Precise Regulation of Carbon Dioxide in Single-use Bioreactors: From pH Stabilization to Cell Metabolism Optimization

In the clean workshop of biopharmaceuticals, rows of columnar Single-Use Bioreactors (SUBs) are running silently. Different from conventional stainless steel reactors, inside these "cell factories" composed of multi-layer polymer films, carbon dioxide (CO₂) is participating in the life activities of cells in an unprecedented way-it is not only an environmental regulator, but also a metabolic designer throughout cell growth.

Technological Revolution

A New Paradigm for CO₂ Control in Single-use Bioreactors

The core breakthrough of Single-use Bioreactors is to push gas transfer efficiency and process flexibility to new heights:

• Gas Permeability of Films: The bioreactor bag uses a three-layer Co-extrusion films (such as polyethylene-vinyl alcohol-polypropylene), and its CO₂ permeability (2.5-4.0 × 10-10 mol/(m·s·Pa)) is significantly higher than stainless steel, but the pressure difference inside and outside the bag needs to be accurately controlled to prevent gas exchange imbalance.

• Dynamic Mixing System: The rocking bioreactor generates surface aeration through rocking, and the airlift reactor uses bottom sparger to achieve circulation. Both achieve dissolution control through the CO₂ partial pressure gradient at the gas-liquid interface.

• In-situ Sensor Technology: Single-use optical pH sensors (such as PreSens) monitor dissolved CO₂ concentration in real time through fluorescent dyes with an accuracy of ± 0. 05 pH units, overcoming traditional sampling delays.

  
  

Table 1. Comparison of CO₂ control between SUBs and conventioanl bioreactors

The Triple Core Mechanism of CO₂ Regulation

Precise Guardians of pH Homeostasis

In a 500 L scale SUB, the buffer system composed of CO₂ and NaHCO₃ faces new challenges:

• High Cell Density Dilemma: When the CHO cell density exceeds 2 × 10⁷ cells/mL, the rate of CO₂ production by metabolism can reach 0.15 mmol/(L·h), which is 5 times the basical level.

• Intelligent Feedback Control: Dynamically adjust the intake air ratio through the dissolved CO₂ probe (such as reducing the CO₂/N₂ mixture from 5% to 3%) to maintain a narrow pH fluctuation of 7.0 ± 0.1.

• Carbonic Acid Crisis Prevention: In the production of mRNA vaccines, pH < 6.8 was found to trigger the aggregation of lipid nanoparticles, reducing the failure rate from 12% to 0.7% by reducing the CO₂ partial pressure.
  

The Invisible Conductor of Cell Metabolism

CO₂ concentration directly affects product expression by regulating energy metabolism pathways:

• TCA Cycle Rebalancing: In HEK293 cells, increasing dissolved CO₂ from 40 mmHg to 80 mmHg can increase citrate synthase activity by 3 times and promote antibody glycosylation.

• Reversal of Lactate Metabolism: When CAR-T cells are cultured, controlling the CO₂ partial pressure in the range of 30-50 mmHg increases the lactate consumption rate by 60% and the maximum viable cell density by 45%.

• Hypoxic Stress Relief: In stem cell microcarrier culture, 10% CO₂ environment reduces HIF-1α expression by 70%, maintaining multi-directional differentiation potential.
  

Molecular Engraver of product quality

CO₂ shapes critical quality attributes (CQAs) of biologics by affecting the cell microenvironment:

Figure 1: Effect of dissolved CO₂ concentration on critical quality attributes of biologics

Cutting-edge Applications: From Gene Therapy to Cultured Meat Manufacturing

Scale Production of Viral Vectors

In the production of adeno-associated virus (AAV), CO₂ regulation has become the key to breaking through production capacity bottlenecks:

• Transfection Stage: 5% CO₂ environment maintains high transfection efficiency of HEK293 cells (> 85%).

• Packaging Stage: A brief increase to 8% CO₂ enhances viral capsid assembly integrity.

• Harvest Stage: Reducing to 4% CO₂ lowers the empty shell rate, and a gene therapy company used this to increase the functional virus titer by 3.2 times.
  

Industrialization of Cultured Meat

In the large-scale culture of muscle stem cells, CO₂ assumes a special mission:

• Myofiber Differentiation: A 10% CO₂ environment increases myosin heavy chain (MyHC) expression by 220%.

• Adipogenesis Regulation: Through alternating 5%/15% CO₂ cycle stimulation, adipocytes are induced successfully to form, solving the problem of cultured meat taste.

• Utilization of Metabolic Waste: Innovatively convert the CO₂ exhaled by cells into bicarbonate buffer to achieve a closed-loop cycle of the culture system.
  

Efficient Exosome Capture

Develop new separation technologies using CO₂ concentration gradients:

• Establish an axial CO₂ gradient in the perfusion SUB (top 50 mmHg → bottom 150 mmHg).

• Exosomes are enriched in the isoelectric point (pH 4.5-5.5) region, and the harvest rate can reach 82%.

• Compared with ultracentrifugation, the purity is 4 times higher, and the film integrity is maintained.
  

Technical Challenges and Intelligent Solutions

Pain Point Breakthrough: Uneven Distribution of CO₂

Intelligent Solutions & Application Cases

In a 2000 L single-use bioreactor, the CO₂ concentration gradient can reach 35%:

1. Computational Fluid Dynamics (CFD) Simulation: Optimize the gas distributor design so that the CO₂ concentration coefficient of variation is < 8%.

2. Pulsed Intake Strategy: 10 seconds of high-flow ventilation every 15 minutes to eliminate local accumulation.

3. Magnetic Levitation Mixing System: Mechanical seal-free design achieves gentle mixing with reduced shear force to < 1 Pa.

Conclusion

CO₂ is an important reaction substrate and metabolite in the central metabolic pathway of mammalian cells. Its concentration affects cell growth, metabolism and even protein production and quality through the effects of pH, HCO₃^-, and osmotic pressure alone and coupled with it.

Changes in CO₂ during cell culture are generally intuitively reflected in changes in pH. Good pH monitoring can eliminate the impact of pCO₂ on cell culture to a certain extent. Real-time monitoring of pH changes helps to better deal with changes in pCO₂.

BioLink’s CytoLinX^® BR Single-use Bioreactor is equipped with sensitive pH control and rapid response. pH changes can be cascaded with CO₂, alkali pump, and acid pump to achieve online monitoring of pH value.

CytoLinX^® BR Single-use Bioreactor

Figure 2: BioLink’s CytoLinX^® BR 10-2000L Single-use Bioreactors

Product Features

• The tank has a good shape and structure and complete functions. A single controller supports the control of multiple vessels by plugging and unplugging, which contributes to significant cost reduces (reaction tank volumes of different sizes can be provided according to process requirements to support process scale-up).

• 50-2000 L Reliable PCS 7 system that meets ISA 88 standard, applicable for whole-plant control.

• The software is designed with user-friendly interface in compliance with 21 CFR part 11.

• Flexible configurations, partly customizable upon customer requirements.

• The main parts are all high-end brands and passed the inspection before leaving the factory to ensure normal operation.

• Single-use bioreactor bags with micro, medium, and macro sparger size available to meet various processing needs.