Keeping pH at healthy biological levels is potentially the most powerful tool in upstream bioprocessing to increase product titers. Bioreactions are expensive and run on long timelines, making it critical to maximize each run.

Mammalian cells are only viable in environments with pH between 7.1 and 7.4. But the cells naturally produce carbon dioxide as a byproduct of respiration, driving pH down. Cells naturally have mechanisms to remove carbon dioxide and prevent toxic buildups, and buffering systems to keep their pH at healthy levels. Bioreactors need to mimic these features to ensure the cells can thrive.

What influences culture pH?

At the beginning of a bioreactor run, the buffer in the culture medium keeps the pH at the desired levels. Carbon dioxide may even be sparged in to keep the pH low. But during the exponential growth phase of the culture, the cells generate large amounts of carbon dioxide as a metabolic waste product. The buffer breaks, and the pH rapidly falls.

At that point, the pH of the culture needs to be raised. Historically, base was pumped into the culture medium. Now, newer bioreactor setups use macro spargers to introduce large bubbles of air or nitrogen. These bubbles don’t dissolve easily, so they strip the carbon dioxide from the culture, bringing it to the headspace of the bioreactor where it is removed.

Recent literature has looked closely at the most effective means of controlling pH in a bioreactor. Here, we rank four approaches to pH control, from least to most effective.

Least effective: Keeping pCO₂ constant

The partial pressure of dissolved carbon dioxide (pCO₂) in a culture is a critical process parameter because of its effect on intra- and extracellular pH. pCO₂ determines the concentration gradient through which intracellular carbon dioxide (generated as a byproduct of cell growth) leaves the cells and enters the culture medium. If pCO₂ is too high, CO₂ can’t leave the cells, so the intracellular pH will drop and the cells will die. If pCO₂ is too low, the cells lose will CO₂ too quickly, their pH will rise, and the intracellular environment will become too basic for the cells to be viable.

pCO₂ effects pH, but they aren’t tightly correlated

Particularly within the buffered pH range, pCO₂ and pH aren’t directly correlated. Therefore, pCO₂ gives much less information about the health of the culture than the pH measurement.

The main reason for this is that with small changes to pH within the realm of cell viability (pH 7.1-7.4), bicarbonate buffer in the (extracellular) medium will effect pCO₂ according to Le Chatelier’s principle.

Also, mammalian cells generate lactate, in addition to carbon dioxide, as a byproduct of respiration. Lactate and CO₂ both make the culture more acidic, but lactate’s effect on pH isn’t reflected in the pCO₂ measurement.

After the buffer breaks and the pH starts to drop significantly, pCO₂ and pH are much more closely related. But it’s still not a strong means of monitoring and controlling pH – which is the variable that actually matters to increasing cell titers.

Less effective: Pumping base into the culture

Cells produce large amounts of carbon dioxide during the exponential growth phase, resulting in significant pH drops. A simple solution is to pump base into the bioreactor using direct feedback from pH sensors.

This method is most effective in:

• Smaller bioreactors, where the effects of uneven mixing and pH localization may be negligible.
• Environments with low pCO₂ and low pH, where cells generate CO₂ which dissolves rapidly, further lowers the pH, and cannot be removed with sparging gases.
• Fermenters fermenters culturing microbial cells, which are hardier than mammalian cells. They can both handle increased agitation and mixing, and are less sensitive to pH fluctuations.

Pumping base doesn’t guarantee a constant pH throughout the bioreactor

For a high product titer, the pH needs to be constant in the entire bioreactor, which requires thorough mixing. Otherwise, there will be localized pockets of particularly high or low pH. But mixing leads to foam and increases shear stress, both of which hurt cell viability.

These problems compound as bioreactor size increases, meaning that pumping base simply doesn’t scale up well. Mixing time and agitation both need to increase with bioreactor size to avoid worsening localization, but this comes at the expense of increased foam and shear stress.

Pumping in base also increases the osmolality (solute per kilogram of culture medium) of the culture. In larger bioreactors, this can decrease cell viability and product titer.

More effective: Combining pumping base and sparging gases

The traditional method of controlling pH involves pumping base into a bioreactor during the early stages of a cell culture and then using sparging gases during the exponential growth phase. Base is particularly useful immediately after the buffer breaks, and sparging gases give tight control, for maximum yields from a culture.

Pumping base has significant downsides, and sparging gases have significant advantages

The most up-to-date setups primarily use sparging gases to control pH. Recent literature has moved away from other methods to focus on optimizing the control loop for sparging gases using feedback from pH and other critical process parameters – including pCO₂.

Most effective: Using only sparging gases

Sparging gases can control pH more tightly than the other methods discussed, primarily because they can react fastest to changes in other critical process parameters. The mix of sparging gases also gives the most precise control and the ability to respond to the largest amount of information from the various sensors in the bioreactor.

Specifically, several factors make this method most effective:

• Sparge rates are limited only by the maximum flow rates of the mass flow controllers that feed the gases to the spargers. The specific micro- and macro- spargers can be engineered and scaled based on desired flow rates and known mass transfer principles.
• Gas bubbles from spargers can be evenly mixed and distributed more quickly than base, and with much less agitation.
• Sparging gases scales very well, because the sparge rate and bubble size often don’t need to change as the bioreactor scales. A 2018 study by Hoshan et al. kept sparge rate constant and simply increased headspace aeration to compensate for increased bioreactor size. They saw titers increased by similar amounts in both 30 L and 250 L bioreactors by simply setting up more optimized control loops between pH and sparged air.

Intelligent sparger engineering and control loop design are critical

If gases flow in too quickly, they introduce shear stress. If gases flow in too slowly, the cells won’t reproduce as quickly as they would otherwise be able, and will not achieve maximum yield.

Well-designed spargers, in contrast, will scale well and produce consistently high titers.

Learn more: Comparing stirred tank, shaker, and rocker bioreactors