pH Control in Bioreactors

Electrochemical glass pH sensors are traditionally used in bioprocessing. These are comprised of an electrode within a buffer solution, encased by a glass membrane. Glass pH sensors measure the potential of H⁺ ions across the glass membrane in millivolts (mV), which corresponds to changes in pH.

• Advantages: The glass membrane material can be adapted to various bioreactor conditions, including extremes in pH or temperature.
• Disadvantages: Glass pH sensors require careful management and can lose accuracy over time, with a need for frequent calibration.

Optical pH sensors use spectrophotometry to measure the pH of the solution. These sensors contain a fluorescent dye, whose fluorescence emission is sensitive to H⁺ ions, due to a shift in the emission or the absorption spectrum. Light emission after excitation is quantified using an optical detector and used to calculate the pH. Eppendorf offers BioBLU® Single-Use Bioreactors with optical pH sensors. The bioreactors are already equipped with the sensors. Therefore, unlike standard glass sensors, they do not need to be sterilized and inserted to the bioreactor by the end-user, which eliminates a possible source of contamination. Excitation and emission light are transmitted to the sensor spot through an optical fiber connected to a module for optical pH sensing, which is part of the bioprocess control station.

• Advantages:
• Optical pH sensors can be used in a non-invasive manner that reduces the contamination risk. This is particularly interesting for cell culture applications.
• Optical pH sensors are inexpensive and disposable.
• Disadvantages:
• The measurement range of optical pH sensors is narrower than the range of electrochemical pH sensors. Eppendorf recommends a measurement range of pH 6.5 to 7.5. This is usually sufficient for cell culture applications but may be too narrow for certain microbial applications.
• Certain chemicals including organic solvents can damage optical pH sensors. Additionally, these sensors can measure a relatively limited pH range.

Digital technology can enhance the reliability and accuracy of pH sensors. A digital sensor memorizes values, which are important to understand the state of the sensor. That includes glass and reference impedance, number of sterilize-in-place cycles (determined by temperature), speed of response, E⁰ pH value, and slope. Digital sensors demonstrate diagnostic capabilities based on these information, meaning that they are capable of monitoring sensor health and predicting the remaining life span of the sensor. This reduces the risk of sensor failure to subsequently minimize the risk of batch loss.

In cell cultures, the most commonly used buffer is sodium bicarbonate. Sodium bicarbonate acts as a buffer system to maintain the pH, reacting with the carbonic acid formed by CO₂ to form an equilibrium. Other synthetic buffers such as HEPES can be used. However, as it is used in the blood, the sodium bicarbonate buffer system is the most physiological one. For cell culture processes, CO₂ gas is used as the acid component. As base, 4 – 10 % bicarbonate buffer is used. Other types of liquid base or even liquid acid can be considered, but as animal cell lines are generally more susceptible compared to microbial cells, care should be taken whenever changing the chemicals in use. As microbial cultures are typically hardier than cell cultures, the addition of basic solutions can be used to increase the pH, including sodium hydroxide, potassium hydroxide and ammonium hydroxide. Alternatively, if the pH needs to be decreased, phosphoric acid or sulfuric acid can be used. In every case, do not use HCl as it is harmful for stainless steel components used in bioreactors. We recommended to check the chemical resistance of the pump tubing used with the supplier. For example, using high concentrations of acid or base with silicone tubing may lead to leaking pump tubing and possible damage to the pump head or controller. Other types of tubing such as Marprene^® are more suitable for higher concentrations of acid and base.

A pH deadband refers to the range in which the pH can deviate without the system responding. This range depends on many factors but is typically less than ± 0.2 from the optimal pH for mammalian and microbial cell cultures. The value of the pH deadband corresponds to the pH range that can be tolerated by cultures while maintaining maximum productivity. Therefore, a pH deadband helps avoid the unnecessary addition of buffers. It minimizes pump oscillation, so the acid and base pump sequentially pumping to maintain the pH setpoint. As a result, using a pH deadband results in smoother operation and pH control in bioreactors.

One-sided pH control uses either base or acid to control the pH setpoint. Two-sided pH control uses both, to counter either too low or too high pH values. One-sided pH control most commonly involves the addition of a basic solution into the culture. It can be sufficient, if the cells are not much affected by a pH value slightly away from the setpoint. In microbial cultures the pH value more likely drops than rises, which is why a one-sided pH control using base is often sufficient.A two-sided pH control loop is common for bicarbonate buffered systems, where the addition of the sodium bicarbonate base is coupled with CO₂ sparging. The dissolved CO₂ turns into carbonic acid and lowers the pH. The sodium bicarbonate is added as a concentrated base solution that quickly mixed in with the medium, to increase the pH.

The sensors are usually successively placed in two or more solutions of known pH, which are typically acidic, neutral and basic solutions. The electrode signal measured in each solution is converted to pH, generating a ‘calibration slope’ which is then programmed into the sensor or the controller. When setting up a bioreactor, sterilization is critical to prevent culture contamination in bioreactors. When using a reusable bioreactor, pH sensors are typically fitted into a bioreactor containing medium or buffer before autoclaving to sterilize the inside of the bioreactor including the pH sensor. When using a single-use bioreactor, the sensor is typically autoclaved separately. In both cases, pH sensors therefore typically require initial calibration prior to sterilization. A second adjustment after sterilization is then necessary to account for any temperature induced changes to the sensor. This is usually achieved by taking offline pH measurements of the culture using another pH meter and adapting the zero factor in the bioprocess control software. Read our protocols for more detailed information on how to calibrate the pH sensor when setting up a cell culture bioprocess or microbial bioprocess .

Adjustment based on offline pH values can be error prone due to changes in pH that can naturally occur during the time between taking the offline sample and pH measurement, as well due to sensor age and other factors that can influence offline pH readings. One technique for in-line pH sensor adjustment has been developed for carbonate-buffered media, measuring the CO₂ concentration within the bioreactor exhaust to infer the pH of the media and recalibrate the pH sensor. To read more about this and learn more about pH sensor best practice, download our ebook .

When using a BioBLU® Single-Use Bioreactor with optical pH sensor its calibration is quite easy. On the bioreactor as well its packaging you find calibration information, which you need to enter in the bioprocess control software.

Frequent pH sensor calibration is critical for reliable and accurate pH control in bioreactors. We recommend pH sensor calibration before each bioprocess run. It can be necessary to adjust the pH sensor during a bioprocess run, because of pH drift. pH drift is a common issue associated with pH control in bioprocessing that means the pH of the culture drifts from the measured pH, reducing product quality and yield. One reason when using glass pH sensors is a difference in ionic strength between the culture and the pH sensor’s internal buffer. The issue of pH drift becomes more important during longer experiments. Within this type of experiment, it is therefore recommended to adjust the pH sensor, using another pH meter, at least once a day.

Temperature plays an important role with regards to sample and buffer pH as well as the electrode's characteristics. The temperature dependency of buffers is known and is shown on the packing. Even if the pH control compensates the influence of temperature on the sensor sensitivity, no compensation can be made for the pH shifts caused by altered reference potentials. If possible, samples, buffers, and sensors should all have the same temperature.

Gel-filled pH sensors might have reached the end of their lifetime after they have been used for various runs and have been autoclaved regularly. If dirt or protein residue is present on the glass surface, you can remove these with either water and a soft cloth or with a pepsin/trypsin cleaning solution. Nevertheless we highly recommend to purchase a replacement sensor. Digital sensors provide important status information helping to decide when a sensor needs to be replaced.