Catherine Shaffer holds a master's degree in biological chemistry and has worked as a research scientist. She is also an award-winning science fiction author and part-time reporter for local public radio.
One of the most critical and frequently monitored parameters measured during bioprocessing workflows is pH. Proteins and other large molecules are sensitive to pH and can lose stability outside optimal pH conditions. When streamlining and scaling up for protein bioprocessing, accurate pH measurements become even more important.
A pH meter determines the acidity or alkalinity of a solution, or its concentration of hydrogen ions, by measuring the difference in electrical potential between two electrodes — the pH electrode, which is sensitive to hydrogen ions, and a reference electrode — which carries a known electrode potential. The typical electrode contains a solution with potassium chloride as the electrolyte. Ideally, pH is calculated from the difference between electrode potentials. In the real world, many factors can interfere with an accurate measurement. Those include the age and condition of the electrode and whether it is properly calibrated.
For a bioprocessing workflow, pH typically is controlled in a very narrow range. There are three main approaches to monitoring pH: off-line monitoring, online monitoring and in situ measurements. Off-line monitoring is performed on samples removed from the process, and in situ monitoring is integrated within the process. Online monitoring is a popular compromise between the two, in which samples are diverted automatically from the flow and rapidly analyzed. In this article, we will touch on the different approaches and applications of pH monitoring in a bioprocessing workflow.
Electrode options
Off-line measurements are a very direct approach to measuring bioprocessing parameters. An off-line measurement is taken from a sample removed from the process. This is generally a very reliable and accurate method for monitoring, but it can be slow and labor-intensive. In situ, or in-line, monitoring is carried out using sensors within the vessel or flow lines. These sensors offer a rapid, real-time measurement, but they can fall short in measurement sensitivity vis-à-vis pH range and durability.
Online monitoring fits somewhere between off-line and in situ monitoring. Typically, online monitoring refers to a process in which a sample is automatically withdrawn and analyzed. This method offers a good compromise between the rapidity of an in situ measurement and the reliability of an off-line measurement.
Off-line pH measurements can be handled with a benchtop pH meter or an automated titration system. For an accurate pH reading, an off-line measurement should be performed rapidly to avoid drifting caused by CO2 off-gassing.
Sample effects on the electrode
Qualities of the sample may also complicate the task of obtaining an accurate pH measurement.
Bioprocessing focuses on large-batch fermentation and purification of proteins. Types of samples that need to be tested include: tissue culture media, bacteria and yeast suspensions, cell lysates, purified proteins and protein and nucleic acid solutions. The functioning of a pH electrode relies on the conductivity of the sample. If a sample lacks conductive ions, it will be difficult to get any accurate pH reading. For example, very pure water is a difficult medium for pH measurement.
At the other extreme are samples containing many large organic molecules. Most bioprocess samples have a lot of these large molecules, including the protein of interest as well as nucleic acids and cellular debris. These solutions can cause clogging of an electrode’s diaphragm, or reference junction, which is the part of the electrode where the system and the solution make electrical contact. A reference junction must be just permeable enough to allow the reference electrolyte to flow out. Reference junctions are available in a range of materials for different applications. A ceramic junction is popular for general applications, but its pores are easily blocked by organics. A platinum junction is not easily clogged, but it has a lower diffusion potential than ceramic. A ground-glass joint is often used with suspensions and other difficult solutions, as it is easy to clean and has a high outflow rate.
“For an accurate and very reproducible measurement, the diaphragm must be really clean. If the diaphragm is blocked or clogged because of large organic molecules, the electrolyte cannot flow through, and your pH reading will be oscillating,” says Hari Narayanan, marketing manager for Metrohm USA. Narayanan recommends Metrohm’s Unitrode electrode for difficult solutions and the Viscotrode for viscous solutions.
Because of the complications of a liquid electrode, in which electrolyte comes in contact with the sample, some bioprocessing researchers prefer a gel electrolyte, which is comprised of potassium chloride and polymer gelling agents to prevent electrolyte from leaking into the sample at the reference junction. The gel makes surface contact with the sample during measurement but does not flow into or mix with the sample.
YSI's TruLine pH 21 and pH 27 electrodes use a polymer electrolyte. According to YSI’s website, these electrodes are low-maintenance, because they don't need to be refilled with electrolyte solution.
In addition to improved electrode technologies, many vendors are offering titration systems with features beyond a simple pH reading. Metrohm's Omnis titrator is a complete system that can perform pH analysis on four samples at once. It includes liquid-handling functionality and software that automates analysis.
Beyond the electrode: optical sensors
Optical sensors are a newer technology utilized in some in situ and online monitoring systems.
Optical sensors use pH-sensitive dyes. A change in pH triggers a change in absorbance or fluorescence that can be detected spectrophotometrically. PreSens Precision Sensing GmbH offers a noninvasive pH sensor for physiological solutions and culture media. SensorSpots are noninvasive optical oxygen sensors that can be adhered to the inner surface of a transparent vessel. According to PreSens’ website, these sensors can be used with culture flasks, tubes, petri dishes and cultivation bags. Flow-Through Cell pH from PreSens utilizes a miniaturized fiber optical chemical sensor that is integrated in a flow-through cell for in-line pH monitoring.
Gernot Thomas John, director marketing and innovation for PreSens, says that customers are often curious about the stability and accuracy of the sensors. “PreSens pH sensor[s] show enhanced stability due to the use of the patented, dual lifetime referencing mechanism. Accuracy is highest with a one-point adjustment, a feature that is supported by most controllers,” says John.
Ocean Optics has developed a sol-gel (wet-chemical fabrication technique) device called an optrode for pH measurement through pH range 5 to 9. The optrode sensors use a pH-sensitive dye that is immobilized in a sol-gel matrix (a colloidal mixture formed by dispersing nanoparticles in a liquid). Hydrogen ions from the solution diffuse into the gel and trigger a color change. According to Ocean Optics’ website, sol-gel sensors have a faster response time than typical polymer gels.
Typically, in-line optical sensors are used in upstream processing, whereas downstream processing requires the precision of an electrode. But this can vary depending on the target product and how the workflow has been optimized.
Whether off-line, online or somewhere in between, pH measurement within bioprocessing workflows is absolutely critical. The structure and kinetics of enzyme molecules can be dramatically affected by small deviations in pH. Most enzyme kinetics are pH-dependent, and if the pH is allowed to deviate in processing, the enzyme activity will be poor or absent. Protein-based pharmaceutical products may be structurally unstable outside of the optimal pH range, and some purification processes may work poorly or fail if the pH is not maintained in a certain range. With a large-batch preparation, a mistake in pH adjustment could be extremely costly in terms of dollars as well as delays to strict production schedules. Finding the best system and best electrode or sensor depends on many process variables. Although pH meters are typically taken for granted in the laboratory, a poorly maintained or dirty electrode will give incorrect readings. So be sure to choose the best tool for your application, and follow use, maintenance and calibration instructions carefully.