pH measurement in bioprocessing
Katja Herter, courtesy of Mettler Toledo | March 29, 2024Bioprocessing is a part of biotechnology that deals with producing and harvesting biological material. Normally, the final product is produced by a living microorganism, such as cells, bacteria, viruses or enzymes. The process can be divided into upstream- and downstream-processing.
Upstream processes (USP) are defined as the entire process from early cell isolation and cultivation over culture expansion of the cells until the final harvest, where the live cell batch gets collected. The upstream process is done in an extremely controlled environment called a bioreactor. Within a bioreactor, the growing conditions can be precisely monitored. The U.S. Food and Drug Administration (FDA) and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) define USP, so-called critical process parameters (CPP), to ensure process quality. A CPP is defined as a process parameter whose variability has an important impact on a critical quality attribute and, therefore, should be monitored steadily. They include pH, dissolved oxygen and carbon dioxide, the oxygen uptake rate, nutrients, metabolites, temperature, pressure and many more.
In order to achieve the perfect outcome, the parameters have to be constantly within range. When the organisms reach the desired density, they are harvested and moved to the downstream section of bioprocessing.
Downstreaming (DSP) refers to the part where the cell mass from USP is processed to meet purity and quality requirements. Downstream is usually divided into these six steps:
1. Separation of biomass with centrifugation. If the final product is intracellular, the biomass will be recovered from processing. On the other hand, if the final product is extracellular, the biomass will be discarded.
2. Cell disruption for intracellular products or second centrifugation of the solid liquid for extracellular products.
3. Concentration of the broth (only for extracellular products).
4. Initial purification of metabolites. Depending on the physio-chemical nature of the product molecule, several methods for recovery are used, mostly different forms of chromatography.
5. De-watering to concentrate the product, which is usually done by vacuum drying or reverse osmosis.
6. Polishing of metabolites as the final step to make the product 98% to 100% pure. The product undergoes formulation depending on its purposes, is packed, and sent out to the market.
Upstream as well as downstream process activities are performed at laboratory, pilot and manufacturing scales and involve process analytical technology (PAT) teams that focus on process optimization, scale-up, and troubleshooting.
pH is a critical parameter in the development and scale-up of cell cultures. Source: Mettler Toledo
Different types of measurement
There are three types of measurements that are dependent on their location.
In-line measurements
Sensors are directly placed in the bioprocessing system. For example, in the bioreactor, several different sensors measuring the critical parameters are directly installed within the system. They build the first wall of quality control.
At-line measurements
With portable instruments, quality control can be done at the bioreactor without leaving the production site.
Off-line measurements
Samples are taken from the production site and brought to the lab. The measurement takes place in a different location; therefore, it is called offline measurement.
pH as a critical process parameter
pH is a critical parameter in the development and scale-up of cell cultures as well as in most downstream applications. Even the smallest bioreactors are equipped with integrated pH sensors. But it does not stop there. In order to achieve the maximal outcome, all buffer solutions used during the process need to be adjusted in pH prior to the actual starting process as well.
Regardless of the size of the production site or laboratory, pH is always present. To ensure the quality of the product, hence the quality of the pH measurement, consider the following points:
Compare measurements at the same temperature
Although modern pH probes come with built-in temperature sensors and automatically correct the slope for temperature changes, it is recommended to compare measurements at the same temperature. The reason for this is that chemical equilibria are temperature-dependent, resulting in different outcomes at different temperatures. Compensating this error is only possible when media characteristics are known, which are typically only available for pH buffer solutions. Therefore, comparing the two temperatures is essential for good quality.
Select the right sensor
Culture and process media have heterogeneous and complex chemical compositions interacting with the sensor reference system. Those interactions are sources of error for pH measurements and can be minimized by selecting the right sensor type.
Recalibrate after sterilization
In-line sensors are commonly sterilized inside the bioreactor. The high temperature affects the pH sensor membrane glass performance. The so-called “process calibration” helps to ensure the correct performance of the sensor. pH is measured in-line and offline, and a sample is taken to the lab. The in-line measurement will be stored in the installed sensor, whereas the off-line measurement will be entered manually into the system to ensure the quality of the in-line measurement. Alternatively, at-line measurements can be done to avoid environmental changes in the sample.
In general, standardized sampling procedures and measurement methods are recommended. With so many variables influencing pH measurement, standardized procedures are the best way to reduce result variability between different operators. Constantly looking out for possible sources of error helps to ensure quality and improve your process outcome.
Find the right sensor for bioprocessing activities here and request a personalized recommendation on Mettler Toledo's Good Electrochemistry Practice Recommendation page to find the most suitable pH system for specific needs.
To contact the author of this article, email pHmatters@mt.com
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