pH Monitoring

pH stands for the "potential of hydrogen" and measures how acidic or basic a solution is. It is a non-dimensional number and is measured on a scale of 0 to 14, with 7 being neutral. Values lower than 7 are considered acidic; values greater than 7 are basic or alkaline.

pH is the the inverse decadic logarithm of the concentration of hydrogen ions in an aqueous solution. The more hydrogen ions present, the lower the pH; conversely, the fewer hydrogen ions, the higher the pH.

Changes in Cellular Morphology and Function

All cells have an optimal pH level that supports their growth. While this can vary some, most cells prefer to be around neutral. When pH levels fall outside of the acceptable range, cell culture growth can become inhibited or altered and the titer of their products reduced. pH levels that are too acidic or alkaline can actually destabilize the genetic material of cells, which can cause mutations and ultimately cell death if not corrected.

Availability of Nutrients

Culture media, also known as growth media, is any solution comprised of amino acids, vitamins, inorganic salts, glucose, and other substrates that support the health and growth of cells in culture. Many also contain serums as a source of growth factors, hormones, and attachment factors.

Depending on the cell type and preferred outcome, nutrient supplementation in cell culture can be used to increase cell viability and genomic stability. The surrounding pH can influence how cells take in these nutrients. Macronutrients such as nitrogen, potassium, calcium, magnesium, and sulfur are more readily available at pH 6.0–6.5, while micronutrients become less available at higher, alkaline levels of greater that 7.0.

Cellular Processes

In bioprocessing, living organisms are used to convert one or several substrate/s into a desired product. These products can influence the pH of your culture environment.

The problem of drifting pH is often very pronounced in microbial cultures, especially in aerobic processes with oxygen limitation, where under low oxygen the cells start to use alternative metabolic pathways, such as mixed acid fermentation, which heavily acidifies the media and leads to strong growth inhibition.

Also microbial growth on complex media can be challenging regarding pH, e.g. if the main carbon source is protein and amino acids, the pH will drift to the basic region due to the accumulation on ammonia.

All that is one of the reasons, why fed batch cultures usually outperform batch-cultures, as you can avoid these pH drifts by feeding the same amount of nutrients in a properly controlled way and thereby preventing accumulation of overflow metabolites.

As cells grow and cell density increases, the problem of acidification increases along with the production of these acidifying molecules.

Culture Environment

The survival of your culture is closely intertwined with environmental factors, e.g. temperature, pressure influencing the solubility and dissociation of pH-active gases (CO₂, ammonia).

Not only does temperature independently play an important role in cell growth as cells have a preferred temperature range, it can also influence the pH levels. For example, under standard conditions (25°C and 1 atm), the pH of pure water is 7. At 30°C, the pH drops to 6.92. This is because at higher temperature, water dissociates into ions more, leading to a higher concentration of hydrogen ions (or H₃O+). A higher concentration of H₃O+ ([H₃O+]) results in lower pH. According to the Bronsted-Lowry acid-base theory, the sample is still in equilibrium and has not changed acidity. We must think about the temperature we are keeping our culture at but also possible temperature changes during handling.

Another consideration is shifts in gas concentration during sample handling. Atmospheric CO₂ can alter the system during handling. Usually the atmospheric CO₂ is lower than in the incubator. During handling, CO₂ leaves the media when exposed to the external air, thereby reducing the buffering capacity and making the pH drift.

Buffering Media

In this traditional method of pH control, a buffer is added to the media to maintain a stable value. A pH buffer acts as either a weak acid or a weak base to ensure that the media will be resistant to changes in pH. This works because the buffer is capable of donating or accepting hydrogen ions which are responsible for establishing pH. The most common base used in mammalian cell culture is sodium bicarbonate (NaHCO₃).

Adding Acid/Base

When pH drifts outside the optimum range, an acid or base can be added to increase or decrease the pH as needed to bring it back to the target value. In a closed-loop setup, the amount added via the integrated injection systems (pumps, LIS, CO₂) is based on feedback from automatic pH sensors. Solution is added to the process and the pH value is closely monitored.

pH Flow Cells For Flow Loops

• Continuously monitor pH in flow loops using fiber optic sensors.

• pH for a variety of applications: 5-7, 6-8, 7-9

• Flexible flow rate range: 5-500 mL/min

• Factory-calibrated and pre-sterilized

• Single-use design reduces risk of contamination

Compatible with:

• Perfusion bioreactors, custom benchtop bioreactors, and small-scale fermenters

• On-line flow loops: Harvest lines, sampling lines, media in/out flow lines, waste removal lines