Measuring consistency from laboratory to process

Ensuring measurement consistency when a process is scaled up from the laboratory to production can be made easier with digital sensor technology.

In the biotechnology industry, analytical sensors are commonly standardised in terms of brand and type during process development. This helps to maintain data consistency when the process is later scaled up. Despite this, companies can still run into considerable problems caused by unreliable sensor signals and disparities concerning the signal algorithm and sensor handling. Digital sensors offer a solution to guarantee data consistency and a way to easy, uniform sensor management.

The importance of measuring consistency from lab to process

It is vital in creating the right conditions in the bioreactor during trials and in the up-scaled process to allow microorganisms or cells to thrive. Correct environmental conditions will ensure that the yield is maximised in a stable and predictable manner.

Two of the most critical parameters during a fermentation process are pH and oxygen, and both need to be controlled carefully. pH and dissolved oxygen values out of specification lead directly to a loss of yield. For some specific cells, typically mammalian cells from humans and animals, the pH value is highly critical and needs to be controlled in a range better then ±0.1–0.2 pH units to obtain the expected yield. The oxygen concentration also gets critical for the batch if it is too low — for example, less than 20–25% — as there is not enough oxygen for respiration. On the other hand, too much oxygen risks the yield as some bacteria tend to grow in size at the expense of the production of the wanted molecules. It is also a waste of expensive sterile oxygen.

When a process is scaled up from the initial laboratory fermentation to pilot and to full-scaled production, it is imperative to keep all conditions unchanged, and it is preferred to keep the identical sensors down to brand and type. This is to ensure that no measuring discrepancies occur when the process is up-scaled that could risk a decrease of process yield.

Measuring behaviour and performance between different sensor brands can occur due to several reasons such as different compensation algorithms, different material performance or different sensor design. Despite standardisation of the sensors, however, discrepancies are still common. They usually can be related to the analytical sensors themselves or to the electrical signals from the sensors.

Below we will explain different reasons behind errors related to the electrical signals and show how those errors can be eliminated by using digital sensors.

Challenges concerning consistency of pH measurements

One of the largest challenges, especially for pH sensors, occurs with the bioreactors in the laboratory. During the autoclaving process, both the glass fermenter and the sensors are exposed to high temperatures in combination with steam. If humidity remains on the sensor contacts this will later result in unreliable and unstable measured values.

It is a well-known fact that the high impedance mV signal from a pH sensor is very sensitive to any humidity or oxides on the metallic cable contacts. Signal drops will result in unpredictable measurement errors and, depending on the environment, they can occur randomly. The biggest challenge is if they only appear occasionally, as this makes them hard to detect.

An ideal pH sensor has a zero point at pH 7.00. In other words, in a pH 7.00 solution, an ideal pH sensor provides a 0 mV signal. In a pH 8.00 solution the same pH sensor will provide a -59.16 mV signal (at 25°C). Under perfect conditions this signal is measured without interference and converted into the pH value by the transmitter. But when corrosion, humidity or oxides are present on the sensor and cable contacts, part of, or in the worst case all of, the -59.16 mV will disappear, and the signal gets closer to 0 mV (pH 7.00). In this case, the signal from the pH sensor would indicate a lower value than there is in reality and the controller in the fermenter would continue to add reagent to increase the pH. The result would be an overdosing of reagents which results in a pH value out of specification and likely a wasted batch.

Comparability of measurements in the laboratory and in the process

During all the development steps it is common to control and even adjust the online pH value in the fermentation by grab-sample analysis, where a relatively small sample is analysed with a laboratory pH sensor. This is the second challenge regarding consistency of the pH-measurement: it is common that measuring discrepancies occur also between measurements in the laboratory and in the process. There may be several reasons for this. But even high-end pH sensors tend to show discrepancies in measurement if the measurements are carried out with sensors of different brands or types.

Typical reasons for this can be:

• Diffusion potentials in the pH sensor due to different reference systems.
• Nonlinearity at high/low pH-values because of different membrane glass.
• Different temperature behaviour dependent on the isothermal point.
• Different compensation algorithms in the pH-transmitter.

Challenges concerning consistency of dissolved oxygen concentration measurements

There are two types of measuring technologies available for dissolved oxygen measurement: the traditional amperometric and optical florescence technology. Amperometric oxygen sensors provide a very small nA signal proportional to the oxygen concentration. Commonly a freshly maintained sensor provides 0 nA at 0 mg/L (%) and 60–70 nA at the saturation point (100%). This small nA current measuring signal requires a sophisticated controller to detect variation in the process.

In contrast the optical measuring principle is based on fluorescence quenching, where oxygen-sensitive molecules are integrated into an optically active fluorescence layer. By applying light energy at a specific wavelength, a response in the form of fluorescent light is received. The decay time and intensity of the response signals are inversely proportional to the oxygen content in the solution.

The optical sensor technology has several advantages compared to the traditional amperometric method:

• No fragile membrane and no electrolyte.
• No polarisation time required.
• Very easy maintenance and handling.
   

The challenge with optical and amperometric oxygen sensors is mainly the interference of air bubbles at the O₂-sensitive membrane when the sensor is top-down mounted. A dissolved oxygen sensor should measure the concentration of oxygen that is dissolved in the solution and that can be employed by the bacteria and the cells. It should not be sensitive to the oxygen of the air bubbles in the solution. The oxygen concentration in the bubbles is completely different to what is dissolved.

The top-down installation of the oxygen sensor in a laboratory fermenter raises the risk of bubbles tending to stick on the membrane. The influence can be minimised with electronic filters and damping of the sensor signal. However, this will slow down the sensor response.

In a pilot and larger fermenter, the oxygen sensors are installed from the side slightly angled from a horizontal line. In this position the influence of air bubbles is negligible. The next challenge arises when values from those two applications are compared. The best solution on the market so far is to use an oxygen sensor with a convex sensor tip. It minimises the risk that bubbles get stuck and also enables top-down installation.

Advantages of digital sensors

Digital sensors can solve the challenges of pH measurement. In a digital sensor the actual sensing part of the sensor is analog and identical to a conventional analog sensor. The difference is that digital sensors include an additional component in the form of a microprocessor that processes measuring signals. Generally, several signals need to be processed and considered in parallel.

The advantage of digital analytical sensors is that they provide 100% signal integrity, improving the reliability of the measurement value. Compared to a measurement loop with analog sensor technology, there is no risk of signal loss between the sensor and the displayed measurement value. Moreover, humidity and oxides on contact surfaces do not cause any issues for the measurement. Either you receive a correct measurement or no measurement at all. This is a great step forward for all fermenter applications in the laboratory, as any remaining humidity on the contact surfaces after the autoclaving process will no longer cause distorted or unstable values.

Measuring consistency means maintaining the same sensor brand and type, as well as keeping the calculation algorithms behind the measurement values unchanged when a process is scaled up from the laboratory to pilot and full process capacity. Standardisation of digital signal processing is much easier between different transmitters when using digital information as opposed to analog signals.

Sensor adjustment and sensor handling

A second great benefit of digital sensors is that sensor handling and sensor adjustment can be standardised between the laboratory and the process. The adjustment of an analog pH sensor can be challenging at the measuring point as buffer and cleaning solutions need to be brought from point to point and additional documentation needs to be done. Digital sensors carry their own adjustment data, which means that they can be cleaned, calibrated and adjusted offline in a stable environment and later installed in process or in laboratory applications.

Sensor adjustment in the laboratory provides several benefits. Beside the time-saving aspect, also the measuring reliability can be improved. The high concentration of protein molecules in the fermentation can easily contribute to clogging of the pH sensors reference diaphragm. This will eventually shorten the sensor lifetime and contribute to measuring errors if it is not cleaned properly.

For reliable measurements from batch to batch, the sensor needs to be carefully maintained with an acid in combination with a pepsin solution. This maintenance can be done more easily in the laboratory compared to the measuring point. The direct result of sensor maintenance in the laboratory is better performance, higher measuring accuracy and in many cases a prolonged lifetime.

By using digital sensor technology that in parallel provides the possibility to use a software for sensor maintenance and management, all handling can largely be standardised and simplified. Digital sensor technology also minimises the risk of discrepancies between the grab sample and the online measurement.

By using the identical digital sensor and signal technology in parallel with appropriate sensor handling, any risk for incorrect values is minimised.

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