September 1, 2010 (Vol. 30, No. 15)

Monitoring and Control of Various Parameters Critical to Safe and Cost-Effective Operations

Compared to chemically produced substances, biotech products are special in two aspects: the molecules are large, and the products are manufactured using living organisms. The main goal of biomanufacturing is to supply the products safely and cost efficiently.

Process analytics during fermentation serves to maintain consistent living conditions for the suspended cells or production microorganism. This includes monitoring and control of the physicochemical environment such as pH, dissolved oxygen, and dissolved carbon dioxide.

All in-line sensors in bioprocess engineering must be sterilizable. The sensors must be stable and reliable over days or even weeks, and they should have low-maintenance requirements in order to be attractive for industrial use.

pH is mostly identified as a key process parameter in fermentation processes because enzymatic activities and cellular  metabolism are sensitive to pH changes. In-line pH measurement has been quite common for many years but is not without its risks. Should the electrode fail during a batch, the pH must be measured with off-line samples, which carries a risk of contamination and is labor intensive.

Mettler Toledo has developed a pH measurement system that not only eliminates the risk of electrode failure during production but also meets all stringent pharmaceutical requirements. The newest generation of Mettler Toledo pH electrodes features Intelligent Sensor Management (ISM) technology. A microchip within the head of the electrode continuously monitors the status of the sensor. 

Can a sensor be used for the next batch?  This is a crucial question for ensuring safe operation. Mettler Toledo’s M700 transmitter displays a sensor network diagram that shows the “health” of the connected sensors. If all corner points on the diagram lie to the extremes of the network the electrode is qualified for next batch operation. Corner points lying closer to the center indicate that maintenance is required. The sensor network diagram allows the status of an electrode to be ascertained without toggling through menus.

The iSense Asset Suite displays how many times an electrode has been sterilized and cleaned.

iSense Asset Suite

The number of sterilization cycles and cleaning cycles that the electrode has been exposed to are automatically counted for easy traceability of all sensors.

The PC-based iSense Asset Suite displays how many times an electrode has been sterilized and cleaned (Figure). In this example the electrode was sterilized 75 times, leading to increased glass membrane impedance. Electrode reactivation or replacement is recommended for safe measurement in future batches.

The microchip includes an analog-to-digital converter, as low impedance, digital transmission of the sensor signal is reliable, even in humid environments. ISM electrodes store calibration data inside the microchip and can be precalibrated outside the clean zone.

iSense Asset Suite stores data on all electrodes and sensors (pH, dissolved oxygen, and conductivity) allowing full traceability. The new ISM pH electrodes can also measure the redox potential simultaneously.

The redox potential is commonly used to maintain anaerobic conditions in a fermentation broth. It can be used to measure trace amounts of dissolved oxygen. Glucose-containing feed medium can be treated as a reducing source in oxidation-reduction of the culture medium. When the oxidation capacity is increased, the redox potential level will elevate to a higher value. Likewise, its value will become lower when the culture broth has a higher reducing capacity.

Beyond temperature, agitation, and pH, dissolved oxygen is another key process parameter. Neglecting control of these parameters could potentially impact final product quality, so in-line measurement can be employed to maintain the culture in its optimal state.

Cell cultures require oxygen for the production of energy from organic carbon sources—e.g., glucose. Given oxygen’s poor solubility in water, the control of oxygen flow is carefully regulated to ensure it does not become a rate-limiting factor in the process.

In contrast, a hyperoxygenated bioreactor air supply can irreversibly impact culture performance. The pO2 value is usually monitored with an amperometric, membrane-covered electrode (Clark type). Given the nature of amperometric DO probes, it is necessary for them to be polarized prior to their use.


Mettler Toledo’s optical DO sensors with built-in ISM provide ease-of-use, process safety, documentation, and maintenance efficiency. The heart of the optical sensor is an oxygen-sensitive layer containing immobilized marker molecules. It absorbs light from a light-emitting diode and is able to release this energy as light of a different wavelength (fluorescence).

The time delay between light absorption and emittance is dependent on the concentration of oxygen present in the medium. Instead of membrane body, inner body, and electrolyte found in amperometric sensors, only one component, the OptoCap, has to be replaced from time to time as a consumable. No prepolarization is needed—the optical probes work out of the box.

The optical-measurement principle offers significant advantages over amperometric technology such as much lower drift, which is important considering mammalian cell cultures ferment over several weeks.

Mettler Toledo optical DO probes also benefit from ISM features such as automatic counting of sterilization and cleaning cycles as well as predictive maintenance information about OptoCap replacement. The optical DO sensor is also fully integrated in the iSense Asset Suite platform.

Dissolved carbon dioxide level can be indicative of the quality of cellular metabolism and is thus routinely monitored as an indicator of culture performance. High pCO2 levels have been reported in the literature as an inhibitor to growth and metabolism and can impact product quality characteristics such as gylcosylation of the protein product.

In fed-batch mode, the dosing of a glucose-containing nutrient can be controlled by a CO2 measuring system, maintaining a safe level of CO2. Mettler Toledo’s CO2 sensor works on a potentiometric principle. CO2 from the process diffuses through a membrane, and the pH change in the internal electrolyte correlates to the partial pressure of carbon dioxide.

Biomass concentration is often a key variable, primarily because it provides information on the growth rate and/or product formation. The optical density (OD) of a cell suspension is widely used. It requires sampling an appropriate dilution of the suspension with a suitable buffer and photometric determination of the turbidity caused by the suspended cells.

Mettler Toledo’s InPro 8000 series provides an alternative to traditional in- or off-line OD measurement limitations. The InPro 8000 series sensors utilize backscattered NIR light to depict true cell mass throughout the entire fermentation. This technology does not need a defined optical path length, and consequently  even measurement at high concentrations is possible without dilution. The backscattered light technique is an ideal method for real-time and continuous measurement in the bioreactor.

Buffer Preparation

Another application for pH and conductivity control is the preparation of nutrient media for inoculum and production fermenters. A similar mixing process is the buffer preparation for downstream unit operations like chromatography, ultrafiltration, and diafiltration. Buffer make-up variability is a significant contributor to variation in almost all production-scale pharmaceutical processes.

In chromatography, buffers are the mobile phase. Buffer accuracy and reproducibility are key leverage variables. pH and conductivity sensors with ISM technology will increase the resolution of chromatography systems, avoiding band broadening and shifting of elution profiles. For chromatography columns, pH and conductivity probes are installed at the inlet and outlet to monitor the performance of the gradient, loading of the column, and regeneration and re-equilibration.

The new ISM series line of pH, dissolved oxygen (optical and amperometric), and conductivity measurement points allows users of process analytical equipment to significantly reduce costs, while increasing the safety of the production process.

Sensors equipped with ISM are continuously monitored for defects and wear. In addition, whereas conventional sensors need careful and time-consuming calibration in the clean zone, sensor replacement with ISM sensors precalibrated with the iSense Asset Suite takes place rapidly.

ISM-enabled sensors are digital, with integrated electronics for signal conditioning in the sensor head. The digital and low impedance signal transmission ensures trouble-free, humidity-insensitive communication with the transmitter.

The iSense Asset Suite is the ideal lab complement for the ISM loop. iSense allows the user to maximize the performance of ISM sensors over their entire lifetime. Users just need to connect the sensor to a PC and get access to various intuitive analysis, calibration, and documentation applications. ISM and iSense provide an easy way to achieve good traceability on all sensors in a production line.

Kurt Hiltbrunner ([email protected]) is market specialist, pharmaceutical industry, at Mettler Toledo.

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