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The importance of [CO.sub.2] control during the manufacture of biopharmaceuticals: Kurt Hiltbrunner, market segment specialist at Mettler-Toledo process analytics, talks to pharma about [CO.sub.2] monitoring and control.

pH and dissolved oxygen control are important during fermentation and cell culturing. Why has in situ [CO.sub.2] control not reached the same level of importance?

To maintain optimal growth conditions to maximize yield, a constant pH, appropriate oxygen supply and sufficient nutrient levels are key. In many processes [CO.sub.2] can act as a stressor or even a toxin to the culture and has to be controlled. [CO.sub.2] is a critical control parameter, mainly in mammalian cell cultures, but the lack of a reliable in situ [CO.sub.2] measuring system had impeded implementation until the first Ingold solution. Off-line sampling and measurement with a blood gas analyser is quite common, but real-time control is not possible with this procedure. Accurate in-line measurement is a must for real-time process control.

What can you tell us about the history of [CO.sub.2] measurement solutions from Mettler-Toledo Ingold?

The first generation of Ingold's [CO.sub.2] sensor, released in 1980, was the only sterilizable [CO.sub.2] sensor on the market. Although it accurately measured [CO.sub.2], it required a calibration procedure, which was not straightforward. So, for the second generation of the sensor, released in 2002, calibration and maintenance were greatly simplified. This sensor shows an excellent correlation if compared to blood gas measurement. The sensitivity in the critical range of 80-200 mbar [CO.sub.2] partial pressure allows accurate measurement and efficient control.

The durability of the CO.sub.2] sensor is highly affected by conditions during sterilization such as temperature and duration. To increase sensor lifetime, the new third generation of sensors feature a redesigned measurement system that includes a microprocessor in the sensor head that digitizes the analog measurement signal. With this technology, which we call Intelligent Sensor Management (ISM), the new sensors are highly tolerable of sterilization procedures. Also, the digital signal is almost immune to electrical interference from other equipment and loss of signal over long cable lengths.

What are the other benefits of ISM?

ISM features advanced predictive diagnostics that mean any maintenance required can be conducted before sensor performance is affected. ISM dramatically increases sensor lifetime and, therefore, the number of batches a sensor's good for. Furthermore, the slope of the internal pH electrode in the new [CO.sub.2] sensor is very stable, even after sterilization. Calibration of the pH electrode prior to sterilization is, therefore, unnecessary. The only calibration step needed is a process calibration after cooling down. If a blood gas analyser is available, just measure a sample of the medium and adjust the transmitter accordingly. This is the easiest way to make a process calibration. Alternatively, sparging and saturation of the medium with a [CO.sub.2] containing gas can be used.


How important is the [CO.sub.2] level during fermentation?

The measurement of [CO.sub.2] produced in mammalian cell fermentations can provide an overall measure of changes in central carbon metabolism and mitochondrial function. High [CO.sub.2] can inhibit cell growth, nutrient utilization, and product formation and in some cases alters protein utilization. Many companies continue to focus on the use of nutrient feeding to increase volumetric productivity, either by increasing cell density or by increasing specific productivity. Basic batch suspension in stirred tank bioreactors has become increasingly less common because nutrient-feeding schemes can reduce waste accumulation and nutrient fluctuations, which allows higher cell densities and productivities to be reached. Recently designed suspension processes are of fed-batch and perfusion types.

How can [CO.sub.2] levels be controlled and what effect on yield can be expected?

In real batch processes, nitrogen or air sparging can be used, together with in-line measurement of [CO.sub.2], to control the level of accumulated dissolved [CO.sub.2] to acceptable levels. In fed-batch processes the [CO.sub.2] level is controlled simply by changing the feeding of the nutrient media. If the [CO.sub.2] level is excessive, just lower the addition of glucose. Our customers have reported an up to 40% increase in yield after implementing such [CO.sub.2] monitoring and control. In addition, control of glucose dosing avoids the inhibition effect that occurs when glucose levels are too high. Conversely, if the [CO.sub.2] level is too low growth rate is not optimal, but this is easily corrected by increasing the glucose feeding rate.


For more information

Kurt Hiltbrunner

Market Segment Specialist

Mettler-Toledo Process Analytics
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Date:Jan 1, 2012
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