Cell cultures in pharmaceutical development react very sensitively to their environment. That’s why the conditions in a bioreactor are subject to precise monitoring. In order to react quickly, online measurement systems became established for monitoring pH, dissolved oxygen and other parameters. Thanks to the Hamilton Incyte and Dencytee sensors, it is now possible to measure cell density online.
Author Dr. Knut Georgy Senior Market Segment Manager Process Analytics, Hamilton Bonaduz
Online measurements of pH or dissolved oxygen have long been established for biological processes. Influencing factors that provide genuinely important information about cell physiology previously required time-consuming and often imprecise offline measurements. Hamilton’s Incyte and Dencytee sensors represent a big step forward. They allow the viable or total cell density to be measured directly in real time for advanced control with never-before instantly available data. Up-to-date information about critical events that could have been missed between offline samples is now immediately recognisable. Thanks to these sensors, production processes can be automated and product developments in research and development departments strategised. The most important information about a culture is obtained during the growth and stationary phases of the cells – i. e. before they die. With the help of online measurements it is now possible to recognise process deviations and quickly make the required adjustments. Detecting when cells begin to die can be difficult simply from looking at the total cell density. Monitoring the viable cells shows an immediate change and, depending on the culture and timing, indicates the next steps to take in order to maximise the yield.
Oxygen and pH measurements
Simultaneous online control of pH is critical in order to generate the right product and maximise yield. Online pH control enables more frequent and smaller adjustments to the pH with the aim of minimising this stress factor and maintaining a constant environment. The dissolved oxygen content is important for the majority of bioprocesses: a lack of oxygen leads to apoptosis (sudden cell death) or anaerobic digestion, reducing the viability and yield significantly. Online measurement and control of dissolved oxygen ensures the optimal amount of oxygen needed to maintain high quality products. All in all, online measurements allow earlier detection of process deviations, reduce labour and the risk of contamination from grab sampling and increase the product yield.
The Incyte sensor was especially developed for use with mammalian cells, yeast and high density bacterial fermentations. Neither changes to the medium nor microcarriers and cell debris influence the results. The functional principle is based on a capacitance measurement: in an alternating electrical field the viable cells behave like small capacitors. Their charging and discharging are measured together with their permittivity. An optimised harvest time, higher yield and lower costs are the key benefits of this technology. Precise control ensures optimum environmental conditions while the very early determination of process deviations forms the basis for the successful use of the Incyte sensor.
The Dencytee sensor, on the other hand, is based on optical technology which measures the total cell density and turbidity of a cell suspension. Near-infrared wavelengths are used for these measurements, making them insensitive to changes in media colour. All particles and molecules that scatter light at 880 nm are detected – both viable and dead cells. This technology is most effective while the cells are growing quickly because that is when almost all cells present are viable. Quick and easy measurements of cell growth, reliable values during the growth phase and the early detection of process deviations are just a few of this sensor’s outstanding strengths.
No more undetected events
The quality and reliability of individual param-eter measurements have been proven by Hamilton in a pilot project along with measurements of various combined factors. The precision of these measurements was evaluated by carrying out additional offline measurements. An optimum cultivation of CHO (Chinese hamster ovary) cells was established in the bioreactor as part of a project to create monoclonal antibodies. In order to attain a high level of reproducibility in the test procedure, all relevant parameters – including pH, dissolved oxygen and the viable and total cell density – were monitored continuously. Even minor process deviations sometimes have a major impact on cell growth in these cultures. The pH and dissolved oxygen measurements are only indirectly linked to cell status and growth.
In conjunction with pH and dissolved oxygen sensors, the capacitive Incyte sensors allow continuous measurements of all relevant bioprocess data. This makes the biological processes significantly more transparent and eases future optimisation. The Hamilton sensors thus provide the online measuring reliability which is essential for optimised process monitoring.
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