Why Use Mass Spectrometry Gas Analysis in Pharmaceutical Fermentation?

Why Use Mass Spectrometry for Gas Analysis in the Pharmaceutical Fermentation Process? Fermentation is the term used by microbiologists to describe generating a product by means of the mass culture of a micro-organism. This product can either be the cell itself (biomass production), the microorganism’s own metabolite or a foreign product. Microorganisms that carry out their metabolism using oxygen are referred to as aerobic microorganisms. Some microorganisms can substitute nitrate or sulfate for oxygen and therefore grow in the absence of oxygen. These micro-organisms are referred to as anaerobic.

Gas Analysis Mass Spectrometers have been used successfully for over 30 years on bacterial and microbial processes. More recently, there has been increasing interest in mammalian cell culture because they offer the prospect of radical advances in vaccines, monoclonal antibodies and gene therapy. Although the requirements of mammalian cell fermentation processes provide challenges to the off-gas analyzer, some mass spectrometers have already proved invaluable in improving understanding and increasing yields of mammalian cell cultures.

In any fermentation it is essential to monitor the state of the culture, since its health determines the conversion rate of nutrients, the formation of unwanted by-products and, in the worst case, the onset of poisoning. Analysis of the respiratory gases being fed into and removed from the fermentor is an ideal way of characterizing the fermentation. It is non-invasive and enables monitoring of the physiological state of the fermentation, including growth kinetics and substrate consumption. It also helps determine the optimum point to halt the process for maximum yield.

Many fermentations are characterized by small changes in oxygen and carbon dioxide concentrations at critical phases of the fermentation, for example, during the lag phase when the micro-organisms exist in equilibrium with the nutrients. It is vital that the method used for measuring off gas is capable of fast, precise analysis. The speed of Mass Spectrometry (MS) makes it ideal for the fermentation application but speed must not be at the expense of precision. It is equally important that precise data is acquired; otherwise small changes in concentration will be lost.

Unfortunately, it is not uncommon to assume that the measurement of oxygen and carbon dioxide in the off gas is all that is required when making the first steps towards process control, and that sufficient accuracy can be achieved by discrete measurement technology. Both of these assumptions are false.

First, the sparge gas is always variable due to external biology—there are humans and animals undergoing respiration, consuming O2 and generating CO2, but during daylight hours there is also plant photosynthesis consuming CO2 and generating O2.

Second, the ubiquitous twin-tower desiccant dryer systems will either absorb or regurgitate CO2 depending on where they are in the regeneration cycle. These effects are illustrated in the figure below which shows an example of the day and night variations in sparge gas levels of CO2 and O2 measured with a process mass spectrometer over 24 hours.

variations in sparge gas levels

Only accurate comparison of sparge gas and effluent gas can provide accurate pre-screening for possible contamination. Accurate comparison is also required in order to calculate real-time information regarding culture respiration and the availability of nutrients.

Two types of MS have been used to monitor fermentation processes: magnetic sector, where charged particles are separated in a variable magnetic field, and quadrupole, where charged particles are separated in a variable RF field. Industry studies* have shown magnetic sector-based analyzers offer the best performance for fermentation off- gas analysis. By combining high speed with excellent stability, the magnetic sector analyzer lends itself ideally to these demanding applications found in the pharmaceutical industry. Magnetic sector mass spectrometers have demonstrated the highest levels of precision for fermentation off-gas analysis and have been successfully monitoring fermentor off-gas at many of the world’s leading biotechnology and pharmaceutical companies for many years.

You can read about the advantages of magnetic sector mass spectrometry, as well as additional details involving gas analysis mass spectrometer applications in fermentation and cell culture process in this application note.

Editor’s Notes:

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*1.David Pollard, Jens Christensen, Vent Gas Analysis, Encyclopaedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology 2010, John Wiley & Sons. *2.Joseph S. Alford, Bioprocess control: Advances and challenges, Computers & Chemical Engineering Volume 30, September 2006.