Advanced online monitoring of cell culture off‐gas using proton transfer reaction mass spectrometry

Abstract

Mass spectrometry has been frequently applied to monitor the O₂ and CO₂ content in the off-gas of animal cell culture fermentations. In contrast to classical mass spectrometry the proton transfer reaction mass spectrometry (PTR-MS) provides additional information of volatile organic compounds by application of a soft ionization technology. Hence, the spectra show less fragments and can more accurately assigned to particular compounds. In order to discriminate between compounds of non-metabolic and metabolic origin cell free experiments and fed-batch cultivations with a recombinant CHO cell line were conducted. As a result, in total eight volatiles showing high relevance to individual cultivation or cultivation conditions could be identified. Among the detected compounds methanethiol, with a mass-to-charge ratio of 49, qualifies as a key candidate in process monitoring due to its strong connectivity to lactate formation. Moreover, the versatile and complex data sets acquired by PTR MS provide a valuable resource for statistical modeling to predict non direct measurable parameters. Hence, partial least square regression was applied to the complete spectra of volatiles measured and important cell culture parameters such as viable cell density estimated (R² = 0.86). As a whole, the results of this study clearly show that PTR-MS provides a powerful tool to improve bioprocess-monitoring for mammalian cell culture. Thus, specific volatiles emitted by cells and measured online by the PTR-MS and complex variables gained through statistical modeling will contribute to a deeper process understanding in the future and open promising perspectives to bioprocess control.

Rapid total volatile organic carbon quantification from microbial fermentation using a platinum catalyst and proton transfer reaction-mass spectrometry

“…Identifying and quantifying headspace metabolites with PTR-MS throughout growth phases may aid in understanding metabolic processes (Bunge et al 2008 ; Crespo et al 2011 ; Luchner et al 2012 ) and improve monitoring in production systems (Schmidberger et al 2014 ). For example, acetaldehyde production appears to be an early marker of impending robust growth in some bacterial and fungal species and can be detected before increases in cell dry weight (Bunge et al 2008 ) or respiratory CO 2 .…”

“…This system has applications in screening VOC producing organisms, VOC yield changes from varying process conditions, identifying appropriate target genes for genetic engineering and verifying increased VOC production in genetically engineered strains. The VOC measurement system could also be used for online process monitoring of bioreactors (Schmidberger et al 2014 ), especially in solid state reactor systems where accurate and immediate online measurements are extremely difficult (Lui 2013 ). Similarly, many novel biological systems under study present challenges to traditional rapid monitoring methods, where complications with standard methods arise from a number of conditions such as complex carbon sources, fungi growing with pellet morphology and biofilms.…”

“…Previously, PTR-MS has been used to explore microbial systems, but estimates of error in VOC quantification can be between 15 and 40 % depending on system conditions (Bunge et al 2008 ; Ezra et al 2004b ; Luchner et al 2012 ; Mallette et al 2012 ; Romano et al 2015 ; Schmidberger et al 2014 ; Singer et al 2009 ). Quantification of total VOC production with PTR-MS can be improved with calibration, but significant uncertainties may remain due to compounds that are not readily detected by PTR-MS (e.g.…”

Advances in process monitoring tools for cell culture bioprocesses

“…Recently, proton transfer reaction MS was used to detect the production of VOCs in cultured cells . For VOC detection in fed‐batch cell culture process, proton transfer reaction in gas phase was reported . The method accomplishes ionization without resorting to fragmentation before MS, thus making it more straightforward to obtain the mass‐to‐charge ratio and abundance level of gas‐phase ions.…”

Progress toward forecasting product quality and quantity of mammalian cell culture processes by performance‐based modeling

“…Some of the techniques mentioned most often are infrared spectroscopy, di‐electric spectroscopy and 2D fluorescence . More exotic analyzers such as proton transfer reaction mass spectrometry or electronic noses also found their way into bioprocessing. In order to correlate the complex signals of most on‐line analyzers with meaningful cultivation parameters e.g., viable cell density or metabolite concentrations, the signals of these analyzers need to be modeled by multivariate statistics.…”