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

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.

Optimizing high-throughput metabolomic biomarker screening: a study of quenching solutions to freeze intracellular metabolism in CHO cells.

Metabolomics is a rapidly emerging tool for studying and optimizing both media and bioprocess development for culturing recombinant mammalian cells that are used in protein production processes. Quenching of the cells is crucial to fix their metabolic status at the time of sampling. Three precooled quenching solutions were tested for their ability to fix the metabolic activity of CHO cells: phosphate-buffered saline (PBS) (pH 7.4; 0.5°C), 60% methanol with 70 mM HEPES (pH 7.4; -20°C), and 60% methanol with 0.85% (w/v) ammonium bicarbonate (AMBIC) (pH 7.4; -20°C). The metabolic activity of the sampled CHO cells was assessed by determining the intracellular levels of ATP using a bioluminescence assay and selected metabolites with LC-MS/MS. We found the precooled PBS (pH 7.4; 0.5°C) to be the optimal quenching reagent for fixing intracellular metabolism. Importantly, the structural integrity of the cell membrane was maintained and highest yields were obtained for intracellular levels of ATP as well as for 18 out of 28 intracellular metabolites. In contrast to the previously reported studies, buffered methanol quenching was not applicable for suspension cultured CHO cells as cellular membrane integrity was affected. We recommend that the cells are quenched and washed simultaneously to keep the sampling time to a minimum and to prevent any further metabolic activity in the cells. We observed that additional washing steps are not required. Our analyses suggest that methanol as quenching solution, even in combination with a buffer substance, appears not suitable for quenching sensitive mammalian cells. The protocol we report herein is a simple cell sampling method that enables high-throughput metabolomic analyses and is suitable for a large number of samples.