High‐throughput nucleoside phosphate monitoring in mammalian cell fed‐batch cultivation using quantitative matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Abstract

Current methods for monitoring multiple intracellular metabolite levels in parallel are limited in sample throughput capabilities and analyte selectivity. This article presents a novel high-throughput method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) for monitoring intracellular metabolite levels in fed-batch processes. The MALDI-TOF-MS method presented here is based on a new microarray sample target and allows the detection of nucleoside phosphates and various other metabolites using stable isotope labeled internal standards. With short sample preparation steps and thus high sample throughput capabilities, the method is suitable for monitoring mammalian cell cultures, such as antibody producing hybridoma cell lines in industrial environments. The method is capable of reducing the runtime of standard LC-UV methods to approximately 1 min per sample (including 10 technical replicates). Its performance is exemplarily demonstrated in an 8-day monitoring experiment of independently controlled fed-batches, containing an antibody producing mouse hybridoma cell culture. The monitoring profiles clearly confirmed differences between cultivation conditions. Hypothermia and hyperosmolarity were studied in four bioreactors, where hypothermia was found to have a positive effect on the longevity of the cell culture, whereas hyperosmolarity lead to an arrest of cell proliferation. The results are in good agreement with HPLC-UV cross validation experiments. Subsequent principal component analysis (PCA) clearly separates the different bioreactor conditions based on the measured mass spectral profiles. This method is not limited to any cell line and can be applied as a process analytical tool in biotechnological processes.

Intracellular CHO Cell Metabolite Profiling Reveals Steady‐State Dependent Metabolic Fingerprints in Perfusion Culture

“…The soft ionization results in mainly singly charged intact molecules that cover a wide m/z range. The simple sample preparation, short measurement time, and straightforward analysis of spectral data make this technique suitable for the monitoring of cell culture processes . In combination with high‐throughput technologies (microarrays for mass spectrometry, MAMS) and labeled internal standards, semi‐quantitative analysis, and improved reproducibility have been shown .…”

“…In combination with high‐throughput technologies (microarrays for mass spectrometry, MAMS) and labeled internal standards, semi‐quantitative analysis, and improved reproducibility have been shown . For example, MALDI‐TOF MS analysis was used to quantify the effect of hypothermia and hyperosmolarity on phosphate metabolism in mammalian cell fed‐batch cultures …”

“…The simple sample preparation, short measurement time, and straightforward analysis of spectral data make this technique suitable for the monitoring of cell culture processes. 22 In combination with high-throughput technologies (microarrays for mass spectrometry, MAMS) and labeled internal standards, semi-quantitative analysis, and improved reproducibility have been shown. 23 For example, MALDI-TOF MS analysis was used to quantify the effect of hypothermia and hyperosmolarity on phosphate metabolism in mammalian cell fed-batch cultures.…”

Simplifying the Preparation of Pollen Grains for MALDI-TOF MS Classification

“…Multivariate methods, such as principal component analysis (PCA) can be applied to mass spectrometric data for several reasons, e.g., for quality control, to reduce complexity of the data, and to emphasize differences within a data set that enable classification [17,18,19,20]. …”

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI‐TOF‐MS

“…In particular, matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometry (TOF‐MS) has become a valuable tool, given its simple sample preparation, data analysis and sensitivity to high‐mass metabolites . In combination with the use of microarrays (MAMS), good reproducibility, and semiquantitative estimation capabilities have been shown in the high throughput analysis of intracellular metabolites in mammalian cell cultures …”