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

• Published in: Biotechnology journal 2015-01, Vol.10 (1), p.190-198
• Main Authors: Steinhoff, Robert F., Ivarsson, Marija, Habicher, Tobias, Villiger, Thomas K., Boertz, Jens, Krismer, Jasmin, Fagerer, Stephan R., Soos, Miroslav, Morbidelli, Massimo, Pabst, Martin, Zenobi, Renato
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Animals; ATP; Automated sample aliquoting; Cell Culture Techniques - methods; Cell Line; Cell Survival; Intracellular Space - metabolism; MALDI-MS; Metabolomics - methods; Mice; Microarrays for mass spectrometry (MAMS); Monoclonal antibody production; Principal Component Analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201400292

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. Biotechnological processes to produce therapeutics are subject of many different optimizations and investigations aiming to (i) ensure process stability and reproducibility, (ii) maximize product yield, and (iii) reduce the overall process time. Monitoring metabolites, i.e. small molecules with prominent roles in the biological network, helps to achieve these goals by giving insights into the biological processes. In this article, the authors describe a workflow for rapid metabolite monitoring based on mass spectrometry without prior separation.