Optimizing High-Throughput Metabolomic Biomarker Screening: A Study of Quenching Solutions to Freeze Intracellular Metabolism in CHO Cells

Abstract

Abstract

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.