NMR-based metabolomics of mammalian cell and tissue cultures

Abstract

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

“…A CHO DG44 cell line expressing a recombinant antibody fusion protein was used for all experiments, and the cell line and process conditions were similar to those previously described . Cell cultures were expanded in a series of shake flasks, rocker bags, and seed bioreactors to generate enough cells to inoculate production bioreactors.…”

“…Moreover, advances in high‐throughput technologies and robotics are driving bioprocess development to even smaller, milliliter scale . Yet, it is known that productivity is generally lower in large (kL) production relative to small (L or less) reactor scales .…”

Metabolomics in the pharmaceutical industry

“…Both NMR [47,48] and mass spectrometry [49] based metabolomic techniques have been developed for this purpose. The effect of dextran sulfate, a commonly used anti-aggregation agent, and volume of the bioreactor were shown to impact specific media nutrients and cellular components [50] and recently, the biochemical reprogramming of two different variants of CHO cells by temperature shift has been reported [51]. This application of metabolomics avoids many of the pitfalls of studying whole animals, since there are fewer concurrent processes and nutrient influx and efflux can be more closely controlled and measured.…”

1H NMR Spectroscopy Profiling of Metabolic Reprogramming of Chinese Hamster Ovary Cells upon a Temperature Shift during Culture

“…These authors reported on the development of a methodology they termed ‘Fermentanomics’ to generate appropriate spectra whereby 50 components were identified [17]. A subsequent study by Aranibar et al used NMR to profile and quantify 30 key metabolites in a CHO cell line engineered to express a model fusion protein demonstrating that the metabolite measurements correlated with currently accepted endpoint based measures, validating the use of NMR for such an approach [18]. NMR has the advantage over other methods such as GC-MS in that labeling/derivatization of the metabolites is not required which reduces the time taken to process samples and in principle, measurements can be made in real time.…”