Advantages of NMR monitoring

As previously mentioned, one advantage of NMR compared with LC-based techniques is the ability to analyze simultaneously more than 50 (up to ca. 100–150) compounds, including amino acids, saccharides, components of the Krebs cycle and vitamins at one time. Specific sets of NMR peaks relate to specific analytes and each peak can be interpreted quantitatively. Therefore, it is possible to identify molecules—polar, nonpolar, volatile and nonvolatile— not previously reachable when using a targeted approach based on techniques such as HPLC or MS. NMR can help in the evaluation of the components that a cell line is consuming, as well as give insight into the production of metabolites and metabolic pathways. For example, formate, acetate, saccharides, and methanol can be accurately and rapidly monitored at the same time using NMR.

NMR is characterized by a suitable limit of detection (1–10 µM), with a linear signal over a broad concentration range (1µ–500 mM) and can provide highly reproducible results. Systematic calibration is not required. As a consequence, results are comparable across machines and research sites.

Stem cells are used in various applications, including in the production of biologics. They have generally been cultured on undefined media, using feeder layers derived from irradiated mouse embryonic fibroblasts or animal-derived serum to provide growth factors. Strict monitoring of culture systems is necessary to prevent undesirable stem cell differentiation.

It can be envisioned that FDA would favor the use of defined media and support the avoidance of animal-based media for future clinical products (3). Biopharma companies have thus already included this aspect in their product development strategy. Defined culture systems, including media and surface substrates, are fast becoming desirable in the production of stem cells to avoid batch-to-batch variation and guarantee the safety of the end-product. This observation is further substantiated by the effort of suppliers to develop and offer new chemically defined products as alternatives to serum-based media products.

Figure 1: Analysis of aspartate and pyridoxine. The difference in consumption between processes using different defined or non-defined media is demonstrated.

Figure 2: Production of two metabolites examined over four days of culture, demonstrating differences in the metabolic pathways taken in the different media types.

In this example, NMR can be used making the fundamental shift from undefined to defined media, as well as for identifying markers to monitor stem-cell culture, and profiling media to avoid batch-to-batch culture variations.