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.
CASE STUDY 1: STEM-CELL CULTURE OPTIMIZATION
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.
NMR monitoring has been used to compare an undefined medium with two different defined media. Figure 1 shows analyses for aspartic acid and pyridoxine. While not present in the nonchemically defined media, pyridoxine appeared
to be consumed when provided in the defined media. Aspartate also appeared to be consumed from the defined media although
it was rather low and stable in the nondefined medium.
Figure 1: Analysis of aspartate and pyridoxine. The difference in consumption between processes using different defined or
non-defined media is demonstrated.
In Figure 2, production of two metabolites has been examined over four days of culture, demonstrating differences in the metabolic behavior
taken in the different media types. The NMR data, combined with cell viability and performance information, enabled a defined
medium to be selected and optimized for a specific stem-cell culture.
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.