Nuclear Magnetic Resonance as a Bioprocessing QbD Application

Published on: 
Pharmaceutical Technology, Pharmaceutical Technology-11-01-2012, Volume 2012 Supplement, Issue 6

The author discusses current expectations in bioprocessing and lays a framework for using NMR to enhance a QbD approach.

Ten years ago this month, in August 2002, FDA published its groundbreaking guidance document, Pharmaceutical cGMPs for the 21st century: A Risk-Based Approach, to encourage a quality- and risk-based, approach to pharmaceutical development and manufacturing. With a growing focus on process analytical technology (PAT), industry quickly began to apply the guidance to production as well as development and the concept of quality by design (QbD) emerged. QbD requires the thorough scoping and understanding of key parameters in process development. An outcome of a QbD approach is that the product attains the desired quality every time it is produced. QbD creates a robust and repeatable framework that ensures that quality is designed-in, not tested for, in the product.

There was initial resistance from some industry parties to the implementation of QbD. Many thought that the process would mean an increase in workload during the development phase. Some noted that implementation would be significantly more complicated with biologics compared with traditional small-molecule drugs. With the understanding that quality and improved efficiency were crucial to all drug products, in 2008, the FDA Office of Biotechnology Products launched a pilot program for QbD to evaluate and identify best practice for the risk-based approach with biologics. Work in this area is now ongoing throughout the industry and this article examines how critical quality attributes (CQA)—a key component of QbD—can be defined using analytical tools, with a focus on nuclear magnetic resonance (NMR).

Aims of bioprocess optimization

The challenge in producing biologics using a cell-line platform is how to develop a robust process with optimal cell-growth conditions within a short timeframe. A product must be delivered which meets market requirements in terms of quality, safety, and cost, and in a manner that anticipates potential issues during clinical and commercial production. A crucial aspect of bioprocess optimization is the development of the media and feed strategy that meets the specific cell-line metabolic requirements. NMR-based methods can provide rapid accurate, quantitative monitoring of more than 50 feed components, contaminants, and metabolites within culture media at any stage of the process framework, and thereby help to meet QbD requirements. This approach necessitates identification of optimal nutritional and cell-growth conditions, as well as resolution of issues related to eventual impurities and causes of variation in the culture-process performance.

Optimization tools. Spent media analysis using high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC–MS) techniques is a common approach to understanding the factors limiting cell growth, and therefore, can help identify which parameters of the cell-culture media should be adjusted to optimize protein production. However, several limitations are associated with these methods. Analyses are generally slow, costly, and restricted in the number of components which may be studied, leading to an incomplete picture of the bioprocess and the factors which may affect scale-up.

Recently, application of NMR spectroscopy to the profiling of complex matrices, such as cell culture media and spent media, has shown major advantages when compared with LC methods (1, 2). NMR spectroscopy, a universal detection method, is characterized by a large dynamic range and measurement of the signal intensity can provide fast acquisition of quantitative results.

NMR and the future of bioprocess optimization

Cell-culture process optimization is crucial if production of recombinant protein products is to be commercially viable. Developments in NMR methodology have provided a new and useful tool allowing the biologics industry to rapidly and accurately analyze culture media, thereby elucidating the concentration profile of multiple components. This technique can be used in designing new media, troubleshooting existing culture media problems, standardizing cell-culture media prior to large-scale production, and examining spent media to highlight efficiency and cost issues.

According to FDA, "Quality by Design [QbD] is understanding the manufacturing process and identifying the key steps for obtaining and assuring a pre-defined final product quality" (3). FDA has identified the use of new analytical methods, such as NMR, to monitor and control processes as important in increasing manufacturing quality through QbD (3). Offering the ability to characterize chemically complex media, NMR techniques have the potential to contribute significantly to an understanding of process-critical parameters, helping to reduce performance variability and minimize the risk of process failure in large-scale biopharma production.


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.

Case study 2: Profiling CHO-culture media

Chinese Hamster Ovary (CHO) cells are often used for the over-expression and production of recombinant proteins. However, each newly generated CHO cell line requires a separate culture optimization process. Process scientists frequently try several different culture media and select the one providing the highest production levels. However, they may have no means of knowing whether the selected conditions are best suited for scaling or whether there is scope for significantly increasing yields.

NMR profiling can provide full visibility of the presence and concentration of feed components, contaminants, and metabolites. Figure 3 shows the concentration of key analytes over the first eight days of a pilot culture of a new antibody-producing CHO cell line. As demonstrated, there was a fast rise in formate and lactate levels, and a rapid decrease in asparagine concentration. Decomposition of the amino acid shown is directly related to an increase in ammonia which is toxic to the cells. The NMR data enabled rapid assessment of the medium parameters which needed to be varied in order to increase cell density and protein production. In addition, comparison of the lactic acid concentration in media from a control culture and one grown under conditions designed to reduce lactate validated the efficiency of the intervention.

Figure 3: The concentration of key analytes over the first eight days of a pilot culture of a new antibody-producing CHO cell line.


With QbD, FDA and other regulatory agencies require companies to have greater control of their bioprocesses. There is an increased need, therefore, for good methodology to monitor the concentration and identity of media components during process development and manufacturing.

In summary, NMR offers a robust, flexible analytical technique that achieves simultaneous feedback on multiple analytes. NMR is further characterized by a large dynamic range, a high specificity and reproducibility, coupled with fast throughput, making it cost efficient. The information that the industry needs for bioprocess development can be obtained with NMR, and NMR can be used across various cellular platforms, including CHO cells, NSO cells stem cells, insect cells, bacteria, yeasts, and algae.

F.C. Girard, PhD, is CEO of Spinnovation Analytical.


1. Spinnovation Case Studies, "Cutting-Edge Analytical Technology to Profile and Manage Optimization of Cell Culture Media" and "Stem Cell Culture: The Advantage of NMR Monitoring," available at

2. Bradley et al., JACS 132, 9531–33 (2010).

3. FDA, "Advancing Regulatory Science at FDA", p.14, available at