Cell culture media optimisation: a new application for NMR
15 Nov 2011 by Evoluted New Media
The traditionally empirical trial and error process of cell culture media selection can be both inefficient and potentially expensive. Now, a new approach using proton Nuclear Magnetic Resonance provides a method of cell culture analysis that will significantly impact this selection process As an analytical technique, Nuclear Magnetic Resonance (NMR) may be one of the most well known, and certainly is one of the most valuable, for elucidating chemical structure and solving analytical problems. Since the discovery and development of NMR throughout the 1940s and 50s, the versatility of NMR has been expanded to encompass a range of useful techniques that give chemists and biologists the ability to look directly into the heart of molecules in order to uncover their structural secrets.
More recently, modern NMR spectrometers (high-field 500 to 900MHz, cryo-cooled probe technology) with greater resolving ability, enhanced sensitivity and improved automation capability have increased the utility of NMR yet again. As a result, NMR now has applications, not only in chemistry, but also molecular diagnostics, food analysis and biomolecular science.
NMR has been recognised as applicable in the analysis of biological fluids and complex biological molecules since the use of 31P NMR to detect metabolites in unmodified biological samples in 19741. Modern NMR spectroscopy is now routinely used for a range of biological analyses, for example, in the study of complex matrices such as urine, plasma or CSF.
Its advantages for biological analyses are that: it does not rely on separation of the analytes; it is non-destructive; it can measure multiple components simultaneously; sample preparation is simple; and it has high analytical reproducibility. As such, NMR comes close to being a universal detector.
Experience in modern biologics manufacture has shown there is an unmet need in cell culture analysis and monitoring of process development. Often the biologics sector uses an empirical approach to setting up cell cultures. Certain cell culture media are used without formal process development work because historically they have provided the highest product yield for the cell-line being cultured. Normally, following a period of trial and error, a culture medium is developed with a composition close to the optimum. Now, because of the expense of biologics manufacture in terms of both time and materials and in light of recent Quality-by-Design directives, it is increasingly important to conduct this development process in an informed and logical manner.
As a result, NMR expertise in the analysis of biological samples is being applied to cell culture media. This novel approach forms the basis of services such as Spedia-NMR (1H NMR) provided by Spinnovation Biologics, an analytical services company based in the Netherlands.
When selecting cell culture media, a good understanding of the factors limiting cell growth and the parameters that can be optimised to increase protein production, is key. In the manufacture of high-value recombinant proteins or antibodies, for example, in-depth knowledge of cell culture components can have a critical bearing on issues such as:
- the cost of time and resources in setting up a large-scale cell culture
- implementation of quality by design (QbD) in biologics processes
- avoidance of out-of-specification batches or significant process variations
- achieving the optimum therapeutics yields
- optimisation of down-stream processing.
Proton NMR provides true multi-component analysis and delivers both qualitative and quantitative data. In a typical cell culture medium, over 50 components can be analysed simultaneously, including feed components, contaminants and metabolites. The technique provides rapid detection and identification of unknown components in non-defined media from plant or animal sources. NMR analysis is not affected by pH or concentration effects, and concentration profiling of batch samples to follow the progress of a biologics process is straightforward and rapid. By automating sample preparation and analysis, modern NMR services deliver rapid throughput and advanced data treatment including pattern interpretation and statistical analysis.
Biologic NMR services for cell culture profiling provide the following advantages:
- a media profile over a determined time period showing the inter-relationship between consumption of feed components , release of metabolites and accumulation of toxic compounds
- accurate concentration data for components (expect limit of detection of around 10µM)
- analysis of multiple components regardless of nature (i.e., polar, non-polar, volatile and non-volatile)
- the ability to deliver component concentration profiles across multiple samples.
To understand the potential of NMR analysis in biologics manufacturing it is best to examine a recent case study. Take, for example, a situation where a Chinese Hamster Ovary (CHO) based cell culture, used for the over-expression of a therapeutic antibody, was not performing as expected.
In this case the concentrations of over 30 components were measured simultaneously using 1H NMR techniques and the data obtained described the concentration profile relationships between crucial feed compounds and metabolites. The complete analysis and results delivery took less than five days.
Analysis of results showed potential for process control improvement in several areas. Firstly the process was limited by the rapid decomposition of the amino acid asparagine into aspartate and ammonia, which became toxic to the CHO cells as levels increased. Adjustment of the culture feed materials allowed this aberration to be compensated and the efficiency was improved. Secondly there were rapid increases of both lactate and formate in the culture and these had to be controlled by the adoption of a new buffering method to provide optimal process conditions.
The use of NMR in this instance provided a rapid intervention that allowed the culture process to be enhanced and the media composition adjusted to ensure an improved yield of therapeutic protein product. In practice 1H NMR is a very robust, flexible analytical technique and its application to the analysis of complex culture media is now a reality, providing a valuable new tool for the biologics industry. With the application of more sophisticated automation and analytical support technologies, the extent of the contribution that proton NMR can make to cGMP, QbD and process analytical technology in biologics manufacturing is expanding and the role of NMR warrants re-evaluation.
Now, proton NMR can be used over a range of different production platforms and cell systems (e.g., CHO, stem cell, hybridoma) to provide insight into the culture process and support upstream process development. Results provide the information necessary to optimise cell culture media making selection processes more rapid and cost effective.
- What is proton NMR? Proton (1H) NMR is a sophisticated spectroscopic technique that measures the frequency of nutation (change in axial rotation) of radio-frequency excited 1H nuclei within a magnetic field. Sample molecules contain protons in different chemical environments and each type has a different resonant frequency and relaxation response. Chemical shift data obtained from the resonant frequency and the shape and size of data peaks are indicators of chemical structure and dynamics allowing the identity and concentration of components in solution to be determined. NMR measurements can be conducted in a number of different modes to supplement structural or identity data particularly for more complex molecules.
Author Dr FC Girard, CEO Spinnovation Analytical
Contact t: +31(0)24-240-3400 e: info@spinnovation-analytical.com w: www.spinnovation-biologics.com
