Over the past two decades, the protein biologics market has exploded and now comprises the fastest growing segment in therapeutics. Chinese hamster cells (CHO) have become the host cell line of choice for the majority of commercial production of protein therapeutics, such as monoclonal antibodies (mAbs), on the market.¹ Other cell lines such as HEK 293 and Vero also play an important role and they are commonly used to produce non-mAb products such globular proteins, viral vector therapeutics, and vaccines.²
The impact of cell culture medium optimization: productivity, quality, and overall success
Early work by Harry Eagle first demonstrated the importance in medium composition in the growth of cells in culture.3,4 Among his findings, Eagle reported that 13 amino acids are essential for cell growth. Richard Ham reinforced the importance of component selection and optimization in development of F12, a fully synthetic, chemically defined, and serum-free medium for single cell cultivation and expansion of CHO cell cultures.5 F12 medium, however, was not well suited to support growth of cell to densities above 105 cell/mL.6 Work by Sato and colleagues created the improved formulation DMEM/F12 via supplementation with hormones and growth factors and by blending 50:50 with Dulbecco’s MEM (DMEM) which provided increased levels of key amino acids and vitamins and additional trace metals.7
Since these early achievements, serum-free medium designed for bioproduction has further advanced and has resulted in vastly improved cell viability, growth, and productivity. A large amount of knowledge has now accumulated, particularly for CHO cells, that demonstrates the importance of medium composition. These efforts have played a major role in increasing mAb titer production from ~ 1 g/L to greater 10 g/L, in some cases.
Media studies have shown that ingredient composition and their respective and relative concentrations can dramatically improve or decrease medium performance. The effect is not limited to energy components (Glucose, glutamine, others) as amino acids, and other ingredients have a key role as well. Groups from MIT and U. Minn. pioneered the concept of stoichiometric analysis of cell growth and amino acid utilization in basal and feed formulations according to nutrient demand; thus preventing depletion of key nutrients while minimizing toxic metabolic waste.8-10 Other approaches such as metabolic, gene analysis, and others have been developed as well. These studies show that both essential and non-essential amino acid composition can have a dramatic effect on cell metabolism as pathways can shift due to the levels of amino acids in the medium.6,11
There are numerous examples in the literature of the importance of amino acid concentration on CHO cell metabolism and production that demonstrate that maintenance of most amino acids at specific concentration ranges is very important for CHO culture [for review see (6)]. Thus, it is not the total amount of amino acids that is critical but rather a balanced composition that meets the needs of the individual cell line6,12,13 or individual cell clone.14
Amino acids can also play a role in other cellular function outside of protein synthesis and energy source. Reports indicate that some amino acids can act as signal molecules, and may influence the rate of apoptosis in cells and other cellular parameters.15 Researchers have also identified that some amino acids at certain concentrations can offer some protection from elevated ammonia6,12 or osmolality.12,16 Chen and Harcum reported that Thr, Pro, and Gly at a level of 20 mM are able to mitigate the negative effects of ammonia.17 McAtee Pereira showed Gln, Glu, Asn, Asp, and Ser levels can reduce ammonia production while preserving cellular carbon flux23. Others have found that care must be taken to avoid excessive amounts of some amino acids such as Asn and Gln, to avoid excess production of detrimental waste.6,11 Note that some amino acids such as Cys, Tyr, Trp, can have low solubility and others can be unstable in some formulations (Gln, Cys), and these need to be carefully monitored in media [for review on solubility see (18)].
Studies have also shown that mAb quality attributes, such as glycosylation pattern, aggregation, and charge variant can be affected by media composition as well.2,13,19-21 Beyond amino acids, additional examples include vitamins, biogenic amines, and others. For example, depletion of the vitamin choline can have a dramatic effect on both mAb titer and mAb aggregation.22,23 Thus, there are ongoing efforts to optimize media formulations to enable production of biotherapeutics at high levels and of high quality. Investment in medium optimization, and the control of media composition, can have a dramatic impact on the overall outcome of a bioproduction effort-as well as shortening the developmental pathway
The important role of medium component analysis
Current chemically defined production media typically contain 50 -100 ingredients, which include sugars, amino acids, vitamins, biogenic amines, metals, buffers, and others.6 A typical medium optimization effort usually involves multiple rounds of optimization through analysis of spent media and monitoring the effect of supplementation or reduction of individual components on the desired outcome of the culture. Optimization can be a complex and a time-consuming experience due to the large number of medium components and the even greater number of possible concentration-dependent combinations. Design of experiment (DOE) can be utilized to reduce the workload somewhat.6,13,24 Approaches to develop a mathematical model (digital twin) will further help in decreasing the number of physical experiments but also require input from the cell culture system.33 Nevertheless, a medium development effort started from scratch can require months to achieve a highly optimized formulation.
Complete optimization of production extends beyond the base medium. Development of a feed-based strategy that presents components at optimized concentrations during the exponential growth phase and production phase can result in significant improvement that sometimes reaches several-fold.13,22,25 Thus, medium composition, medium optimization, and the degree of monitoring and control26 can have profound effects on cell metabolism, cell health, and product production and quality.
The medium also needs to support scale up efforts from single cell cloning, to clone selection to expansion in mini-bioreactor27 (Ambr© type), to bench-top bioreactor, to large scale. This can include perfusion bioreactors at the N-1 or in the production vessel, which use medium exchange to achieve increased cell densities and output by removal of toxic metabolites.28,29 The effort to optimize and monitor media throughout development and production phases can result in large benefit at each step of the process development.
The need for at-line and efficient analytical capability to monitor medium composition is central to the effort to develop, optimize, and control media formulations. This desire for robust analytical methods is compounded by the fact that each cell line/clone can a have different metabolic demand, and thus compounds the need in a multi-line production environment. Sadly, much of current bioproduction is accomplished without fully optimized formulations due to poor workflows, cost, or time constraints. Formulations are also often under-optimized due to sampling media from only one or two time points in the cell growth curve. Examples of such are end-of-growth-phase or end-of-culture, both of which typically lie outside of the most productive phase of the cell growth curve. Early phase development is focused on speed and often uses a ready-to-go commercial formulation. Late phase development focuses on further optimization but cost and lack of accessible analytics can prevent a full optimization. Thus, many media currently in use are not fully optimized to due to lack of a robust analytical solution.
The limitations of classical analytical methods
The most common method of quantitating medium components is by classical methods; typically, HPLC. This method requires experienced operators, optimization of conditions, the use of amino acid labelling reagents, and specialized columns for each type of component. While HPLC methods are effective they can be expensive as dedicated facilities may be required. Often sample turn-around is less than desired due the complexity of the task or because of a backlog at the testing facility. These delays, in turn, negatively impact the process development timeline. Moreover, the sample size requirements for these classical methods may not allow frequent analysis of samples from smaller-scale developmental platforms such as microbioreactors.
Outsourcing quantification is an option, however it is expensive and time consuming as it involves shipping of frozen samples and ultimately analysis is performed using the same time-consuming classical methods. The additional time for shipping further extends the timeline of media optimization. Thus, these classical strategies typically do not provide actionable data, as it can take days to weeks to receive testing results.
To address the current testing problems, the REBEL analyzer, by 908 Devices, uses microchip capillary electrophoresis coupled with high pressure mass spectrometer (CE-HPMS) to streamline media analysis. The Rebel can analyze a panel with multiple amino acids, vitamins, and bioactive amines in minutes. This new, affordable, easy-to-use technology provides multiple benefits and can substantially reduce timelines for medium and feed development. The affordability of this analytical method available at the point of need supports analysis of more time points within a production time course. In addition, the near real-time capability, and small footprint of the Rebel enables at-line analysis, that in turn, can support process analytical technology (PAT) strategies and the potential employment of mitigation strategies, if used at-point during a production campaign.
The three general methods available for media development
Generally, there are three options to implement a media development effort for bioprocessing applications which consists of: 1) use of-the-shelf commercial media and feeds, 2) outsourcing media development, or 3) in-house media development.
Off the shelf media
There are numerous off-the-shelf media and feeds available on market. These have the limitation in that they were developed using the cell line/clone of the vendor and vendor’s developmental system (bioreactor and process). Thus, commercial formulations are optimized to someone else’s cell line, rather than your own.13
This can result in vast underperformance of these formulations in your own production line(s) and may require extensive screening of media from several vendors to identify an appropriate formulation. Furthermore, the variation in nutrient demand between clones of the same cell line (example CHO) can further complicate the robustness of off-the-shelf solutions. It has been shown that amino acids can be easily depleted in some off-the-shelf media.13
Using off-the-shelf media does not eliminate developmental efforts, as typically several media and feeds must be screened to maximize desired outcome.13 Another limitation with this approach is that you do not know the complete formulation and list of ingredients. Thus, therapeutic producers lack comprehensive knowledge of their cell system and their process, and cannot be certain that their production is fully optimized and controlled. Sometimes, a therapeutic producer will find that there are no satisfactory off-the-self options that provide the desired outcome.
The main advantage of utilizing off-the-shelf strategies is savings of time and expense. Use of pre-formulated solutions can reduce time to market through a shortened developmental timeline. They can also reduce costs, particularly short-term cost, with possible consequence of greater overall cost.
Outsourcing media optimization
Outsourcing media optimization has resulted in the advancement of custom institution-specific “platform media” that can offer increased performance over off-the-shelf-solutions. However, this solution also has limitations in that the medium is only optimized for the cell line/clone that is sent. Full optimization of a medium may take months, even with experienced service providers. Furthermore, the therapeutic producer will not be able to gain first-hand knowledge of their cell line from the developmental process.
Since these outsourced formulations are developed to a clone that is provided to them, in their production vessel, there are often problems with use during culture scaling. Cells early in the selection and amplification process can have a vastly different nutrient demand than small-scale culture, and again from cells grown in high-density multi-1000 liter bioreactors. This problem can be mitigated somewhat by development of specific media for each phase of need. It is not uncommon for producers to develop several formulations and feeds for the production phase, which are then screened and further optimized, similar to off-the-shelf solutions, in order to mitigate in-house clonal variation.
In-house media development
In-house development has distinct advantages over other options as the therapeutic producer gains knowledge of their cell line, will know the formulation, and can be reasonably certain that an optimization technique is carried out to a high level. However, this approach has limitations, such as the need to acquire expert personnel and analytical capability. The timeline can be longer than with other solutions if a complex build out of equipment is required. Nevertheless, acquiring medium analytical equipment with at-line capability can provide long-term benefits, as it can be employed during product production as part of a process analytical technology (PAT) strategy.26,30
Often a combination of strategies can be considered such as outsourcing development with a limitation for off-the-shelf components, rather than the creation of a highly optimized custom media. Other combination strategies may include using off-the-shelf solutions early in production to hasten speed to market and later using fully optimized media to improve efficiency and lower long-term costs.
Media QC testing
QC testing of cGMP media is usually performed by the service provider and QC of non-GMP developmental media can usually be performed for an additional fee. However, it is a smart approach to self-validate any acquired media, so possible mistakes in development or bioproduction can be avoided. Studies have found wide variation in expected levels of ingredients in off-the-shelf media. Some results showed as much as 2.5x expected levels and others were not present at all.31 For this purpose, the Rebel analyzer is also of great value as it can analyze a robust panel of dozens of media ingredients onsite in just a few minutes. This provides the upmost confidence that the media purchased is composed as promised and that additional lots will perform as expected, thereby reducing any possible variability in performance.
Cell culture media analysis and process analytical control (PAT)
The drive towards an in depth-understanding of a production process effect on product quality is partly driven by the US Food and Drug Administration’s (FDA) process analytical technology (PAT) initiative that was introduced in their Guidance to the Industry document in 2004.32 The PAT framework seeks to ensure the quality of pharmaceutical products through real-time monitoring of the process.26 Identifying the sources of variability in a process, variability management, and determining whether product quality may be affected are components of PAT.26
Many bioreactor conditions such as DO, temperature, conductivity, pH, pO2, pCO2, metabolites, cell viability, density, and productivity can currently be measured by either in-line sensors or near-line analysis of culture fluids with bioanalyzers. For a more in-depth analysis of medium conditions, media analyzers such as the Rebel can provide near real-time analysis (7 minutes) of >30 different amino acids, vitamins and biogenic amines.
The small footprint of the Rebel also enables use at-line where the analyzer can be placed on bench or countertops along with other analytical equipment or placed on a cart for movement to different locations. Furthermore, this device requires a sample volume of only 10 µL, which is just a fraction of the sample already taken for other biochemical analyses. Other aspects that support integration into a PAT initiative include: simple operation (load and push a button), 24/7 availability (unlike many in-house analytical facilities), software that is designed for cGMP compliance with support of 21 CRF Part 11 for digital documentation, and .CSV file format for integration into other software systems.
Additional benefits of at-line medium analysis
The advancement of optimized media and feed strategies for fed-batch culture and optimized media for perfusion bioreactors has played a large role in advancing volumetric productivity into the multi-gram/L range. A properly constructed medium strategy can potentially increase productivity several fold. One reason these strategies work so well is because they ensure that media components are present and maintained at an optimal concentration. Thus, near real-time, at-line analysis can be used to monitor whether your feed or perfusion strategy is operating as expected.
In addition to monitoring and control, at-line medium analysis may enable a mitigation pathway for adverse events due to unexpected medium or feed imbalance. This in turn, could increase the overall efficiency of the production campaign.
Much of the increase in productivity since the start of the bioproduction era decades ago has been due to optimization of cell culture media to support cellular health and to maximize product productivity and quality. With this advancement in media formulation and feed strategies, there is increased need for robust analytical tools. Many of the classical methods of media analysis were burdensome, slow, expensive, and limiting.
New microfluidic technologies, like the Rebel, have advanced over classical analytical methods by providing near-instantaneous readings, at-line, with a simple to operate device within a small footprint. These advances in medium analysis should support the industry into the future with expanded capabilities that ensure high productivity and consistent product quality.
1. Jayapal, K. P., Wlaschin, K. F., Yap, M. G. S. & Hu, W. S. Recombinant Protein therapeutic from CHO cells-20 years and counting. Chem Eng Prog 103, 20-47 (2007).
2. Kunert, R. & Reinhart, D. Advances in recombinant antibody manufacturing. Applied microbiology and biotechnology 100, 3451-3461 (2016).
3. Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science (New York, N.Y.) 122, 501-514 (1955).
4. Eagle, H. Amino acid metabolism in mammalian cell cultures. Science (New York, N.Y.) 130, 432-437 (1959).
5. Ham, R. G. CLONAL GROWTH OF MAMMALIAN CELLS IN A CHEMICALLY DEFINED, SYNTHETIC MEDIUM. Proceedings of the National Academy of Sciences of the United States of America 53, 288-293 (1965).
6. Ritacco, F. V., Wu, Y. & Khetan, A. Cell culture media for recombinant protein expression in Chinese hamster ovary (CHO) cells: History, key components, and optimization strategies. Biotechnology progress 34, 1407-1426 (2018).
7. Bottenstein, J. et al. The growth of cells in serum-free hormone-supplemented media. Methods in enzymology 58, 94-109 (1979).
8. Xie, L. et al. Gamma-interferon production and quality in stoichiometric fed-batch cultures of Chinese hamster ovary (CHO) cells under serum-free conditions. Biotechnology and bioengineering 56, 577-582 (1997).
9. Xie, L. & Wang, D. I. Stoichiometric analysis of animal cell growth and its application in medium design. Biotechnology and bioengineering 43, 1164-1174 (1994).
10. Zhou, W., Rehm, J. & Hu, W. S. High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding. Biotechnology and bioengineering 46, 579-587 (1995).
11. Duarte, T. M. et al. Metabolic responses of CHO cells to limitation of key amino acids. Biotechnology and bioengineering 111, 2095-2106 (2014).
12. Kishishita, S. et al. Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells. Journal of bioscience and bioengineering 120, 78-84 (2015).
13. Reinhart, D., Damjanovic, L., Kaisermayer, C. & Kunert, R. Benchmarking of commercially available CHO cell culture media for antibody production. Applied microbiology and biotechnology 99, 4645-4657 (2015).
14. Pan, X., Streefland, M., Dalm, C., Wijffels, R. H. & Martens, D. E. Selection of chemically defined media for CHO cell fed-batch culture processes. Cytotechnology 69, 39-56 (2017).
15. Paudel, S., Wu, G. & Wang, X. Amino Acids in Cell Signaling: Regulation and Function. Advances in experimental medicine and biology 1332, 17-33 (2021).
16. deZengotita, V. M., Abston, L. R., Schmelzer, A. E., Shaw, S. & Miller, W. M. Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality. Biotechnology and bioengineering 78, 741-752 (2002).
17. Chen, P. & Harcum, S. W. Effects of amino acid additions on ammonium stressed CHO cells. Journal of biotechnology 117, 277-286 (2005).
18. Salazar, A., Keusgen, M. & von Hagen, J. Amino acids in the cultivation of mammalian cells. Amino acids 48, 1161-1171 (2016).
19. He, L. et al. Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses. Biotechnology journal 13, e1700254 (2018).
20. Gawlitzek, M., Estacio, M., Fürch, T. & Kiss, R. Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells. Biotechnology and bioengineering 103, 1164-1175 (2009).
21. Torkashvand, F. et al. Designed Amino Acid Feed in Improvement of Production and Quality Targets of a Therapeutic Monoclonal Antibody. PloS one 10, e0140597 (2015).
22. Kuwae, S., Miyakawa, I. & Doi, T. Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content. Cytotechnology 70, 939-948 (2018).
23. McAtee Pereira, A. G., Walther, J. L., Hollenbach, M. & Young, J. D. (13) C Flux Analysis Reveals that Rebalancing Medium Amino Acid Composition can Reduce Ammonia Production while Preserving Central Carbon Metabolism of CHO Cell Cultures. Biotechnology journal 13, e1700518 (2018).
24. Jordan, M. et al. Cell culture medium improvement by rigorous shuffling of components using media blending. Cytotechnology 65, 31-40 (2013).
25. Whitford, W. Fed-batch mammalian cell culture in bioproduction. BioProcess Intl. April (2006).
26. Maruthamuthu, M. K., Rudge, S. R., Ardekani, A. M., Ladisch, M. R. & Verma, M. S. Process Analytical Technologies and Data Analytics for the Manufacture of Monoclonal Antibodies. Trends in biotechnology 38, 1169-1186 (2020).
27. Elliot, K. et al. Spent media analysis with an integrated CE-MS analyzer of Chinese hampster ovary cells grown in an ammonia-stressed parallel microbioreactor platform. BioProcessing J. Feb. 29, 2020. Accessed Jan: Retrieved from https://bioprocessingjournal.com/index.php/article-downloads/882-vol-19-open-access-2020-spent-media-analysis-with-an-integrated-ce-ms-analyzer-of-chinese-hamster-ovary-cells-grown-in-an-ammonia-stressed-parallel-microbioreactor-platform.
28. Brau, C. et al. Poster: Optimization and evaluation of perfusion medium in high cell density mammalian cell culture. Accessed July 20, 2021: Retrieved from https://assets.thermofisher.com/TFS-Assets/BPD/posters/optimization-evaluation-perfusion-medium-high-cell-density-mammalian-cell-culture-systems-poster.pdf.
29. Mayrhofer, P. & Kunert, R. Perfusion Medium Development for Continuous Bioprocessing of Animal Cell Cultures. Accessed July 20, 2021: Retrieved from https://www.americanpharmaceuticalreview.com/Featured-Articles/562913-Perfusion-Medium-Development-for-Continuous-Bioprocessing-of-Animal-Cell-Cultures/.
30. Anderson, J. L. et al. Poster: Rapid at-line nutrient profiling from an ammonia stressed CHO cell line utilizing an intergrated benchtop analyzer. Accessed July 20, 2021: Retrieved from https://908devices.com/wp-content/uploads/2019/08/BioProc-Summit-2019-GAH-poster_finalv2-2.pdf.
31. Gavin, C., Anderson, J. L., Blakeman, K. H., Miller, S. & Harris, G. A. Poster: An at-line analyzer capable of quantitating amino acids, vitamins, amines and dipeptides from cell growth media. Accessed July 20, 2021: Retrieved from https://908devices.com/wp-content/uploads/2019/08/BPI-2020-Poster_final.pdf.
32. US FDA. Guidance for Industry PAT-A framework for innovative pharmaceutical development, manufacturing. Accessed July 20, 2021: Retrieved from https://www.fda.gov/media/71012/download. Cell Culture Media Analysis for Cell Therapy Applications
33. Demesmaeker M, et al. Bioprocessing 4.0 — Where Are We with Smart Manufacturing in 2020? Pharm. Outsourcing, September 2020; https://www.pharmoutsourcing.com/Featured-Articles/568001-Bioprocessing-4-0-Where-Are-We-with-Smart-Manufacturing-in-2020