Cell-culture media formulations comprise the ingredients required by cells to survive, grow, and function as needed for the intended cell-culture application. The exact formulation depends on the nature of the cell-culture process, the type of cell and specific cell line employed, and the class of biologic and target properties of the biomolecule being produced. Very small changes in the chemical composition of cell-culture media can cause significant changes in performance. Consequently, there are significant benefits to fine-tuning the media formulation for any given bioprocess.
Numerous components
Cells rely on many different chemicals and materials to stay healthy and active. Those used for biopharmaceutical manufacturing must also be able to express the target product in high yield and quality. To do so, cells require glucose and other carbon-based sugars as energy sources, amino acids, and peptides to support protein production; vitamins to act as cofactors for enzyme functions; lipids to serve as basic components for cell membrane construction; salts to achieve osmotic balance and enable many natural biologic processes; and trace elements used as enzyme catalysts, redox reaction components, and for other cellular functions, according to Kenneth Low, global head of media R&D in Lonza’s Bioscience.
In general, observes Kimberly Schrag, R&D manager, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany, modern cell-culture-media formulations are chemically defined (i.e., do not contain hydrolysates that often cause lot-to-lot variability) and do not contain any animal-derived components. She notes that in addition to amino acids, vitamins, trace elements/minerals, lipids, pH-buffering agents, and carbon sources, they can also contain recombinant growth factors, antioxidants, and some components for specific functions, such as poloxamer 188, which reduces shear stress for suspension cultures.
Amino acids, as well as serving as building blocks for proteins, are a major nitrogen energy source important for cell proliferation, adds Fei Chen, director of cell-culture process development at WuXi Biologics. Nucleotides and polyamines are also often included in media formulations to meet the needs of specific cells.
Proprietary media formulation development processes available
Despite the complexities associated with media formulation, it is possible to develop “standard” development processes that are sufficiently flexible to allow for tailoring and optimization. Both WuXi Biologics and MilliporeSigma have adopted this strategy.
“A key consideration for media formulation development is to find a common formulation for most clones and host cell lines to serve as a good starting point for medium and process development,” explains Hang Zhou, senior vice-president and head of bioprocess research and development at WuXi Biologics.
WuXi Biologics has developed a medium suitable for most molecules in various host cells with robust cell growth and high productivity for a lower cost of goods. Starting with this basic formulation, medium formulation screening and optimization are pursued. “Our media development platform provides fast and intelligent formulation development tools to support customized formulation design according to the needs of different drug molecule in various host cells, including a high-throughput screening platform from a large media library, intelligent formulation customization with digital tools, and intracellular metabolomics-based formulation optimization,” Hang comments.
Using the media formulation development platform, WuXi Biologics can simulate and predict formulation performance prior to experimentation, allowing for more precise screening to modify attributes with just two or three rounds of studies and limited conditions (<10 for each round), Fei Chen, director of cell-culture process development at WuXi Biologics, says. In one case, productivity was increased by 78% and media cost/gram-protein reduced by 37% from an original process in just three rounds of formulation development.
In addition, WuXi Biologics has characterized several mainstream host-cell lines with highly representative cell performance, according to Hang. Universal media formulations have been designed using the WuXi Biologics media development platform for each of these modular clones.
MilliporeSigma, meanwhile, uses a proprietary media development process based on multivariant data analysis of measured cell responses to media component concentration changes. As an example, Kimberly Schrag, R&D manager, MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany, observes that for development of HEK293 media for adeno-associated viral (AAV) production, cell growth, transfection efficiency, and AAV critical quality attributes are used for the analysis, which reveals which components impact each response (e.g., titer, growth, %-full capsids). This information is then used to optimize the medium to meet project goals. “Initially created for CHO [Chinese hamster ovary] media formulation development, this methodology has been adapted for other cell lines by incorporating media panels built for specific cell lines and biologics,” she comments.
Maintaining balance essential
One of the keys to successful media formulation development, says Jana Mahadevan, R&D Manager, the Life Science business of Merck KGaA, Darmstadt, Germany, is maintaining the balance of these many components, as having the right composition directly impacts performance. “Imbalances in glucose and amino acid levels have been observed to result in heightened production rates of lactate and ammonium, affecting cell-specific metabolic processes,” she notes. Specifically, elevated glucose concentrations have been associated with the inhibition of cellular respiration and oxidative phosphorylation, a phenomenon known as the “Crabtree effect,” leading to increased glycolysis rates and sustained lactate production. “Meticulously balancing all media formulation ingredients, which provide vital nutrients to support cell growth, metabolism, energy production, and ensure osmotic balance and pH regulation, fosters consistent and reproducible cell-culture conditions,” Mahadevan concludes.
The type of product is important
Generation of different types of biomolecules via cell culture involves different biological processes, which in turn dictate the needs of the cells involved. “Different bio-therapeutics, such as monoclonal antibodies (mAbs), other bioactive proteins, viral vectors [adeno-associated viral (AAV) and lentiviral (LV)], and exosomes have very different compositions and therefore may place very different nutritional requirements on the cells,” comments Josi Buerger, associate principal scientist involved in bioscience R&D at Lonza. For example, she notes that cells genetically engineered to produce mAbs and bioactive proteins may require higher amounts of amino acids in the culture media compared to cells bio-engineered to produce large amounts of exosomes.
There is a very important difference between processes producing a biopharmaceutical protein that gets produced by the cell (antibodies, enzymes, and various other proteins), viruses that get produced by the disruption of the cell (for vaccines or gene therapies), and if the cell itself is the product (cell therapy, but also non-pharma applications like cultured meat), Schrag agrees.
For instance, while media formulations for viral vector production are similar to those for recombinant protein and mAb production, the media for all products should be able to support rapid (low doubling time), high-density cell growth, and high-titer production, observes Mahadevan.
Consequently, media for AAV and LV vector production are formulated to be compatible with plasmid transfection for transient expression and thus have subtle differences from media for protein manufacturing, says Schrag. She goes on to note that even for AAV versus LV vectors, media must be formulated to meet different nutritional requirements because these two types of viral vectors differ significantly in composition and have different modes of infection (i.e., lytic vs non-lytic).
Not only do media formulations need to be adjusted to meet specific needs for growth and production, they must also be adjusted for the right product quality attributes (PQAs) to ensure both drug efficacy and safety, according to Zhou Hang, senior vice-president and head of bioprocess research and development at WuXi Biologics.
For most therapeutic proteins (mAbs, bispecific or multi-specific antibodies, antibody fragments, antibody-drug conjugates [ADCs], etc.), purity is always important due to its potential impact on drug safety, efficacy, and stability. Meanwhile, post-translational modification attributes, such as glycosylation profiles, and charge-variant distributions may be related to drug potency. “Each molecule may have different challenges in PQA tuning. For example, maintaining a higher purity for a bispecific or fusion protein is usually challenging, and thus more attention needs to be paid to media and process development. For biosimilars, PQA profiles must be finely tuned to demonstrate comparability to their originator molecules,” Hang explains.
Cell type is also a key determinant
Another reason different media formulations are required relates to the use of different types of cells. Different cell types may have very different basal nutritional needs that must be considered within the context of maximizing cell growth, product yield, stability, quality, and correct protein folding/conformation, remarks Alexis Bossie, director of media R&D in the Bioscience business at Lonza.
There are different nutritional needs for primary cells vs. immortalized cell lines, according to Bossie. “Primary cells from human or other animal sources require serum, growth factors, and any other support that in vivo systems deliver. In vitro culture of primary cells will require all these elements to be present in the culture medium,” she says. Immortalized cell lines, however, can propagate in the absence of high concentrations of serum, cytokines, and other growth factors in the culture media.
For biopharmaceutical proteins, Chinese hamster ovary (CHO) cells are well established, and media and processing technologies are mature, according to Schrag. Consequently, high-performing media is readily available off-the-shelf.
The variety of production hosts for vaccines and gene therapies has expanded and now includes HEK293 and other mammalian cells, as well as insect cell lines. “For these platforms, media and feeds exist but the technology is not as mature as for CHO cells,” says Schrag. For example, she notes that some of the commercially available media are not chemically defined or even require animal-derived components.
With cell therapies, it is important to maintain the quality parameters of the cells to ensure they retain their therapeutic potential, as the cell is the product, Schrag observes. Unlike for other biologics, several growth factors are needed, creating an interesting challenge for media development. “Only when these molecular biochemistry needs are met, the classical media development and optimization of nutrients can be initiated, and constant checks on the cells’ quality are needed,” she explains.
Variability within cell lines derived from the same lineage is an added challenge, according to Mahadevan. “The choice of specific cell line used for producing different biomolecules can impact the media requirements, as each cell line may have specific nutritional needs and sensitivities,” she explains. As is the case for different proteins, media formulations must often therefore be tailored for specific cell lines to ensure proper post-translational modification and folding to support production of stable products with therapeutic efficacy.
Process conditions also matter
Cell-culture processes can also be run under several different conditions, all of which impact the optimal composition of media formulations. Adherent vs. suspension and fed-batch vs. perfusion are just two examples.
Adherent media formulations tend to be leaner because the cell densities are lower compared to suspension cultures, and adherent media contains higher levels of components that enable attachment, like Ca/Mg, says Schrag. In addition, while there are some chemically defined, animal-component-free media for attached cultures, she points out that many attached processes (e.g., virus production for vaccines) still use a basal medium plus fetal bovine serum (FBS).
Suspension media are more concentrated than adherent media because suspension processes operate at higher cell densities. They also often contain components such as poloxamer 188 and anti-clumping agents, according to Mahadevan. Perfusion media are even more concentrated than conventional suspension media and contain much higher levels of components that are exhausted during growth and recombinant protein production, including amino acids and glucose.
In addition to these reasons for modifying media formulations, there are other drivers for adjustment to meet specific process requirements. Even for the same cell line, says Chen, the transfection medium, cell-passage medium, and production (fed-batch or perfusion) medium are quite different. Transfection media must be formulated considering how they can impact the transfection process. “Metal ions such as iron (III) are reported to inhibit transient transfection due to their effect on polyethyleneimine transfection reagents,” she observes. For cell-passage processes, cost-effective formulations that support desirable cell growth rates and ensure clone stability take precedence.
Nutrient-rich basal media, meanwhile, are typically preferred for both fed-batch and perfusion production processes, according to Chen, although for fed-batch processes, supplemental feeds are used to boost cell growth and enhance productivity, while for perfusion processes more attention is paid to maintaining cell viability. “Fed-batch platforms often require feeds to keep cell growth, titer, and product quality high, whereas perfusion/continuous processing platforms require media formulations that will keep the cells at a steady state for longer periods of time once the desired growth to a critical mass is achieved while maintaining high product quality,” Bossie agrees.
Do not forget cost considerations
In addition to the complexity created by diverse molecule formats, cell types, and process conditions, cost considerations are another big challenge to media development. “The goal is to develop the best media formulation that maintains high cell growth, product titer, stability, and quality (correct protein folding, assembly, and glycosylation) at the lowest possible cost,” states Low.
For instance, he notes there is a definite trend toward developing fully chemically-defined media formulations with components that are of non-animal origin and are devoid of expensive ingredients such as growth factors or hormones. He adds, however, that it can be difficult to get cells that exhibit robust growth and produce bioproducts at high titers while maintaining high product quality and stability to be less dependent on these expensive components.
Development of a small-scale and high-throughput model for media development is also crucial, Hang observes. “Such models need to be sufficiently representative while also cost-effective,” he emphasizes. As an example of a problematic approach for intensified perfusion processes, Hang points to the spin tubes widely used for media screening and optimization. “These tubes show some limitations in process control and mass transfer in certain culture modes, including perfusion, and therefore are not adequate small-scale models for media formulation development,” he says.
Multipronged strategies best
Successful media formulation requires a deep understanding of the specific biologic drug substance, cell type/line, and process involved, including metabolic demands and support for specific-post-translational modifications. “This knowledge is employed using statistical experimental design methods to systematically evaluate and optimize media components and conditions,” says Schrag.
Computational and experimental tools used to attain the desired cell growth, protein expression, and productivity include design-of-experiment (DoE) studies with systematic varying of media component concentrations, computational modeling (multi-factorial analysis) for optimizing nutrient utilization, machine-learning algorithms for predicting complex interactions between media components and cellular responses, high-throughput screening for identifying optimal conditions, development of media supplements and feed strategies, and other mathematical models for predicting optimal compositions, according to Mahadevan. “Employing a combination of these tools and approaches enables systematic optimization tailored to specific cell lines, processes, and desired outcomes,” she concludes.
For instance, starting from media screening, Chen explains that key components can be identified and optimized using various statistical analysis methods. In this manner, selected key subgroups or components in the formulation can be identified and their concentrations optimized based on the statistical data. “Due to the complexity of cell-culture-media-formulations, however,” Fei cautions, “it is not practical to use statistic approaches to simultaneously optimize all components, as the time and workload involved would be too onerous.”
Use a systematic approach
The best strategy for media formulation development for biologics manufacturing often involves a systematic and iterative approach tailored to the cell line’s specific needs, according to Buerger. Media development should also focus on ease-of-use, adds Ioanna Zormpa, associate principal scientist supporting biologics media R&D in Lonza’s Bioscience business.
There are four main steps in media formulation development, including basal media formulation, media optimization, performance evaluation, and scale up. The starting basal media formulation should include sufficient components for the specific application, as well as supplemental feeds if appropriate, with regulatory requirements for sourcing media components taken into consideration, Buerger says.
Optimization studies should be in concert with process development to facilitate ease of integration into downstream manufacturing processes. Evaluation of the selected optimized media formulation for growth rate, productivity, and product quality requires appropriate media analytical capabilities, some of which may require development of new analytical methods. Buerger therefore recommends developing an overall strategy adaptable to these differing needs. Scale-up studies ensure that performance of the cell line and media formulation remain consistent as processes move from the lab to the production plant.
The development of media formulations that are stable over time is also essential for maintaining consistent cell-culture performance and product quality. Therefore, conducting appropriate expiry studies to determine media shelf life is another key part of this process, according to Zormpa, as it helps to ensure effectiveness and safety over a specified period.
Throughout these steps, the production of easy-to-use media products should be kept in mind, as they can simplify the manufacturing process, reduce the potential for errors, and increase efficiency for end users, Zormpa adds. As an example, she points to single powder formulations, which simplify media preparation, lower the risk of component omission, and enable straightforward water reconstitution protocols, make them a practical and efficient choice for biologics manufacturing.
“These practices collectively contribute to a more streamlined, reliable, and effective media development process,” Zormpa concludes.
About the author
Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.
Article details
BioPharm International®
Vol. 37, No. 6
June 2024
Pages: 10-15
Citation
When referring to this article, please cite it as Challener, C.A. Addressing the Complexities of Media Formulation Development. BioPharm International 37 (6).
