Optimized Design Elements of Cell Culture Media for Industrial Applications

Developing a robust cell culture process necessitates the selection of an optimized cell culture media that not only contains all the nutrients required for cell growth and protein expression but also promotes cell health, viability, metabolism, product quality, manufacturability, and regulatory compliance. It is essential to optimize formulas based on supply chain and intellectual property considerations. The ideal cell culture media offers the best solution for a specific process or product while supporting high protein titers and specific productivity. For these reasons, the adequate optimization of mammalian cell culture media is an extremely complex and resource-intensive endeavor, as extensive experiments are required to fine-tune the concentrations of various media components. These components must align with the desired attributes of the final formulation and the performance of the media.

Industrial cell culture processes necessitate media that can support large-scale production and preparation. Consequently, both dry and liquid media formulations should be optimized for attributes that influence manufacturability, including component solubility, media filtration, sterilization, storage stability, production scalability, and feedstock consistency, in order to minimize batch variation. Media optimization efforts for fed-batch processes often concentrate on developing nutrient replacement solutions to enhance growth and productivity. However, for industrial applications, manufacturability attributes and engineering considerations, such as preparation time, shear protection, and foam reduction, are equally important.

In contrast to batch processes, where cells are cultivated in a single media until nutrients are depleted, fed-batch processes utilize at least two types of media: a basal media to support initial growth and one or more feed media. Feed media can be introduced at specified intervals or continuously later in the process to replenish depleted nutrients and promote growth and protein expression during the stationary phase. In its simplest form, the feed media may consist solely of glucose, the primary carbon source necessary for energy production in cell growth. More commonly, the feed media resembles a concentrated version of the basal media, with concentrations of specific components reaching up to 15 times that of the basal media. Feed media can be formulated based on data from batch culture experiments, which identify nutrient depletion and depletion rates. Nutrients are strategically divided between the basal media and the feed media to provide cells with optimal nutrient levels while avoiding solubility limits or inhibitory concentrations. Certain nutrients, such as glucose, can be added separately to allow for more precise control of concentrations in the media and to prevent the accumulation of unwanted metabolites, such as lactate.

In an industrial setting, different media may be utilized during the early stages of cell line development, such as transfection and single-cell cloning, compared to the basal media employed later for inoculum expansion and basal production. While using a distinct media initially may facilitate the adoption of commercial media optimized for sensitive processes like transfection or cloning, it may necessitate a longer adaptation period to transition to richer basal and feed media formulations. This adaptation process can prolong the project timeline and expose the cells to additional stress, potentially leading to the selection of unstable subpopulations. The ideal cell culture process would employ a single media throughout—from transfection and cloning to the production basal media—requiring minimal adaptation and featuring only minor differences between the cloning and basal media.

Most modern cell culture production processes utilize chemically defined, serum-free, and animal component-free media. This approach mitigates potential contamination risks from adventitious microbial or viral pathogens associated with serum or animal-derived components, while also addressing consistency and reproducibility challenges linked to inadequately defined media components, such as hydrolysates. Although chemically defined media have been employed for some time and numerous effective formulations are readily available, further optimization is often necessary to establish a robust, high-titer process tailored to a specific clone. Growth patterns, metabolite production, and nutrient consumption rates may vary between clones. Consequently, the objectives of optimization efforts include increasing titer, maintaining desired nutrient levels, optimizing trace elements, salts, and osmolarity, minimizing the accumulation of metabolic waste products, preventing apoptosis and viability issues, and avoiding cell aggregation or clumping.

Manufacturability is a critical aspect of optimizing culture media for industrial cell culture processes. Mammalian cell culture media are highly complex. In an industrial setting, this complexity poses challenges in ensuring a scalable and reproducible manufacturing process that delivers consistent performance, from small-scale process development to large-scale clinical and commercial production. Consequently, it has become common practice to prepare a ground and mixed dry powder formulation that includes all or most of the components. Grinding is conducted using various types of equipment, such as ball mills and pin mills. Premixed powders can be produced reproducibly in both small-scale and large-scale production, are easy to transport, have an acceptable shelf life, and generally require only reconstitution with water before use, followed by pH adjustment and filter sterilization. While media suppliers typically offer liquid culture media formulations or concentrates, these products are more expensive to ship in large quantities and have a shorter shelf life.

For dry powder formulations, it may be necessary to divide the entire formulation into multiple premixed powders due to the solubility limitations of certain ingredients. Generally, a culture media consisting of one to two premixed powders is preferable, as it simplifies the preparation process. Other ingredients can be added individually or as additives, depending on their solubility, stability, and sterilization requirements. The optimization of culture media formulation should take into account the availability of raw materials, solubility, permeability, scalability, room temperature stability, wettability of dry powder ingredients, and the consistency of the final ground and mixed dry powder or dissolved liquid culture media.

Certain media components present challenges regarding stability and solubility. Degradation or precipitation of these components can lead to fluctuations in nutrient availability and redox balance. To mitigate degradation of light-sensitive or heat-labile components, liquid media are typically stored in cold conditions and shielded from light. Some amino acids, such as glutamine, tyrosine, and cysteine, exhibit stability and/or solubility issues that hinder their incorporation at the required concentrations in culture media. Additionally, certain growth factors, including insulin, IGF, and multivitamins, may also be unstable in liquid culture environments. To address these limitations, the culture media can be optimized by administering unstable components as separate supplements—such as glutamine and growth factors—added just prior to use. These components can also be continuously supplied as part of the rehydration media to prevent depletion. Furthermore, amino acids can be delivered in the form of small peptides or dipeptides, which can enhance their concentrations in the culture media while improving stability and solubility.

Osmolarity is a critical factor in the design of cell culture media. All ions, including salts, amino acids, buffers, and fatty acids, contribute to osmolarity. High osmolarity (greater than 450 mOsm/kg) leads to reduced cell growth, titer, and cell viability, as well as increased cell size and doubling time. Below 450 mOsm/kg, the effects of increased osmolarity on cell growth are limited. In fact, during the production phase, higher osmolarity (up to approximately 400 mOsm/kg) has been shown to enhance titer. However, raising osmolarity to 450 mOsm/kg also results in increased production of ammonia and lactate, which can indirectly impact culture performance. Consequently, production and expansion media for recombinant protein expression in CHO cells are typically formulated to maintain relatively low osmolarity (250-350 mOsm/kg), while feed media are adjusted to higher osmolarity later in the production process.

Engineering challenges are a critical aspect of media design. Shear stress resulting from mechanical agitation, gas sparging, and foam formation can harm cells. This issue is particularly pronounced in chemically defined and serum-free media, likely due to their lower overall protein content. Foam presents several concerns in bioreactors. In addition to adversely affecting cell viability and productivity, severe foaming can lead to plugging, overpressurization, and fouling of exhaust filters, which can have catastrophic consequences for the process. To mitigate shear-related damage and control foaming, protective polymers and surfactants, such as Pluronic F-68 and defoamers, are often incorporated into chemically defined media. Optimizing the levels of Pluronic and defoamers in the media can enhance protection against shear and foaming while minimizing negative impacts on oxygen mass transfer and kLa values in the bioreactor environment.

Virus control during media preparation is another critical manufacturability issue. Inactivation methods commonly employed include ultraviolet (UV) irradiation, gamma irradiation, heat treatment, extreme pH adjustments, and exposure to solvents or detergents. For chemically defined media, ultrafiltration and short-term high-temperature treatments have been utilized to mitigate the risk of viral contamination. Although effective, short-term high-temperature treatment can lead to precipitation or the formation of insoluble particles in cell culture media. Research has demonstrated that adjusting the concentrations of calcium salts and inorganic phosphates in the basal media formulation can significantly reduce precipitation resulting from heat treatment. An alternative approach is to implement virus-retaining filtration, which has broad applicability and may require only minimal special considerations for chemically defined media.

At Duoning, we have developed four major culture media platforms: CHO cell culture media, HEK293 cell culture media, insect cell culture media, and vaccine-producing cell culture media. Leveraging our pioneering serum-free culture media technology, we enable the efficient and high-yield cultivation of various cell lines. Additionally, we offer customized culture media development services, drawing from our extensive library of basal and feed culture media. This process is enhanced by Design of Experiments (DoE) for nutrient consumption analysis, metabolic engineering design, parameter optimization for target product quality attributes, and ultimately, process scale-up validation. Our customized services can deliver tailored products suitable for customer applications within 60 to 120 days.

In addition, we offer a comprehensive bioreactor technology and product line, ranging from parallel benchtop bioreactors for Design of Experiments (DoE) to large-scale single-use and stainless-steel bioreactors. Among these, the DuoBioX® Explore stands out as a bioreactor system featuring a cutting-edge design, exceptional performance, comprehensive functionality, and user-friendly operation. Users can control up to eight glass tanks with a single computer, allowing them to operate independently or in parallel, significantly accelerating the process development timeline. The DuoBioX® Explore system is ideal for the development and research of cell culture processes within the biopharmaceutical industry.

Also, we offer DuoWave® wave-type bioreactors, which employ a unique mixing method that fosters a gentler growth environment for cells. The newly designed process control software is feature-rich and features a simple, intuitive interface that is easy for users to navigate. The working volume of DuoWave® wave-type bioreactors ranges from 500 ml to 25 L, making them suitable for the rapid expansion of various cell types, seed preparation, culture process development, and the efficient preparation of laboratory samples.

The DuoBioX® Pro single-use bioreactor features a bottom-stirring tank design that enables continuous scale-up of bioprocesses from 50 liters to 2000 liters. When combined with the 3D single-use cell culture bags developed and manufactured by Duoning, it offers significant advantages in biocompatibility. Its flexible manifold design accommodates a variety of complex upstream process requirements. Additionally, the unique integrated stirring and ventilation system not only minimizes shear stress but also ensures excellent mass transfer and mixing performance. The process consistency before and after scale-up is commendable, making it suitable for various stages, from biopharmaceutical research and development to GMP production.