For the production of cell-based biological products, the fundamental requirement is a culture media that supports cell growth, which is closely linked to process performance and safety. The primary environmental conditions necessary for cell survival and proliferation include an appropriate temperature, optimal pH, suitable osmotic pressure, essential chemical nutrients, and the effective removal or dilution of toxic waste. This article will briefly discuss the culture media-related issues that should be avoided or mitigated in cell culture to achieve the desired product yield and quality by implementing a robust cell culture process.

Extracellular Matrix: Problems encountered during the preparation and storage of cell culture media can lead to cell death. To better understand the critical functions of culture media in cell culture and the effects of chemical imbalances and fluctuations, it is essential to consider the factors that induce stress in cell cultures, ultimately resulting in cell death. The three primary morphological characteristics of cell death include necrotic cells in passive cell death and apoptotic or autophagic cell morphology in programmed cell death. Limited nutrient and oxygen delivery, the accumulation of metabolic byproducts, and elevated osmotic pressure levels are the main contributors to cell death. For cell lines utilized in industrial applications, the predominant pathway of cell death is apoptosis; therefore, cell line engineering methods can be employed to reduce susceptibility to cell death. Generally, eukaryotic cells isolated from tissues require an extracellular matrix as a critical factor for cell survival. The extracellular matrix is a complex three-dimensional macromolecular network in which all cells of tissues and organs reside. If this connection is abruptly disrupted, the cell will undergo programmed cell death.

A significant advantage of Chinese Hamster Ovary (CHO) cells in bioproduction is their ability to adapt to suspension culture conditions. Various culture protocols have been developed to acclimate cell lines to serum-free media, while most anchorage-dependent cell lines tend to detach when serum is removed. The adaptation to specific chemical conditions renders these cells highly sensitive to standard cell culture practices; therefore, the selection of chemically defined media components must be approached with great care to prevent cell death during the acclimation process. This adaptation can lead to substantial changes in cell phenotype and the underlying gene transcription. Chemically defined media must effectively maintain these cellular properties as the cells successfully transition to suspension and serum-free culture conditions.

pH: In bioproduction, the antibody productivity per unit cell of CHO cells is influenced not only by the specific clone but also by the culture conditions. Within the pH range of 6.8-7.8, the consumption rates of glucose and glutamine, as well as the production rates of lactate and ammonium, increase with rising pH levels. Additionally, pH affects amino acid consumption and production rates. CHO cells can phenotypically adapt to prolonged exposure to low pH conditions (pH 6.6), exhibiting a slower doubling rate, a 0.12 unit increase in intracellular pH, and modified thermotolerance. In bioreactors, pH is maintained within a defined range through the addition of a base when the pH falls below the lower control limit. However, pH gradients that may arise in large-scale cultures can impact cell physiology and overall process performance, even with brief exposure of a small portion of the bioreactor contents to alkaline pH. Consequently, a critical function of chemically defined media is to provide adequate buffering capacity at the target pH to minimize pH stress in cell culture. Typically, chemically defined media for CHO cell culture are optimized to maintain pH levels between 6.6 and 7.4. The primary factors influencing the pH of the culture media in bioprocessing include gas diffusion during preparation and storage, as well as the addition of alkali.

Osmotic pressure: Proper regulation of cell volume is essential for cell survival and function. Osmotic stress occurs when changes in osmotic pressure cause water to move across the membrane, driven by osmosis. Hypoosmotic stress leads to cell swelling and triggers various cellular responses, including the disassembly of the actin cytoskeleton. In contrast, hyperosmotic pressure results in cell shrinkage and causes molecular crowding within the cell. Both hypo- and hyperosmotic stress can initiate programmed cell death, exhibiting features of apoptosis, or can induce autophagy. An effective chemically defined media must maintain an osmotic pressure that is sufficiently high to support cell proliferation during the growth phase while remaining low enough to allow for an increase in osmotic pressure during the fed-batch process. For CHO cell culture, the osmotic pressure of the chemically defined media is typically adjusted to a range of 260 to 320 mOsm/kg.

Shear/hydrodynamic stress: Increased gas flow rates and agitation in the sparger induce shear stress in the cell culture, which can lead to cell death characterized by a necrotic phenotype. At energy dissipation rates exceeding 10^6 ~ 10^8 W/m3 caused by bubble collapse, CHO cells undergo necrosis, resulting in the release of lactate dehydrogenase. Although CHO cell growth and productivity have demonstrated resistance to aqueous hydrodynamic stress levels up to 6.4 x 10^6 W/m3, cell physiology is still altered. Another related issue is foam generation in the bioreactor, which adversely affects nutrient supply and interrupts gas transfer from the culture broth to the reactor headspace. While aeration rate and impeller speed are media-independent parameters in cell culture processes, they can still significantly influence the suppression of shear stress and foam formation by incorporating appropriate media supplements, such as Pluronic F-68.

Oxidative stress: Studies on CHO cell metabolism have demonstrated that these cells metabolize a significant amount of glucose through the pentose phosphate pathway, leading to an increased production of oxaloacetate. This altered cellular metabolism is regarded as an adaptation to oxidative stress in cell culture. Experiments measuring gas exchange rates in CHO cell cultures within stirred tank bioreactors have revealed that an increase in the dissolved oxygen (DO) set point further induces oxidative stress. Intracellularly, antioxidant mechanisms influence mitochondrial function and metabolism, ultimately resulting in reduced recombinant protein expression. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the primary compounds responsible for oxidative stress. These reactive species can originate from either endogenous or exogenous sources. It is important to note that free radicals are byproducts of normal cellular metabolism and can have beneficial effects at low levels of ROS/RNS. However, at elevated concentrations, an excess of reactive species can lead to cellular stress, surpassing the protective capacity of both non-enzymatic and enzymatic antioxidants. ROS and RNS consist of oxygen and nitrogen species, including both free radicals and non-free radicals, which can readily generate free radicals. Free radicals possess at least one unpaired electron and can exist independently, allowing them to attack various biomolecules and alter the normal oxidation state essential for biological function. When oxidative stress exceeds the threshold that cells can tolerate, apoptosis is triggered. This process is typically initiated by receptor and caspase activation, mitochondrial dysfunction, DNA damage, or structural and functional abnormalities in specific proteins. Furthermore, oxidative stress and apoptosis are considered closely related physiological states, as ROS and cellular redox changes can play a role in the signaling pathways associated with apoptosis. The positive effects of antioxidants in cell culture must be interpreted with caution when compared to physiological systems. Often, the positive effects observed in cell culture do not accurately reflect in vivo conditions because the culture media typically lacks antioxidants. The sources of oxidative stress in cell culture are varied; for instance, cells in culture are frequently exposed to higher oxygen concentrations than those found in physiological conditions. More specific contributors to oxidative stress include elevated glucose levels and insufficient antioxidants in the culture media, such as tocopherol, ascorbic acid, and vitamin E. Additionally, providing cells with adequate selenium can be challenging. If selenium is not supplied in sufficient amounts, cells may experience oxidative stress due to the dysfunction of the selenium-dependent antioxidant system. One of the primary reasons for the pro-oxidative properties of culture media is the high concentration of transition metals, particularly iron and copper. While these metals are essential for cellular function, if they are not complexed with the physiological iron transporter transferrin, they can catalyze various reactions that lead to the production of reactive oxygen species (ROS). All instances of oxidative stress highlight the necessity of developing culture media with a balanced redox state, which can protect cells from damage caused by high dissolved oxygen (DO) levels or excessive formation of ROS /RNS during metabolic processes.

Nutrient consumption and waste accumulation: The primary function of the culture media is to provide essential nutrients to the cells. All compounds required by cultured cells that cannot be synthesized from precursors must be supplied by the culture media in a bioavailable form, achieved through an appropriate rehydration strategy. The composition of the culture media significantly influences cell metabolism and gene expression. A key consideration is the accumulation of lactate and ammonium during the process, which can limit performance. Although these compounds are not directly cytotoxic, they can inhibit cell growth by lowering intracellular pH and disrupting metabolic processes.

Modern cell culture necessitates the development of strategies to prevent the accumulation of waste products. Some studies utilize lactate rehydration to facilitate a metabolic shift towards lactate utilization, while others identify copper as essential for this metabolic transition. The correct composition of chemically defined media and a balanced formulation are fundamental to the robustness of cell culture processes. Any deviations caused by degradation, formulation errors, or issues during preparation and storage can be critical. For instance, a deficiency of asparagine, the primary source of intracellular nitrogen, results in growth arrest and a significant increase in pyruvate uptake. Similarly, a lack of serine negatively impacts cell growth and may trigger its de novo synthesis. In conditions where glucose or glutamine levels are intentionally kept low to minimize the accumulation of lactate or ammonia, hybridoma cells have been observed to induce apoptosis through mitochondrial and death receptor pathways. Comparable responses occur in CHO cells when they experience nutrient deprivation during extended culture periods, leading to the accumulation of waste products. Research has indicated that rehydration strategies can mitigate apoptosis and autophagy at the conclusion of cell culture, as further nutrient deprivation may precipitate cell death. Apoptosis in CHO cells is not solely attributed to the absence of organic nutrients; it can also result from deficiencies in calcium (Ca2+) and magnesium (Mg2+).

Culture media influences recombinant protein quality: Culture media plays a fundamental and significant role in cell culture performance, influencing cell growth, lifespan, and productivity. Equally important is the effect of the culture media on the recombinant protein—the final product itself. mAb heavy and light chain expression cassettes are typically cloned with an N-terminal signal sequence that directs the nascent protein through the endoplasmic reticulum via a co-translational translocation pathway. Once the transmembrane transport across the endoplasmic reticulum membrane is complete, the protein can enter the secretory pathway through vesicular transport and be excreted from the cell into the extracellular matrix. Therefore, the cell culture media, where nutrients are gradually depleted and metabolic waste products accumulate over time, represents the first extracellular environment that recombinant proteins encounter. However, the quality of a product is not solely determined by the chemical properties of the extracellular matrix; intracellular protein expression is also significantly influenced by the conditions of the culture and the culture media. It is important to note that changes in the concentration of culture compounds that impact product quality may arise not only from the addition or removal of components during the development of the culture media but, more critically and unpredictably, from fluctuations in impurity concentrations, the adsorption of compounds (e.g., filter media), precipitation, or other chemical reactions that render certain compounds or elements unavailable for bioavailability.

Duoning Media C series consists of animal-free, protein-free, and chemically defined basal media that are suitable for batch, fed-batch, and perfusion cultures of CHO cells in the development and production of therapeutic protein products. This media series is compatible with the DN feed series and does not contain L-glutamine. The recommended concentration of L-glutamine is between 4 mM to 6 mM.

Product features:

?   Chemically defined (CD) culture media, animal and protein-free

?   Support requirements for high-density suspension culture and high yield

?   Universal culture media for various CHO cell types (CHOK1, CHOS, DG44, CHOZN)

?   Compared to other products on the market, the antibody titer can be increased by 100%, with the highest yield reaching 12g/L

?   Comprehensive technical support

?   Simple powder reconstitution (three-step method) & GMP production

?   Independent intellectual property