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Lactate Metabolism Mechanism and Regulation Strategies in CHO Cells

Chinese Hamster Ovary (CHO) cells are the predominant host cells for producing therapeutic proteins such as monoclonal antibodies in biotechnology. The metabolism and regulation of lactate are critical to enhancing the yield and quality of target products during CHO cell culture. Lactate accumulation not only impairs cell proliferation and protein expression but also acidifies the culture environment, further compromising cell growth and metabolism. Therefore, elucidating lactate metabolism mechanisms and implementing targeted regulation strategies is of great practical significance.

Mechanism of Lactate Formation

Most cells generate energy for vital activities primarily through aerobic glycolysis rather than mitochondrial oxidative phosphorylation. During the exponential growth phase of cell culture, approximately 60%–80% of glucose and 16%–25% of glutamine are converted into lactate. As cells transition from the rapid growth phase to the stationary phase, the metabolic pattern shifts from lactate production to lactate consumption.

Two key proteins mediate the lactate consumption pathway:

Monocarboxylate Transporters (MCTs): These proteins facilitate the co-transport of lactate and hydrogen ions across the cell membrane.

Lactate Dehydrogenase (LDH): This enzyme catalyzes the conversion of lactate to pyruvate, which subsequently enters the tricarboxylic acid (TCA) cycle.

The metabolic switch of lactate is modulated by the intracellular and extracellular lactate concentration gradient as well as the transmembrane proton gradient.

Three typical lactate metabolic phenotypes are observed in industrial cell culture: lactate-consuming, lactate-accumulating and lactate-recycling phenotypes. Empirically, a lactate concentration below 20 mM is acceptable, while levels exceeding 40 mM exert adverse effects on cell growth and specific productivity. Lactate accumulation inhibits cell proliferation and causes a drop in culture pH. To compensate for acidification, alkaline solutions are routinely added, which in turn elevates osmotic pressure and ultimately deteriorates the culture microenvironment.

Accordingly, the lactate-consuming phenotype (lactate metabolic shift) is regarded as the optimal metabolic characteristic. Timely lactate consumption stabilizes culture pH, reduces the demand for alkaline supplements, and maintains osmotic pressure at a low level. This allows additional nutrients to be supplemented to meet cellular metabolic requirements.

As a characteristic metabolite and alternative nutrient for cells, lactate serves as a key indicator of metabolic abnormalities. Comprehensive assessment combining pH, osmotic pressure, partial pressure of carbon dioxide (pCO₂), glucose consumption and protein expression levels is required to diagnose abnormal cellular metabolism, rather than relying solely on lactate profiles.

Major Strategies for Lactate Metabolism Regulation

Lactate metabolism is a sophisticated biological process closely associated with cell lines, culture media, cultivation processes and bioreactor scales. The following sections elaborate on strategies to mitigate lactate production in mammalian cell cultures from the perspectives of culture medium optimization, cultivation condition control and cell line engineering.

1. Culture Medium Optimization for Improved Lactate Metabolism

Alternative carbon and nitrogen sources

Replace glucose and glutamine with slowly metabolized carbon and nitrogen substrates to alleviate lactate accumulation. Common alternatives to glucose include fructose, maltose and galactose; glutamine can be substituted with glutamate, pyruvate and TCA cycle intermediates. Studies have demonstrated that galactose replacement markedly reduces lactate formation, yet it lowers the specific growth rate and peak viable cell density. To address this issue, a biphasic feeding strategy with alternating glucose and galactose utilization is applied in fed-batch cultures, which effectively cuts down lactate accumulation and optimizes cellular metabolism.

Maintenance of low glucose concentration

Maintaining glucose at a low level reduces glycolytic flux and subsequent lactate generation. Meanwhile, potential impacts of glucose limitation on protein glycosylation must be taken into account. HyClone offers glucose-free Cell Boost 7A feed medium to facilitate glucose concentration control in fed-batch cultures.

Lactate Supplementation and Adaptation (LSA) technology

As reported by Freund et al., customized basal media and feeds are designed to drive metabolic equilibrium toward the lactate-consuming phenotype. In this approach, CHO cells are adapted to media supplemented with sodium lactate, which supplies energy for cells and stabilizes culture pH. When applied to fed-batch cultures, this technology reduces lactate concentration by 8-fold and achieves a viable cell density up to 35 million cells per milliliter.

Copper ion supplementation

Copper ion supplementation in copper-deficient culture systems effectively alleviates abnormal lactate accumulation. However, excessive copper triggers increases in basic variants, Man5 glycans and protein aggregates, so the dosage must be strictly controlled.

Commercial culture medium screening

HyClone provides prototype media PSL A01 and PSL A02, which are formulated to relieve lactate accumulation and enhance product titer.

2. Cultivation Process Optimization for Improved Lactate Metabolism

High pH-based DO-controlled Glucose feeding (HIPDOG)

This strategy regulates glucose feeding rate according to pH fluctuations. When lactate metabolism shifts from accumulation to consumption, culture pH rises. At this point, slow glucose feeding induces moderate lactate production and a subsequent pH decline. Glucose feeding is then halted to lower extracellular glucose concentration, prompting lactate consumption and pH recovery. Fed-batch cultures adopting the HIPDOG strategy achieve significant improvements in product yield and process stability via efficient lactate control.

Reduced culture temperature and pH

In fed-batch cultures, the specific lactate production rate shows a linear correlation with culture pH and temperature settings. Lowering temperature decelerates overall cellular metabolism, thereby decreasing glucose and glutamine consumption as well as lactate formation. A slight reduction in pH alters the activity of glycolytic enzymes and membrane potential, leading to reduced glucose uptake and lactate synthesis.

Nevertheless, suboptimal temperature and pH negatively affect cell proliferation, protein productivity and glycosylation profiles. Hence, biphasic regulation of temperature and pH requires targeted optimization: pH is adjusted within a narrow range, while the timing and magnitude of temperature reduction are fine-tuned, on the premise of preserving product yield and quality.

Enhanced mass transfer

In large-scale bioreactors, impaired lactate metabolic transition is often attributed to excessive pCO₂ accumulation, which compromises mitochondrial function. Optimizing mass transfer mitigates CO₂ buildup and facilitates the shift from lactate production to consumption. Meanwhile, shear stress caused by excessive agitation and aeration should be avoided during mass transfer improvement.

3. Cell Line Replacement and Genetic Engineering to Remodel Lactate Metabolic Pathways

Cell line selection

Lactate metabolic performance must be evaluated during monoclonal cell line screening to mitigate potential risks in process scale-up.

Inhibit glucose consumption and lactate production

Downregulate lactate dehydrogenase (LDH) gene expression, or upregulate the anti-apoptotic gene BCL-2A to enhance mitochondrial activity, so as to reduce glucose uptake and lactate synthesis rates.

Promote pyruvate flux into the TCA cycle

Upregulate the activity of pyruvate carboxylase (PYC) and pyruvate dehydrogenase (PDH), inhibit pyruvate dehydrogenase kinase (PDK), or overexpress malate dehydrogenase (MDH) to redirect pyruvate toward the TCA cycle.

Optimize alternative sugar metabolism

Overexpress the fructose transporter GLUT5 to accelerate cellular fructose utilization, enabling fructose to serve as a viable substitute for glucose.

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Lactate Metabolism Mechanism and Regulation Strategies in CHO Cells

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