Effects of Growth Factors and Culture Medium Additives on the Metabolism and Productivity of IgA-Producing CHO Cells

Research Background

Most existing research on mammalian cell expression systems and therapeutic antibodies has focused on IgG. In recent years, interest in the research and development of therapeutic IgA antibodies has continued to grow. Recombinant monoclonal antibody biologics are primarily produced using CHO cells, which are also the mainstream host cells in the pharmaceutical industry. IgA is a key molecule in the activation of mucosal immunity. Compared to IgG, IgA antibodies demonstrate superior efficacy in killing tumor cells: IgA can bind to the FcaRI receptor on the surface of myeloid cells and eliminate tumor cells through the effector functions of its Fc domain. IgA is considered a highly promising antibody for tumor immunotherapy, which is also a major advantage of IgA-based therapeutics. This study selected cell lines expressing IgA1 and IgA2, which belong to different subtypes; therefore, optimizing the recombinant IgA production process is of practical significance. Investigating cellular processes, nutrients, and metabolic patterns can guide the development of culture media and improve the production levels of recombinant proteins.

Adherent cell culture media often contain fetal bovine serum, which naturally contains growth factors and various active components; however, serum-free media must be used for suspension cells in the production of therapeutic proteins. Growth factors are important signaling proteins in the body. Supplementing culture media with growth factors helps maintain high cell viability, prolong cell survival, and regulate growth rates; some growth factors can also activate proliferation, induce differentiation, and regulate cellular signaling pathways and physiological functions. Fibroblast growth factor (FGF) promotes the proliferation, differentiation, and metabolism of epithelial cells and participates in cell regeneration. FGF-2 exhibits different effects on the survival cycles of stable cell lines expressing IgG and IgA. Epidermal growth factor (EGF) is a typical mitogen capable of promoting the proliferation of various cell types, including fibroblasts and epithelial cells. Nerve growth factor (NGF) promotes the growth of human cells, maintains cell health, and provides stable, reproducible conditions for in vitro mammalian cell experiments.

Glucose and glutamine are the most important carbon and nitrogen nutrient substrates in cell culture. Glutamine metabolism affects cellular productivity, and rapid cell proliferation depends on vigorous glucose metabolism; glutamine is involved in cellular energy supply, growth, redox balance, and pH regulation. Certain amino acids also participate in cellular metabolism and can serve as alternative carbon and nitrogen sources, either synergizing with or replacing glutamine to supply cells. The addition of glutamine substitutes can alter metabolic indicators such as glucose consumption, lactate production, and ammonium ion accumulation, thereby improving CHO cell growth and increasing recombinant protein yield. Glutamine serves as a secondary carbon source for the TCA cycle (Figure 1); however, during the late exponential growth phase, cells preferentially uptake lactate and aspartate and no longer prioritize the utilization of glutamine. Some studies indicate that under specific conditions, cells can switch metabolic modes to utilize lactate for more efficient energy metabolism.

Figure 1: The Tricarboxylic Acid (TCA) Cycle and Glycolysis Metabolic Pathways

Glucose is the primary carbon source for the cellular TCA cycle; insufficient glucose consumption by cells can induce protein glycosylation and glyoxylation, thereby compromising the quality of therapeutic antibodies. Ammonium ions and lactate generated through cellular metabolism are toxic byproducts that can alter the pH and osmolarity of the culture system, inhibit cell growth, reduce protein yield, and disrupt antibody glycosylation.

CHO cell metabolism is phase-specific: during the logarithmic growth phase, cells exhibit high-glucose glycolysis and produce large amounts of lactate; once glucose is depleted, cells undergo a metabolic shift from lactate production to lactate consumption, switching to a highly energy-efficient metabolic mode that enhances recombinant protein expression levels. By replacing glutamine and downregulating lactate dehydrogenase activity, the accumulation of harmful metabolites can be reduced, cell growth conditions improved, and antibody expression uniformity enhanced.

Glycosylation is a core quality attribute of antibodies. The IgA1 and IgA2m1 cell lines exhibit distinct differences in structure, glycosylation characteristics, and growth performance, which significantly impact cell metabolism and antibody production efficiency. This study aims to investigate the effects of epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF)—either individually or in combination with glutamine substitutes—on optimizing CHO cell growth and IgA antibody production capacity by regulating cellular metabolism.

Results and Discussion

This study investigated the regulatory effects of epidermal growth factor (EGF), basic fibroblast growth factor (FGF-b) , and nerve growth factor (NGF) on the growth, metabolism, and productivity of the stable IgA1 and IgA2m1 cell lines (Figure 2). All experiments used a uniform concentration of 12.5 ng/mL of the three recombinant human growth factors to compare their effects on the live cell density and viability of the two IgA subtype cell lines.

The results showed that, compared with blank DMEM/F12 basal medium, the addition of growth factors increased the culture density of both IgA cell lines by an average of 1.4–2.9-fold. Among them, the proliferative effect of hNGF was significantly stronger than that of hEGF and FGF“b.” By day 10 of culture, the cell densities of IgA1 and IgA2 in the hNGF group had increased to 3.2-fold and 2.8-fold, respectively, compared to the control group, reaching a maximum of 9.0 × 10⁵ cells/mL and 1.0 × 10⁶ cells/mL, with a more pronounced effect on the IgA2 cell line. Both hNGF and hEGF maintained high cell viability, with cell viability remaining above 80% on day 10, effectively extending the culture period; in contrast, FGF-β exhibited a weaker growth-promoting effect, with cell density and viability on day 10 nearly halved compared to the hNGF group, and this disadvantage was more pronounced for the IgA1 cell line.

This study further examined the synergistic effects of individual growth factors in combination with zinc sulfate. The results showed significant differences in the effects of various combinations on the two IgA cell lines. The combination of hEGF and zinc sulfate had no significant effect on the IgA1 cell line but effectively increased the cell density of the IgA2 cell line. The combination of hNGF and zinc sulfate simultaneously promoted the proliferation of both cell lines, with the IgA2 cell density increasing 1.5-fold, while the IgA1 cell density showed no significant change. During the early stages of culture, IgA1 cells grew more slowly but maintained viability for a longer period, whereas IgA2 cells exhibited higher viability in the later stages of culture. This synergistic effect may stem from the specific regulation of cell proliferation and survival by hNGF and zinc sulfate, and is also related to structural differences between the two IgA subtypes. Zinc ions can maintain cellular expression capacity by stabilizing mRNA structure and promoting stable protein binding; in this study, both the maximum live cell density and the final cell viability were significantly increased.

Figure 2: Effects of growth factor addition on cell density (A, B) and cell viability (C, D)

1. Effects of growth factors, alone and in combination with additives, on glucose consumption

High glucose levels are a means of increasing recombinant protein yield, but they inhibit cell growth, disrupt nutrient metabolism, and compromise protein quality. In this study, static culture in six-well plates was used to measure residual glucose levels in IgA1 and IgA2m1 cells under different treatment conditions. During the late exponential growth phase (5–10 days), the glucose consumption rates of the two cell lines were similar when only growth factors were added; the combination of growth factors with zinc sulfate reduced glucose consumption by approximately 45%. Among these, the combination of zinc sulfate with hNGF or hEGF yielded the best results, with residual glucose levels in the medium significantly higher than in the groups treated with growth factors alone; the IgA2 cell line exhibited the highest residual glucose level in the hNGF + zinc sulfate group, reaching 287 µM.

The results indicate that hEGF and hNGF, when combined with zinc sulfate, can reduce glucose consumption while promoting cell proliferation and maintaining high viability, thereby altering the levels of metabolites in the culture system.

Figure 3: Effect of Growth Factors on Glucose Consumption in Stable Cell Lines Expressing Recombinant Monoclonal Antibodies of the IgA1 and IgA2m1 Subtypes

2. Effect of Metabolic Changes Induced by the Combination of Growth Factors and Zinc Sulfate on the Production Capacity of Different IgA Subtypes

In this study, DMEM/F12 was used as the basal medium. Multiple treatment groups were established, including single-factor additions and combinations of growth factors with zinc sulfate, with a group receiving no additional components serving as the control. IgA1 and IgA2m1 cells were cultured statically in six-well plates. Samples were collected on days 5 and 10 to measure LDH activity and ammonium ion concentration in the supernatant, and the relationship between lactate and ammonium production and antibody yield was analyzed.

The FGFβ group exhibited the highest accumulation of lactate and ammonium ions. Treatment with hNGF or hEGF reduced these metabolic byproducts, and a synergistic effect was observed when these factors were combined with zinc sulfate; the ammonium ion level in the IgA2m1 group was significantly lower than that in the IgA1 group. Compared to FGFβ, hNGF and hEGF downregulated LDH activity, with a greater improvement observed in IgA2m1 cells.

When used alone, hNGF and hEGF increased IgA2m1 antibody production by 1.5 times compared to the FGFb group; when the growth factors were combined with zinc sulfate, the yield increased to twice that of the FGF b group. The combination of hNGF/hEGF with zinc sulfate can reshape cellular metabolism, reduce lactate and ammonium production, and increase antibody yield; this synergistic effect stems from the growth factors’ regulation of cell growth and survival, while zinc stabilizes mRNA, further enhancing product expression.

Figure 4: Effects of FGF-b, hNGF, and hEGF on metabolite levels and productivity in stable IgA1 and IgA2 cell lines on day 10 of static culture

3 Comparison of the effects of combined growth factors and succinate on the metabolism of cells of different IgA subtypes

Glutamine dominates early cellular metabolism, while succinate participates in the TCA cycle when glutamine is deficient. In this study, 4 mM succinate was used to replace glutamine, and experiments were conducted in combination with different concentrations of hNGF and hEGF. The results showed that succinic acid increased lactate levels but promoted antibody synthesis; when combined with hNGF or hEGF, IgA2m1 production increased by 25% on day 10, and production doubled when accompanied by reductions in lactate and ammonium ions.

hNGF and hEGF exhibited dose-dependent effects on antibody expression; when growth factors were increased to 25 ng/mL, lactate production during IgA2m1 production decreased. The highest IgA2m1 yield—up to 0.487 mg/mL—was achieved under conditions combining hNGF and succinate; the same treatment had a minimal effect on IgA1, with its yield being only 1/1.5 to 1/2 that of IgA2m1.

The combination of growth factor and succinic acid optimizes IgA metabolism in CHO cells, with IgA2m1 showing more significant benefits. Conditions characterized by low metabolite levels, such as low lactate, are conducive to scale-up, high-density cultivation, and feed addition process development, and can provide a basis for the development of IgA cell lines, particularly IgA2m1.

Figure 5: Effects of Cell Culture Medium Additive Formulations on Cell Metabolism and IgA1 and IgA2m1 Antibody Yield

Conclusions

This study indicates that replacing glutamine with succinic acid can reduce lactate levels, improve the maintenance status of IgA2 cells, and increase their productivity. Epidermal growth factor and nerve growth factor can accelerate the growth of IgA1 and IgA2 cells, boosting antibody production while reducing lactate production; The addition of these two types of growth factors promotes the uptake of glucose, glutamine, and succinic acid, potentially leading to metabolic reprogramming in which cells switch to consuming lactate. Succinic acid is less effective than glutamine at generating pyruvate via the tricarboxylic acid cycle, which also contributes to the reduction in lactate levels.

Basic fibroblast growth factor exacerbates the accumulation of lactate and ammonium ions, which is detrimental to the synthesis of products in both types of IgA cells. Replacing glutamine with succinate downregulates LDH activity, reduces lactate levels, and increases IgA antibody production. In summary, a culture medium formulation that combines growth factors with succinate as a substitute for glutamine can improve CHO cell metabolism, reduce harmful metabolites, and holds practical value for the industrial production of recombinant IgA antibodies.

As a supplier of biopharmaceutical equipment, we provide bioreactors tailored for CHO cell culture. These bioreactors can precisely match the optimized culture medium process described above, stably regulate the culture environment, and fully leverage the process advantages of substituting glutamine with succinate and combining growth factors. This approach reduces the accumulation of harmful metabolites, facilitates the transition of recombinant IgA antibody production from pilot-scale to industrial-scale operations, and enhances the batch-to-batch consistency of cell culture and antibody production capacity.