Regulation And Mechanism Of Culture Temperature And Cell Density On CHO Cell Growth And TNFR-Fc Expression

The human tumor necrosis factor receptor-IgG Fc fusion (TNFR-Fc), has been successfully used for the treatment of rheumatoid arthritis, and therefore it has great economic value and broad market prospects. Optimization of large-scale animal cell culture for improving the quality and quantity of TNFR-Fc in the industrial production process is critical for implementing industrialization and enhancing competitiveness in domestic and international markets. Previous results showed that nutrients supplyment, metabolic by-products accumulation, environmental parameters control and cell physiological state affected GS-CHO cell growth and product expression. Therefore, in this study based on understanding of the properties of GS-CHO cell growth, metabolism and product biosynthesis, effects and mechanisms of environmental parameters on product expression was investigated in these respects including cell density, culture time and specific antibody productivity, and on the basis an economic and efficient cell fed-batch culture was developed for high cell density and high quality and quantity of TNFR-Fc.First of all, batch cultures at 37℃were performed to recognize the characteristics of CHO cell growth, metabolism and TNFR-Fc expression. It was found that in batch cultures the specific cell growth rate (μ) was 0.025 h-1, the maximum cell density was 3.19×106 cells/ml, and TNFR-Fc concentration and specific productivity rate (qTNFR-Fc) were only 56.83 mg/L and 3.94 pg/cell/d, respectively. Specific consumption or production rates of glucose and lactate were 3.24 mmol/109cells/d and 4.15 mmol/109cells/d, respectively. At the last phase of culture glucose was depleted and cell death appeared in large quantities. Besides glucose, amino acids were also consumed at different levels, and specific consumption rates of glutamine and glutamate were 0.388 mmol/109cells/d and 0.072 mmol/109cells/d, respectively. In the course of the study we found that culture temperature and cell growth density had a significant effect on TNFR-Fc production, and therefore batch and biphasic fed-batch cultures in the bioreactor were carried out to investigate effects of culture temperature and maintaind cell density in fed-batch cultures on CHO cell growth,qTNFR-Fc and sialic acid content of TNFR-Fc. As a result, lowering temperature could obviously suppress cell growth, extend the survival time of cells and increase qTNFR-Fc.Compared with those at 37℃, at 30℃theμ(0.002 h-1) was decreased by 92%, culture time was prolonged for 7 days, and especially qTNFR-Fc (15.65 pg/cell/d) was significantly increased by 3.3-fold. In addition, the results showed that culture temperature also affected the sialic acid content of TNFR-Fc. When cell viability was above 95%, sialic acid content of TNFR-Fc was achieved 77μg/mg at 37℃which was obviously higher than that at 30℃, but sialic acid content of TNFR-Fc was decreased to 43μg/mg with the decrease of cell viability at the late phase of culture, while the decreased cell viability did not affect the sialic acid content of TNFR-Fc at 30℃, which was maintained at a level of 60μg/mg. With the biphasic fed-batch cultures, we found that maintained cell density affected the qTNFR-Fc and siclic acid content of TNFR-Fc. At fed-batch culture of maintained cell density (6×106 cells/ml) the qTNFR-Fc was only 4.51 pg/cell/d, which was decreased by 68% in comparison to that at maintained cell density (2×106 cells/ml), resulting in that the final TNFR-Fc concentration of 296 mg/L was decreased by 33%. "High cell density and low TNFR-Fc production" was just the basic characteristic of CHO cell growth and TNFR-Fc expression. Althouth the decrease in cell viability did not affect the siclic acid content of TNFR-Fc at the maintained phase of fed-batch cultures, it was achieved 70μg/mg at maintained high cell density which was higher than that (60μg/mg) at maintained low cell density.Secondly, the mechanism of the effects of culture temperature on TNFR-Fc production was investigated in the respects of gene level, protein level and cell level. As a result, lowering culture temperature improved metabolic efficiency of glucose, decreased the production and accumulation of lactate and specific consumption of amino acids. At reduced temperature, CHO cell biomass, intracellular protein content and the proportion of cells in G1 phase were significantly increased, and those were beneficial for TNFR-Fc production. By analysis of TNFR-Fc transcription, it was found that lowering temperature increased TNFR-Fc mRNA level, and the alteration of TNFR-Fc mRNA level was correlation with the change in qTNFR-Fc, indicating that the increased qTNFR-Fc at reduced temperature was related to the increase in TNFR-Fc mRNA level. It was further found that the increased TNFR-Fc mRNA level at reduced temperature was mainly due to improving the mRNA stability of TNFR-Fc, and the half-life of TNFR-Fc mRNA was 5.55 h at 30℃, whereas that was only 3.69 h at 37℃. Based on previous studies, a dynamic model was developed for describing TNFR-Fc post-translational process, and the assembly rate and transport rate from ER to Golgi was accelerated by lowering temperature. In summary, optimization of cell metabolism, improvement of physiological state, increase in TNFR-Fc mRNA content, and acceleration of post-translational rates were responsible for the increased qTNFR-Fc at reduced temperature in combination. In addition, at 37℃the higher expression and activity ofα-2,3-sialyltransferase was beneficial for sialic acid synthesis, but with the culture progress sialidase was accumulated in the culture medium due to the cleavage of CHO cells, and at 37℃sialidase activity was achieved 7.54 nmol/h/ml at 168 h, which was 6.5-fold of that at 360 h at 30℃. As a result, the sialic acid of TNFR-Fc was significantly degraded at 37℃, but not at 30℃.Likewise, the mechanism of effects of maintained cell density at fed-batch cultures on TNFR-Fc production was investigated from gene level, protein level and cell level. As a result, increasing cell density did not change the specific consumption or productivity rates of major nutrients (glucose and amino acids) and by-products (lactate and ammonia). Physiological factors including cell biomass, total intracellular protein and cell cycle distribution were not affected by increasing cell density, however, the intracellular pH (pHi) of CHO cells was declined at high cell density (The pHi was 7.05 and 6.82 at low and high cell-density culture, respectively). Differences in pHi provided useful information for understanding the mechanism of effects of maintained cell density on TNFR-Fc production, and it was further found that the decrease in pHi mainly resulted from the increased CO2 partial pressure (PCO2), which was achieved 100 mmHg at maintained high cell density being far more than that (34 mmHg) at maintained low cell density. In addition, the qTNFR-Fc (5.85 pg/cell/d) was decreased by 59% in cultures of maintained low cell density (2×106 cells/ml) under high pCO2 (90 mmHg), indicating that the pCO2 and pHi was correlation with the qTNFR-Fc.By analysis of TNFR-Fc gene transcription and post-translational process, we found that the decrease in transcriptional rate of TNFR-Fc resulted in the reduced TNFR-Fc mRNA content of CHO cells at maintained high cell density, and the assembly rate and transport rate from ER to Golgi were also decreased by increasing cell density at maintained phase. Although maintained cell density did not affect the expression and activity ofα-2,3-sialyltransferase, its activity was improved by 1-fold by increasing maintained cell density, because of the increased pCO2 and the decreased pHi at this condition. Therefore, sialic acid content of TNFR-Fc obtained at maintained high cell density was higher than that at maintained low cell density. On the other hand, sialic acid content of TNFR-Fc was not changed at maintained phase due to the suppressedα-2,3-sialyltransferase activity at reduced temperature.Finally, a biphasic fed-batch culture at maintained high cell-density was established to optimize TNFR-Fc production by lowering culture temperature and falling upon pCO2, based on the characteristics of CHO cell growth and metabolism. Compared with the initial batch culture at 37℃, at the above biphasic fed-batch culture the culture time was prolonged for 144 h, the maximal viable cell density (10.96×106 cells/ml) and the qTNFR-Fc (15.44 pg/cell/d) was increased by 1.7-fold and 3.2-fold, respectively. As a result, the final TNFR-Fc concentration (1019 mg/L) was significantly increased by 14.6-fold. In addition, the sialic acid content of TNFR-Fc was 52μg/mg and meet product quality requirements.According to the above research work, the economic and efficient biphasic fed-batch culture was developed for laiding the foundation for industrial production of TNFR-Fc. In addition, the research method and the optimized strategy used in this study, together with the understanding of the effects of temperature and maintained cell density on TNFR-Fc production, provided valuable reference to process development and optimization for other mammalian cells producing antibodies or recombinant proteins in industry.