| The human tumor necrosis factor receptor linked to the Fc portion of human IgG1, named TNFR-Fc, has been used for the treatment of rheumatoid arthritis successfully. And the demand for TNFR-Fc for therapeutic applications was rising constantly. However, the limitation in the production capacity was unable to meet the demands for TNFR-Fc in the world. Thus, there was an urgent need to improve the TNFR-Fc production efficiency by mammalian cell culture. The depletion of essential nutrients, the accumulation of toxic byproducts and the low specific antibody productivity were considered as the principal problem limiting cell growth and the final product concentration, resulting in the low production capacity. Therefore, systematical studies on cell growth, metabolism and antibody synthesis, besides responses of cell physiological states to culture environment factors were the key step for process development and optimizaition.Firstly, the characteristics of GS-CHO cell growth, metabolism and antibody production were investigated in bioreactor batch and fed-batch cultures. The result showed that the concentration of glucose in the medium played an important role in the cell growth, metabolism, and antibody synthesis. It was found that underfeed a culture (glucose concentration< 5 mmol/L), resulting in reduced cell growth (<0.5 day-1), lactic acid accumulation (only 1.01 mmol/(109cells-day)) and antibody production (only 1.84 mg/(109cells-day)); overfeed a culture (glucose concentration> 15 mmol/L), leading to rapid lactic acid production (>9.99 mmol/(109cells-day)) and increased osmolality; and have a relatively wide range of glucose target values (5-15 mmol/L), resulting in sustaining the high cell growth rate (0.7 day-1), improving antibody production (5.67 mg/(109cells-day)), maintaining nutrient consumption rate constantly, extending culture longevity, and minimizing byproduct (lactic acid) production. In addition, analyses of the nutrients in the medium, such as amino acids and phosphate, revealed their different degrees of utilization by GS-CHO cells. Leucine, isoleucine, lysine, proline, threonine, valine and phosphate were consumed at high rates. And the mass of nutrients from the unbalanced basal medium could not match their requirements in the later fed-batch and perfusion cultivations, because of the high cell concentration and the long duration.Secondly, batch and biphasic fed-batch cultivations of GS-CHO cells were carried out at three different temperatures (30℃,33.5℃and 37℃) with the aim to investigate the effect of temperature on cell growth, metabolism, and antibody production. It was found that the cell growth indicated a dependency on the culture temperature:it attained a maximal level at 37℃and was completely arrested at 30℃. And the sub-physiological temperature cultivation of GS-CHO cells resulted in the increased percentage of cells in the G1/G0 phase, dry cell weight, and the mass of protein, carbohydrate, nucleic acid, and lipid per cell. Results also showed that the specific nutrient (glucose, glutamine, glutamic aicd, and phosphorous) consumption rates and the specific byproduct (latic acid, ammonia, and alanine) production rates were decreased at 30℃. The specific arginine and threonine consumption rates were increased by 2.7-fold and 0.9-fold than those at 37℃, respectively. Further metabolic flux analysis on GS-CHO cells at different temperature levels showed that:(1) the absolute flux of glucose flux flowed into glycolysis pathway and the need of glucose for biomass synthesis were reduced, only 1/5 and 1/20 compared with those of GS-CHO cells cultured at 37℃, but the proportion of glucose flowed into TCA cycle was increased by 125%; (2) an abrupt reduced in the flux of lactic acid formation pathway (>90%) was observed with the temperature down-shift, from 0.765 mmol C/(109cells·day) at 37℃to 0.053 mmol C/(109cells·day) at 30℃; (3) more than 200-400% amino acids were used in antibody synthesis at 30℃than those at 37℃, comparing with the reduced absolute flux of amino acids flowed into biomass synthesis. The results of energy metabolism analysis also indicated that GS-CHO cells were in the state of high energy production at 30℃. These results led us to the conclusion that the reduced temperature resulted in efficient cell metabolism, higher nutrients flux flowed into antibody production, and inhibited byproducts (latic acid, ammonia, and alanine) accumulation.In addition, specific TNFR-Fc productivity, qTNFR-Fc, increased as culture temperature decreased, and the maximum qTNFR-Fc of 17.3 mg/(109cells-day) was obtained at 30℃, which was 3-fold higher than that at 37℃. Real-time quantitative PCR analysis revealed that the relative TNFR-Fc mRNA content increased by lowering culture temperature like qTNFR-Fc, indicating that the increased transcription level of TNFR-Fc was responsible in part for the enhanced qTNFR-Fc at low culture temperature. However, our subsequent studies also showed that the specific antibody productivity was a function of maintained viable cell density in the sub-physiological temperature cultivation, named the cell density-dependent antibody production. Increasing maintained viable cell density to above 2.2×106 cells/ml led to a significant decrease in the qTNFR-Fc at 30℃. Taken together, lowering temperature could improve qTNFR-Fc, while the negative effect of increasing maintained viable cell density on qTNFR-Fc at 30℃could compromise the benefical effects of the increased integral of viable cell concentration with time and the increase in qTNFR-Fc by the temperature down-shift, resulting in even reduced final TNFR-Fc concentration. Moreover, we confirmed that the cell density-dependent antibody production was not attributed to the nutrients limitation, the byproducts (lactic acid and ammonia) inhibition, the elevated osmolality, and the variation of cell cycle distribution.Finally, a computational biphasic dynamic fed-batch/perfusion model was established based on the characteristics of GS-CHO cell growth, metabolism, and antibody production. The model that we describe in this work had three elements:(1) the well-formulated and balanced feed medium was designed by rational design to add nutrients at appropriate stoichiometric rates equal to their consumption rates; (2) the robust, metabolically responsive feeding strategy was based on the offline measurement of glucose with the aim of supplying sufficient nutrients to match their consumption, simultaneously minimizing the accumulation of byproducts (lactic acid and osmolality); (3) base on the results of the effects of temperature and maintained viable cell density on antibody production, enhancement of productivity of TNFR-Fc was achieved by the temperature-shift from 37℃to 30℃and controlled maintained viable cell density (2.2-3.4×106 cells/ml). The processes under the direction of this model not only provided balanced nutrients, but also minimized the accumulation of byproducts, resulting in increased specific TNFR-Fc productivity, extended culture duration, and enhanced final TNFR-Fc concentration. Compared with batch cultivations, the biphasic dynamic fed-batch cultivation generated the greater increase in specific TNFR-Fc productivity (17.37 mg/(109cells·day)) and the longer culture duration (22 day). The increase of specific TNFR-Fc productivity combined with the prolonged culture time resulted in a 11-fold increase of final TNFR-Fc concentration (574 mg/L),2.4-fold increase of TNFR-Fc yield, respectively. Similarly, enhancements of specific TNFR-Fc productivity, final TNFR-Fc concentration, and bioreactor volumetric TNFR-Fc productivity were also achieved in the biphasic dynamic perfusion cultivation.As a result of above research work, the economic and efficient biphasic dynamic fed-batch and biphasic perfusion cultivations for GS-CHO cells producing TNFR-Fc were developed. This generic and high-yielding biphasic dynamic fed-batch and biphasic perfusion cultivations would shorten development time, and ensure process stability, thereby facilitating the manufacture of TNFR-Fc by GS-CHO cells. On the other hand, the research methods and the control strategies used in the study, together with the understanding of growth, metabolism, and antibody production of GS-CHO cells, provided valuable reference to process development and optimization for other GS-engineered cell lines producing antibodies in industry.
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