Process Development And Optimization For The DHFR-CHO Cells In Fed-batch Cultures

Biological drugs, including antibodies, recombinant proteins, nucleic acids, peptides and other drugs belong to pharmaceutical industry with rapid development and promising prospects. The global sales of biological drug in2010reachedS140billion, accounting for16%of the global pharmaceutical market, half of which came from the contribution of antibody industry. With the increasing demand for antibody drugs, as well as more and more new products into clinical research, animal cell culture technology, as the core technology of modern bio-pharmaceutical industry, has achieved unprecedented development in the past decade. How to quickly and efficiently establish production processes with high-density cell growth and high-yield-quality product expression, is not only an important task for countries to develop antibodies, recombinant protein drugs, and other bio-products for pharmaceutical industry, but also has become core competency for innovation and development of international bio-pharmaceutical industry. Therefore, it is of great significance for developing China’s bio-pharmaceutical industry and improving its international competitiveness to study the key technologies of process development and optimization for animal cell culture and further establish efficient production process for antibodies, recombinant protein drugs and other biological products. In this paper, the relationship between nutrient metabolism and byproducts accumulation with cell growth and production will be studied for DHFR-CHO cells producing anti-CD20monoclonal antibody. And the material and energy metabolism with lactate metabolism at the core as well as their important roles in cell growth and production will be studied deeply through the design and development of medium composition and feeding strategy. On this basis, the cost-effective fed-batch culture will be designed and developed with a view of effective regulation of lactate metabolism, improvement of utilization of nutrient, and reduction of lactate (as metabolic by-product), so as to improve the productivity and economy of animal cell culture process, and lay the foundation for industrial production of anti-CD20monoclonal antibody.Firstly, the characteristics of cell growth, metabolism and monoclonal antibody production were investigated in batch culture. It was found in batch culture that the maximal viable cell concentration reached3.6×106cells/ml, and the final antibody concentration was95mg/L. Glucose was not exhausted in the end of batch culture with initial concentration of40 mmol/L, but above58%consumed glucose was converted into lactate in batch culture and the peak concentration of lactate reached54mmol/L. It was observed that glutamine was depleted in the end of cell growth stage, and the peak concentration of ammonium reached3.3mmol/L. In addition, other amino acids like aspartate and cysteine were also exhausted in the end of cell growth stage. The deficiency of these nutrients might have adverse influence on cell growth and productivity. Moreover, the unsatisfied supply of amino acids in the current medium might also cause the unbalanced concentrations during the culture process, with various consumption and production rates of different amino acids, which further inhibited cell growth and productivity.On the basis of atch culture results, through the study of glucose supply approach, it was found that when g(?)cose was fed and maintained at mediate concentration, cell growth was significantly impr(?)ed and the utilization efficiency of glucose was increased remarkably with reduced acc(?) nulation concentration of lactate. Through Plackett-Burman and central composite design method, the formulation of amino acids in the initial medium was then optimized to obtain serum-and protein-free medium PFIA. When verified in batch culture carried out in bioreactor, cell growth and productivity were both improved with maximal viable cell concentration of4.6×106cells/ml and the final antibody concentration of180mg/L, better than two commercial medium, demonstrating that PFIA could be used as the initial medium for fed-batch culture. Furthermore, by investigating the different influence of components in feeding medium on cell growth and productivity, the relative concentrations of amino acids and vitamins in FM-0was determined based on the top-down approach.With PFIA as the initial medium and with feeding of glucose and FM-0as feeding medium in corresponding feeding strategy, the fed-batch culture was carried out in bioreactor for validation and evaluation. It was found in fed-batch culture that maximal viable cell concentration reached6.5×106cells/ml, and the final antibody concentration reached270mg/L. However, it was also observed that the short cell maintenance and low specific production rate of antibody were the major deficiency of the current fed batch culture process. Further investigation of nutrient metabolism indicated that the supply of nutrient was not balanced enough with great accumulation of most amino acids during the later cultivation. Meanwhile, lactate was found significantly produced and accumulated, and the osmotic pressure was also remarkably increased.To further explore the deficiencies of current fed-batch culture process and provide the direction for further optimization, a series of separate experiments were carried out to investigate the influence of metabolic by-products i.e. lactate and ammonia, and the osmotic pressure on cell growth and productivity. It was found that when the accumulation concentration of lactate reached above35mmol/L during the cultivation, cell growth and productivity was greatly inhibited. The results of fed-batch culture in bioreactor and relative experimental verification indicated that it was essential and necessary to study and understand material and energy metabolism with lactate metabolism at the core in the fed-batch culture process and finally realize the regulation of lactate metabolism for further development and optimization. Therefore, the production of lactate could be reduced for better culture environment, and cell growth, maintenance and production of monoclonal antibody could be further improved.In order to provide reasonable basis for regulation of lactate metabolism in the follow-up fed-batch culture process, an in-depth study of material and energy metabolism with lactate metabolism at the core, as well as their relationship with cell growth and productivity was carried out in this paper. Four conditions were firstly designed according to the concentration of glucose and category of carbon sources to investigate the metabolic characteristics of lactate and pyruvate. Then cell growth, productivity, glycolysis, amino acids and energy metabolism was furtuer explored under two distinct metabolic characteristics of lactate. It was found during the later cultivation, accompanied by the metabolic shift of lactate metabolism from production to consumption, the efficiency of cellular material and energy metabolism were both improved and cell productivity as well as cell maintenance was kept stable. Moreover, the relationship among glucose, pyruvate and lactate was also investigated via addition of glucose or pyruvate at the beginning or midway of cultivation with metabolic shift of lactate metabolism from production to consumption. It was found that the concentration of pyruvate played critical role in the metabolic shift of lactate metabolism. The depletion of glucose caused limited supply of pyruvate and finally led to the consumption of lactate from production. Therefore, inadequate pyruvate from the dried-up supply of glucose was the prerequisites for metabolic shift of lactate from production to consumption.Finally, based on the understanding of lactate metabolism of DHFR-CHO cells, a combined glucose and galactose feeding strategy was designed and optimized through adjusting the ratio of glucose and galactose and feeding time. Meanwhile, the formulation of amino acids and vitamins in the current feeding medium FM-0was also optimized to obtain feeding medium FM-Opt suitable for the combined glucose and galactose feeding strategy. With feeding of FM-Opt and with the combined glucose and galactose feeding strategy, a fed-batch culture process was established with regulation of lactate metabolism at the core. Due to the effective regulation of lactate metabolism as well as the balanced supply of nutrients, cell maintenance was remarkably prolonged with IVCC value of64(109cells·day/L), increased by170%compared with batch culture, and increased by45%compared with the initially-developed fed-batch culture; antibody productivity was improved with specific production rate of antibody of14.8mg/(109cells·day), increased by97% compared with batch culture, and increased by143%compared with the initially-developed fed-batch culture. The final concentration of anti-CD20monoclonal antibody reached at944mg/L, greatly increased by4.2-fold compared with batch culture, and increased by2.5-fold compared with the initially-developed fed-batch culture.Through this research, a fed-batch culture process was established with regulation of lactate metabolism at the core, laying the foundation for eventual industrial production of anti-CD20monoclonal antibody. Based on the study of metabolic characteristic of lactate during the animal cell culture process, the importance of lactate metabolism in process has been pointed out, which enriched and deepened people’s understanding of lactate as metabolic by-product. Moreover, the development approaches for the fed-batch culture process in this paper also provide important reference for other production processes of antibody drugs.