Effects Of Glutamine And Asparagine On CHO Cell Growth,Metabolism And Antibody Production

With the increasing market demand for antibody drugs, how to efficiently establish production processes with high-yield-quality monoclonal antibodies become the core competencies of bio-pharmaceutical industry development. Glutamine and asparagine are two of evident nutrients that affect the efficiency of large-scale animal cell culture processes. Both are the most important providers of nitrogen unit and essential for cell growth and antibody production. However, both produce metabolic by-products ammonia in the metabolic processes. The accumulation of ammonia will seriously affect antibody expression and quality.Therefore, this paper will focus on glutamine and asparagine depletion and by-product ammonia accumulation that become critical technical problems in animal cell culture process, dhfr-CHO cells producing monoclonal antibody with broad market prospects will be studied. We will begin with improving the utilization of glutamine and asparagine based on the basic theories and methods of metabolic engineering and process optimization. Firstly, we rapidly and efficiently developed a serum-free medium for dhfr-CHO cells high-density-yield antibody expression through Simplex Lattice mixture design, then we optimized amino acids followed by Plackett-Burman design, Design of Expert analysis of variance showed that asparagine had a significant positive effect on antibody expression, while glutamine was not conducive to antibody expression, other amino acids had not significant influences.Secondly, we designed adding seperately, total supplement concentration and different proportion experiments for glutamine and asparagine in basal medium and feed medium and investigated the effect on cell growth, metabolism and antibody production. The results showed that:(1) Specific growth rate were only0.26and0.09day-1with either glutamine or asparagine depletion, it meant that both were essential for normal growth of dhfr-CHO cells. Only the concentration of glutamine and asparagine reached4mmol-L-1simultaneously could afford to cells normal growth.(2) Adding same concentration of glutamine and asparagine (take8mmol·L-1for example), cells growth rate and maximum cell densities for glutamine adding were1.13and1.26times than asparagine adding. It indicated that glutamine was greater demand in cell growth phase;(3) Asparagine made larger contribution for antibody synthesis. As the proportion of asparagine and glutamine increased from0.5to2, the final antibody concentration improved up to1.13times. Increased the proportion of asparagine and glutamine in feed mediums also obtained similar results;(4) Antibody production continued to improve when total concentrations of glutamine and asparagine increased, however, when the concentration reached13.5mmol-L"1, antibody production had no more significant increase.(5) The production and accumulation of by-product ammonia were determined by cumulative total concentration of glutamine and asparagine, its yield coefficient YAmm/(Gin+Asn) was changed between1.13±0.10mmol-mmol"1Finally, based on these above results, we investigated the metabolic characteristics of dhfr-CHO cells under different proportions (Asn/Gln as0.5and2), the results of metabolic flux analysis in high proportion (Asn/Gln as2) compared to low proportion (Asn/Gln as0.5) showed that:(1) Key metabolic flux nodes in TCA cycle increased which improved up to1.21;(2) Acetyl Co A producted from pyruvic acid, leucine and lysine aminotransferase pathway or deamination increased up to1.17;(3) Metabolic flux of by-product lactate and alanine decreased by13%and10%, indicating that nutrition were metabolized more effectively. It meant that high proportion of asparagine and glutamine process (Asn/Gln as2) had sufficient energy supplement、exuberant lipid metabolism and higher utilization of nutrients which contributed to high expression of monoclonal antibody.Through this research, we provided the basis and foundation for glutamine and asparagine supplement in the development and optimization of serum-free mediums. In addition, we learnt about the effect of glutamine and asparagine on cell growth, metabolism and antibody production. Therefore, it had an important reference for nutrition supplement in the design and development of serum-free mediums. Furthermore, it established the foundation on the road of monoclonal antibodies industrialization.