Improvement of rCHO Cell Density and Heterologous Protein Production: Effect of Culture Conditions

Physico-chemical parameters have a great impact on mammalian cell growth. In this study, the effects of a number of amino acids, precursors, serum, glucose, osmolality and temperature on the recombinant Chinese hamster ovary (rCHO) cells producing the recombinant human coagulation factor VIII (rhFVIII) were investigated, so as to improve the rCHO cell density in the packed-bed bioreactor. The results showed that while the concentration of ammonium, as the main by-product, increased by more than 6mM, the rCHO cell density decreased significantly. On the other hand, adding certain amino acids and precursors mitigated the stress caused by ammonium and consequently increased cell density. Proline and glycine increased cell density up to approximately 28%in comparison with the negative control. Nucleic acids,as the main precursors, had a significant impact on rCHO cell growth, which increased by up to 20%. Higher amounts of glucose (3.5 g/l) supplied the carbon and energy requirements of the cells, inhibited by-products formation and sequentially increased heterologous protein production. The precursors allowed a 14%improvement in rhFVIII activity at high glucose concentrations, which was 1.5 fold greater than the negative control. Both hyperosmotic pressure and temperature downshifts had remarkable adverse effects on the rCHO cell density, being reduced to 2.7×106 cells/ml. The acquired suitable culture conditions were applied to the packed-bed bioreactor, and consequently an rCHO cell density of 18×108 cells/l was achieved during 120 h of cultivation. The results of this study indicated that the rCHO cells essentially need supplementary materials to deal with inappropriate culture conditions; especially in a serum-free medium.