Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture

Abstract

In an effort to reduce the accumulation of ammonia in culture medium, three different TCA cycle intermediates, (alpha-ketoglutarate (α-KG), citric acid and succinic acid) along with glutamic acid for a comparison, were examined as a substitute for glutamine with rCHO cell line producing a Fc-fusion glycoprotein. Among them, α-KG showed the best production performance. When cells were cultivated with 4mM α-KG, the final ammonia concentration did not exceed 3mM, which is less than one fourth of that with 4mM glutamine. The replacement of glutamine increased the lag phase and reduced cell growth. However, it increased the specific productivity by 2.7-fold, resulting in a 1.3-fold increase in the maximum product concentration. Furthermore, the sialic acid content of the Fc-fusion protein with 4mM α-KG was higher than that with 4mM glutamine in all cultures, most likely due to the lower ammonia concentration. The results of Western blotting and activity assays of intracellular α-2,3-sialyltransferase and extracellular sialidases are in good agreement with tests done to assess the sialic acid content of the Fc-fusion protein. Taken together, the data obtained here demonstrate that α-KG is a potential substitute for glutamine for improved glycoprotein production in rCHO cells.