کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
23214 | 43420 | 2014 | 7 صفحه PDF | دانلود رایگان |
• The TCA cycle intermediates replaced glutamine in recombinant CHO cell cultures.
• Among them, α-ketoglutarate (α-KG) had the best production performance.
• Replacement of glutamine by α-KG reduced the accumulation of ammonia significantly.
• Replacement of glutamine by α-KG better sustained the sialylation of Fc-fusion protein.
• α-KG is a potential substitute for glutamine in CHO cell cultures.
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 4 mM α-KG, the final ammonia concentration did not exceed 3 mM, which is less than one fourth of that with 4 mM 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 4 mM α-KG was higher than that with 4 mM 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.
Journal: Journal of Biotechnology - Volume 180, 20 June 2014, Pages 23–29