Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture.
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.
Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
Tae Kwang
TK
Ha
Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: gyunminlee@kaist.ac.kr.
Gyun Min
GM
Lee
D000641:Ammonia / Q000032:analysis / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002952:Citric Acid Cycle / Q000187:drug effects / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry* / Q000494:pharmacology; D005973:Glutamine / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D003470:Culture Media; D007141:Immunoglobulin Fc Fragments; D007656:Ketoglutaric Acids; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D000641:Ammonia; D012799:Sialyltransferases; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
Alpha-ketoglutarate; Ammonia; CHO cells; Glutamine; Sialylation