Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells.
Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.
Kochanowski
N
N
Laboratoire des Sciences du Génie Chimique, UPR CNRS 6811, ENSAIA-INPL-2, avenue de la Forêt de Haye, 54 505 Vandoeuvre-lès-Nancy Cedex, France.
Blanchard
F
F
Cacan
R
R
Chirat
F
F
Guedon
E
E
Marc
A
A
Goergen
J-L
JL
D000818:Animals; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D006031:Glycosylation; D007371:Interferon-gamma / Q000096:biosynthesis*; D011994:Recombinant Proteins; D044967:Serum / Q000378:metabolism*; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D014530:Uridine Diphosphate / Q000032:analysis / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000032:analysis / Q000378:metabolism*
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D014530:Uridine Diphosphate; D007371:Interferon-gamma; D012799:Sialyltransferases; D014544:Uridine Triphosphate; D005690:Galactose