Sialylation enhancement of CTLA4-Ig fusion protein in Chinese hamster ovary cells by dexamethasone.
The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half-life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4-Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases alpha2,3-sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU-486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5-L controlled bioreactors. Addition of 1 microM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N-linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4-Ig fusion protein expressed in CHO cells.
Jing
Ying
Y
Biologics Process and Product Development, Technical Operations, Bristol-Myers Squibb Company, East Syracuse, New York 13057, USA.
Qian
Yueming
Y
Li
Zheng Jian
ZJ
D000069594:Abatacept; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003907:Dexamethasone / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D018796:Immunoconjugates / Q000378:metabolism*; D015735:Mifepristone / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors; D012799:Sialyltransferases / Q000378:metabolism
D018796:Immunoconjugates; D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D000069594:Abatacept; D003907:Dexamethasone; D012799:Sialyltransferases; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid