Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells.
Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.
Sung
Yun Hee
YH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-Dong, Yusong-Gu, Daejon 305-701, South Korea.
Song
Yeon Jung
YJ
Lim
Seung Wook
SW
Chung
Joo Young
JY
Lee
Gyun Min
GM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002087:Butyrates / Q000494:pharmacology*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012964:Sodium / Q000494:pharmacology*; D013926:Thrombopoietin / Q000096:biosynthesis* / Q000235:genetics*
D002087:Butyrates; D011994:Recombinant Proteins; D013926:Thrombopoietin; D012964:Sodium