Lee, Chung-GeunOh, Myung JinPark, Seung-YeolAn, Hyun JooKim, Jung Hoe2018-06-272018Lee, Chung-Geun, Myung Jin Oh, Seung-Yeol Park, Hyun Joo An, and Jung Hoe Kim. 2018. “Inhibition of poly-LacNAc biosynthesis with release of CMP-Neu5Ac feedback inhibition increases the sialylation of recombinant EPO produced in CHO cells.” Scientific Reports 8 (1): 7273. doi:10.1038/s41598-018-25580-9. http://dx.doi.org/10.1038/s41598-018-25580-9.http://nrs.harvard.edu/urn-3:HUL.InstRepos:37160090Sialylation of recombinant therapeutic glycoproteins modulates their pharmacokinetic properties by affecting their in vivo half-life. N-glycan branching on glycoproteins increases the number of potential attachment sites for sialic acid. Here, we introduce a new approach for increasing the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells by modulating poly-N-acetyllactosamine (poly-LacNAc) biosynthesis. We did not observe an increase in rhEPO sialylation, however, until the feedback inhibition by intracellular cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), which is a limiting factor for sialylation, was released. Thus, we found that a combined approach inhibiting poly-LacNAc biosynthesis and releasing CMP-Neu5Ac feedback inhibition produces the most significant increase in rhEPO sialylation in metabolically engineered CHO cells. Furthermore, a detailed analysis of the resulting N-glycan structures using LC/MS revealed increased tri- and tetra- sialylated N-glycan structures accompanied by a reduction of di-sialylated N-glycan structures. These results validate our new approach for glycosylation engineering, and we expect this approach will be useful in future efforts to enhance the efficacy of other therapeutic glycoproteins.en-USJournal Article2018-06-2710.1038/s41598-018-25580-9