원문정보
초록
영어
Sialylation modulates pharmacokinetic properties of recombinant therapeutic glycoproteins affecting their in VIVO half-life. N-glycan branching on glycoproteins provides the potential attachment sites for sialic acid. Here, we introduce a new approach for increasing the sialylation of recombinant human erythropoietin (rhEPO) produced m CHO cells by modulating poly-N-acetyllactosamine (poly-LacN Ac) biosynthesis. Initially, we found that repressiOn of the f33gnt2 increases tri- and tetra- N-glycan structures. We did not observe an mcrease in rhEPO sialylation, however, until the feedback inhibition by intracellular cytidinemonophosphate-N-acetylneuraminic acid ( CMP-N eu5Ac), which is a limiting factor for sialylation, was released. Thus, we found that a combined approach inhibiting poly-LacN Ac biosynthesis and releasing CMP-N eu5Ac 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.