Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy—which we call GlycoDelete—that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.
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We thank G. Pontini, Y. Poitevin, L. Bernasconi, D. Schrag and S. Raimondi (NovImmune) for their help with anti-CD20 generation and characterization; S. Savvides (Ghent University) for providing the 293SGnTI(−) clone expressing the Flt3 receptor extracellular domain; E. Van Damme (Ghent University) for providing ConA; and E. Dirksen and K. Nooijen (Merck) for the LC-MS analysis of anti-CD20. L.M. and M.B. are supported by predoctoral fellowships, and N.F. by a postdoctoral fellowship, of the Fund for Scientific Research-Flanders (FWO). F.S. and S.D. are supported by predoctoral fellowships of agentschap voor Innovatie door Wetenschap en Technologie (IWT) Flanders (Strategic Basic Research fellowship nos. 101456 and 111252). This research was supported by VIB, Ghent University Industrial Research Fund (UGent-IOF) Advanced Grant no. 041 (N.C.), FWO research project grant no. G.0.541.08.N.10 (N.C.) and Hercules Foundation grant no. AUGE019 (B.D.).
L.M. and N.C. are inventors on patent applications covering the GlycoDelete technology (WO/2010/015722).
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Meuris, L., Santens, F., Elson, G. et al. GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins. Nat Biotechnol 32, 485–489 (2014). https://doi.org/10.1038/nbt.2885
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