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GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins


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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Figure 1: GlycoDelete strategy and cell-line characterization.
Figure 2: GlycoDelete glycan characterization.
Figure 3: Functional and immunological characterization of GlycoDelete anti-CD20.


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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.).

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Authors and Affiliations



L.M.: took the GlycoDelete technology from concept to reality: cell engineering, cell characterization, GM-CSF analytics, and thermofluor assays and co-wrote the manuscript. F.S.: made key contributions in the full exploration of the GlycoDelete technology: GM-CSF analytics, anti-CD20 analytics, and pharmacokinetics experiment, and co-wrote the manuscript. G.E.: made key contributions to the exploration of GlycoDelete technology for modulating antibody properties: experimental design, and supervision and interpretation of anti-CD20 bioanalytics experiments, and manuscript correction. N.F.: bioactivity, immunogenicity ELISA and pharmacokinetics experiments, contributed to the manuscript. M.B.: gene expression experiments and data processing. A.D.S.: ADCC, ELISA FcγR binding and CD20-binding assays. S.D.: LC-MS/MS experiments and analysis. F.R.: anti-CD20 expression construct. (This plasmid is not explicitly mentioned in the text and is an unpublished resource that was made available to us through the work of F.R.) E.P.: assisted with immunogenicity ELISA experiments. E.H.: pharmacokinetics experiment. P.M. and G.M.: SPR and BLI experiments. L. Cons and L. Chatel: pharmacokinetics experiment. B.D.: supervision of LC-MS/MS experiments. N.C.: conceived the GlycoDelete technology, initiated the project, assisted in experimental design and interpretation, and co-wrote the manuscript.

Corresponding author

Correspondence to Nico Callewaert.

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Competing interests

L.M. and N.C. are inventors on patent applications covering the GlycoDelete technology (WO/2010/015722).

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Supplementary Figures 1–15, Supplementary Tables 1–8 and Supplementary Notes 1–6 (PDF 4124 kb)

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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).

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