Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 May;32(5):485-9.
doi: 10.1038/nbt.2885. Epub 2014 Apr 20.

GlycoDelete Engineering of Mammalian Cells Simplifies N-glycosylation of Recombinant Proteins

Affiliations
Free PMC article

GlycoDelete Engineering of Mammalian Cells Simplifies N-glycosylation of Recombinant Proteins

Leander Meuris et al. Nat Biotechnol. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing financial interests statement

The authors declare not to have competing financial interests beyond their inventorship (L.M. and N.C.) on patent applications covering the GlycoDelete technology (WO/2010/015722).

Figures

Fig. 1
Fig. 1. The GlycoDelete strategy & cell line characterization.
(a) In mammalian cells with intact glycosylation machinery (a; upper part), oligomannose glycans entering the Golgi are trimmed by class I mannosidases (ManI) to Man5GlcNAc2. They are committed to hybrid or complex type N-glycans after modification by N-acetylglucosaminyltransferase 1 (GnTI) with a β-1,2-N-acetylglucosamine on the α-1,3-mannose. Several glycosylhydrolases and glycosyltransferases further model complex type N-glycans through many biosynthetic steps (black arrows; enzymes not shown), leading to substantial heterogeneity. In GlycoDelete cells, (a; lower part) GnTI is deleted and endoT is targeted to the Golgi, resulting in hydrolysis of oligomannose N-glycans by endoT. The resulting single GlcNAc stumps can be elongated by Golgi-resident galactosyl- and sialyltransferases. (b) Concanavalin A selection directly selects for the desired GlycoDelete glycan phenotype, as deglycosylation of cell surface glycoproteins by endoT results in the absence of ConA ligands, rendering cells resistant to ConA. The parental 293SGnTI-/- cells die when treated with ConA. (c) Growth curve for 293SGnTI-/- and 293SGlycoDelete cells in 6-well culture plates, counted every 24 hours. Error bars are standard deviations (S.D.), n=3. Numerical data for this graph are in Supplementary Table 2. (d) Scatterplot of average (n=3) gene expression values of 7344 genes for 293SGlycoDelete versus 293SGnTI-/- cells. The correlation coefficient is 0.9865. Significantly differentially expressed genes (P<0.01) are labeled with their names. Microarray signal intensities lower than 8 on the represented scale were too low for reliable detection.
Fig. 2
Fig. 2. GlycoDelete glycan characterization.
(a) SDS-PAGE of 293S, 293SGnTI-/- and 293SGlycoDelete GM-CSF samples. Each sample was treated with PNGaseF, sialidase or both enzymes, analysed on an SDS-PAGE gel and stained with coomassie brilliant blue. The non-cropped can be found in Supplementary Fig. 15. (b) MALDI-TOF-MS spectra of GM-CSF samples. Peaks are labeled with their m/z values. The spectrum of the 293SGnTI-/- GM-CSF reveals the presence of Man5GlcNAc2 and fucosylated Man5GlcNAc2 on the glycopeptide containing N37 (top spectrum; left and right glycans, respectively). These glycoforms are absent in GlycoDelete GM-CSF (2nd spectrum). New peaks at m/z values corresponding to HexNAc, Hex-HexNAc and Sia-Hex-HexNAc modified glycopeptides are detected. Spectra of exoglycosidase-digested GlycoDelete GM-CSF N-glycans with α-2,3-sialidase or both a broad spectrum sialidase and β-1,4-galactosidase are shown. These spectra show that N-glycans on GlycoDelete GM-CSF N37 are Neu5Ac-α-2,3-Gal-β-1,4-GlcNAc and Gal-β-1,4-GlcNAc. (c) Thermofluor assay of 293S, 293SGlycoDelete and E. coli produced GM-CSF. We observed similar average (n=3) melting curves for all GM-CSF glycoforms (Tm is approximately 60°C). (d) Bioactivity of 293S and 293SGlycoDelete produced GM-CSF as measured in a TF1 erythroleukemia cell proliferation assay (n=3). E. coli produced GM-CSF serves as a non-glycosylated control sample. The error bars are S.D. Numerical data for this graph are in Supplementary Table 3. (e) ELISA analysis of anti-glycan antibody titers in GlycoDelete GM-CSF immunized rabbit serum. Removal of sialic acid and galactose monosaccharides from the GlycoDelete glycan does not reduce serum antibody recognition. Numerical data for this graph are in Supplementary Table 4.
Fig. 3
Fig. 3. Functional and immunological characterization of GlycoDelete anti-CD20.
(a) Anti-CD20 SDS-PAGE. Gl. Del: 293SGlycoDelete anti-CD20. HC: antibody heavy chain. LC: antibody light chain. The non-cropped gel is in Supplementary Fig. 15. (b) LC-MS/MS in SRM mode of GlycoDelete anti-CD20 glycopeptides. Peak labels state LC elution times. Peak color-codes: trisaccharide- (red), disaccharide- (blue) or monosaccharide-modified (yellow) glycopeptides. Exoglycosidase digests with sialidase and β-1,4-galactosidase illustrate identical glycans as observed for GM-CSF. (c) CD20-binding by anti-CD20 as assessed by flow cytometry. Numerical data for this graph are in Supplementary Table 5. (d) Average melting curves (n=3) as determined in a thermofluor assay for untreated or PNGase-digested 293S and 293SGlycoDelete anti-CD20. (e) Competition ELISA (top three graphs) and ADCC assay to assess effector function of the anti-CD20 Fc. Concentration series of 293S and 293SGlycoDelete anti-CD20 were compared in their competition with a coated anti-Fc antibody. The error bars indicate standard error of the mean (S.E.M.) with n=3. The 4th graph shows specific lysis in an ADCC assay. The error bars indicate S.D. and n=3. Numerical data for this graph are in Supplementary Table 6. (f) Anti-glycan antibody ELISA analysis of 293SGlycoDelete anti-CD20 immunized rabbit serum. Anti-CD20 recognition by antibodies in the serum of rabbits immunized with GlycoDelete GM-CSF was analysed. Anti-CD20 samples were treated with sialidase, sialidase and galactosidase or no enzyme. Error bars show S.D. and n=3. Numerical data for this graph are in Supplementary Table 7. (g) Anti-CD20 pharmacokinetics in mice. Blood concentrations of anti-CD20 were measured over time after intravenous injection of 293S or 293SGlycoDelete anti-CD20. Error bars are S.E.M. and n=4. Numerical data for this graph are in Supplementary Table 8.

Comment in

  • Simply better glycoproteins.
    Lepenies B, Seeberger PH. Lepenies B, et al. Nat Biotechnol. 2014 May;32(5):443-5. doi: 10.1038/nbt.2893. Nat Biotechnol. 2014. PMID: 24811516 No abstract available.

Similar articles

See all similar articles

Cited by 34 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback