[en] Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.
Herman, Xavier ; Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium
Far, Johann ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie analytique inorganique
Peeters, Marie; Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
Chaumont, François; Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium
Navarre, Catherine; Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium
Language :
English
Title :
In vivo deglycosylation of recombinant glycoproteins in tobacco BY-2 cells.
Publication date :
02 June 2023
Journal title :
Plant Biotechnology Journal
ISSN :
1467-7644
eISSN :
1467-7652
Publisher :
Blackwell, Oxford, Gb
Peer reviewed :
Peer Reviewed verified by ORBi
Name of the research project :
GlycoCell
Funders :
SPW DGO6 - Service Public de Wallonie. Economie, Emploi, Recherche
Funding number :
WALInnov-Glycocell-1810010
Funding text :
FRIA FNRS (X. Herman). Supported by the European Union's Horizon 2020 Research and Innovation Program under grant agreement No. 731077 (EU FT-ICR MS project, INFRAIA-02-2017) and from the European Union and Wallonia program FEDER BIOMED HUB Technology Support (No. 2.2.1/996) for the funding of the SolariX XR 9.4T. Software development is also supported by the European Union's Horizon 2020 program (EURLipids Interreg Eurogio Meuse-Rhine). The UPLC and the Q-Exactive were financed by the FEDER and Wallonia Region.
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