In vivo deglycosylation of recombinant glycoproteins in tobacco BY‐2 cells

Author:

Herman Xavier1ORCID,Far Johann2,Peeters Marie1,Quinton Loïc2,Chaumont François1,Navarre Catherine1

Affiliation:

1. Louvain Institute of Biomolecular Science and Technology UCLouvain Louvain‐la‐Neuve Belgium

2. Mass Spectrometry Laboratory‐MolSys Research Unit ULiege Liège Belgium

Abstract

SummaryProduction 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.

Funder

European Regional Development Fund

Fonds De La Recherche Scientifique - FNRS

Horizon 2020 Framework Programme

Service Public de Wallonie

Publisher

Wiley

Subject

Plant Science,Agronomy and Crop Science,Biotechnology

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