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
Subject
Plant Science,Agronomy and Crop Science,Biotechnology
Cited by
2 articles.
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