Engineering N-glycosylation profile of pharmaceutical glycoproteins produced in Nicotiana tabacum BY-2 cells
(2023)
Files
Thesis_XavierHerman_withoutcover.pdf
Open Access - Adobe PDF
- 4.74 MB
Details
- Authors
- Supervisors
- Abstract
- Nicotiana tabacum BY-2 cells are among the most used plant cell lines for producing biopharmaceutical glycoproteins. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is poorly controlled. N-glycan composition is a concern for therapeutic or vaccine glycoproteins because it can alter their activity and lead to batch-to-batch variability. To tackle these issues, we are developing a set of glyco-engineered BY-2 cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Generation of glyco-engineered cell lines is a powerful tool to produce recombinant therapeutics with fine-tuned biological activities but also to broaden our understanding of the impact of N-glycans on their properties. We first generated a cell line producing glycoproteins devoid of complex N-glycans. It was obtained using CRISPR/Cas9-mediated edition of N-acetylglucosaminyltransferase I (GnTI) genes, whose activity is a prerequisite for the formation of all complex N-glycans, as well as α1,3-fucosyltransferase genes. Absence of complex N-glycans on secreted glycoproteins was confirmed by Western blotting and mass spectrometry. Next, we generated cell lines producing in vivo deglycosylated proteins. It was obtained by expressing the fungal endoglycosidase T (EndoT) in GnTI/FucT knockout cell lines. EndoT cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. Finally, an immunoglobulin G and the glycoprotein B of human cytomegalovirus were expressed in the GnTI knockout background expressing or not EndoT. Glycoproteomic mass spectrometry analysis of the secreted antibody and in vitro enzymatic mobility shift assay of glycoprotein B revealed that each cell type generates glycovariants with the anticipated N-glycan structures.
- Affiliations
Citations
Herman, X. (2023). Engineering N-glycosylation profile of pharmaceutical glycoproteins produced in Nicotiana tabacum BY-2 cells. https://hdl.handle.net/2078.5/237001