Biologically active, magnICON®-expressed EPO-Fc from stably transformed Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures

• Published in: Journal of biotechnology Vol. 160; no. 3-4; pp. 242 - 250
• Main Authors: Nagels, Bieke, Van Damme, Els J.M., Callewaert, Nico, Zabeau, Lennart, Tavernier, Jan, Delanghe, Joris R., Boets, Annemie, Castilho, Alexandra, Weterings, Koen
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 31-08-2012
• Subjects: biopharmaceuticals; biotechnology; chemiluminescence; Erythropoietin; Erythropoietin - biosynthesis; Erythropoietin - genetics; immunoassays; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - metabolism; In vitro activity assay; Molecular farming; N-glycosylation; Nicotiana - physiology; Nicotiana benthamiana; patients; plant proteins; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Polysaccharides - genetics; Polysaccharides - metabolism; Protein Engineering - methods; Transformation, Genetic - genetics; XylT; FucT; Molecular farming; Nicotiana benthamiana; Erythropoietin; LR; rhEPO; LC–ESI MS; In vitro activity assay; GnT; N-glycosylation; EPOR
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2012.03.003

► EPO-Fc was transiently expressed in Nicotiana benthamiana plants with humanized N-glycosylation. ► Plant-expressed EPO was shown to be active in vitro. ► N-glycan analysis of EPO-Fc revealed the presence of multi-antennary N-glycans.
In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized.
Here, we report the transient magnICON® expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants.