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

Abstract 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.