Production of O-glycan modified biopharmaceuticals in plants

Many recombinant biopharmaceuticals that are approved for human use are glycoproteins. Glycosylation of recombinant therapeutic proteins affects their structural and functional properties. Control of glycosylation is therefore a critical step to enhance the efficacy of therapeutics and eliminate unwanted side effects resulting from non-authentic glycosylation. Current production technologies for recombinant glycoproteins are mainly based on expression in mammalian cells, which do not allow the efficient manipulation of glycosylation and thus contain heterogeneous glycans. This project aims to provide a plant-based expression platform for the production of recombinant glycoproteins with a customized glycosylation profile. Recent developments in modification of the N-glycosylation pathway have shown that green plants are a versatile expression system for the generation of therapeutic proteins with a defined human-type N-glycosylation. We have shown that engineered Nicotiana benthamiana plants can very efficiently produce recombinant biopharmaceuticals like human erythropoietin with homogenous N-glycans carrying sialic acid. In this project, we aim to achieve the generation of distinct O-glycan structures on recombinant proteins using our established plant-based production platform. To this end, we will express all proteins required for the initiation and extension of human mucin-type O- glycosylation in N. benthamiana plants together with different O-glycosylated therapeutically relevant proteins. All generated glycan structures will be validated and tested for their influence on the biological activity of the recombinant biopharmaceuticals. Moreover, we will combine our technologies for N- and O-glycan engineering to develop a production system for recombinant glycoproteins with controlled human-type N- and O-glycosylation, which should advance the development of novel protein-based therapeutics.

We have generated a plant-based expression platform (Nicotiana benthamiana plants) for the expression of recombinant glycoproteins with humanized N- and O-glycans. Proper N- and O-glycosylation of recombinant proteins is important for their biological function and their therapeutic efficacy. Plants are attractive hosts for engineering of mammalian-type O- glycosylation steps, as they contain no endogenous glycosyltransferases that perform this type of glycosylation. Here, we produced different types of human type O-glycans by transient expression of the required mammalian biosynthesis pathway in N. benthamiana. O- glycosylation sites from recombinant proteins like human erythropoietin or immunoglobulin A1 were efficiently modified with defined glycan structures. We successfully combined N- and O-glycan modification strategies to generate a production platform equipped with a fully functional human-type N- and O-glycosylation machinery. Importantly, the glycan engineering steps were all well-tolerated without any adverse effects on plant growth or productivity. Collectively, our data demonstrate that N. benthamiana plants are amenable to extensive engineering of the O-glycosylation pathway which greatly expands the potential of plants as novel expression hosts for the production of recombinant glycoproteins with customized glycans.