In vitro glycan modification of pharmaceutical proteins produced in Nicotiana benthamiana

Pharmaceutical proteins like protein-based vaccines and antibodies are of great importance for an effective treatment of patients. The majority of pharmaceutical proteins are glycosylated and the efficacy of these proteins is in many cases affected by the glycoform attached to the protein. Recombinant protein production systems are able to produce glycosylated proteins, but these systems often produce a wide range of glycoforms of which many lead to a reduced efficacy or no efficacy at all. Nicotiana benthamiana has been proven to be a versatile expression system for glycosylated proteins, but has difficulties in generating an exact mimic of the natural eukaryotic glycoforms critical for a high efficacy for the treatment of patients. For example, it is challenging to generate a terminal β1,4-galactosylated N-glycan due to the trimming of plant native galactosidases. In vitro modification of these plant produced N-glycosylated proteins can lead to a homogeneous glycosylated protein with the desired glycan structure. In our research, we focus on the heterologous expression of Golgi-localized glycosyl transferase (GT) and glycoside hydrolase (GH) enzymes lacking their insoluble transmembrane region in Escherichia coli. These heterologous expressed glycozymes are used in in vitro reactions to modify N-glycans of recombinant expressed proteins produced in Nicotiana benthamiana, with the goal of generating a pharmaceutical protein with the desired glycan structure.