Co- and posttranslational engineering of erythropoietin

Erythropoietin is the most important drug to treat different types of anaemia. Glycosylation accounts for 40% of its molecular weight and is very important for the protein stability. Until now, glycoproteins are produced in eukaryotic cells resulting in a heterogeneous glycosylation pattern. This structural heterogeneity of N-linked oligosaccharides encumbers the correlation of glycan structure with glycoprotein function. Herein, a semi-synthetic approach is presented for the production of EPO with well-defined glycan structures at specific positions. E. coli cells were used to incorporate non-natural amino acids into EPO by amber stop codon suppression methodology. These non-natural amino acids bear a specific bio-orthogonal chemical function. After expression and purification, EPO could be coupled to oligosaccharides or PEG chains by copper-catalysed cycloaddition between alkynes and azides. The generated EPO variants were characterised biophysically and the biological activitywas investigated by differentiation and proliferation assays. First steps were undertaken towards elucidating the impact of glycosylation on the function of a therapeutic glycoprotein.