Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris

► We report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. ► We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. ► In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp® (darbepoetin). ► Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp®. ► Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.