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

• Published in: Journal of biotechnology Vol. 157; no. 1; pp. 198 - 206
• Main Authors: Nett, Juergen H., Gomathinayagam, Sujatha, Hamilton, Stephen R., Gong, Bing, Davidson, Robert C., Du, Min, Hopkins, Daniel, Mitchell, Teresa, Mallem, Muralidhar R., Nylen, Adam, Shaikh, Seemab S., Sharkey, Nathan, Barnard, Gavin C., Copeland, Victoria, Liu, Liming, Evers, Raymond, Li, Yan, Gray, Peter M., Lingham, Russell B., Visco, Denise, Forrest, Gail, DeMartino, Julie, Linden, Thomas, Potgieter, Thomas I., Wildt, Stefan, Stadheim, Terrance A., d’Anjou, Marc, Li, Huijuan, Sethuraman, Natarajan
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.2012; Elsevier
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cell Proliferation - drug effects; Darbepoetin alfa; Erythropoietin; Erythropoietin - analogs & derivatives; Erythropoietin - biosynthesis; Erythropoietin - blood; Erythropoietin - genetics; Erythropoietin - pharmacokinetics; Erythropoietin - pharmacology; Female; Fundamental and applied biological sciences. Psychology; Glycoengineered Pichia pastoris; Glycosylation; Humans; Male; Mice; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Polyethylene Glycols; Polysaccharides - chemistry; Protein Engineering - methods; Rats, Sprague-Dawley; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Erythropoietin; Glycosylation; Glycoengineered Pichia pastoris; Ascomycota; Fungi; Production; Pichia pastoris; Optimization
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2011.11.002

► 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. Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, 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). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. 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.