title: Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris.
abstract: 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.
authors.affiliation: GlycoFi, Biologics Discovery, Merck & Co. Inc., USA.
authors.forename: Juergen H
authors.initials: JH
authors.lastname: Nett
authors.forename: Sujatha
authors.initials: S
authors.lastname: Gomathinayagam
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Hamilton
authors.forename: Bing
authors.initials: B
authors.lastname: Gong
authors.forename: Robert C
authors.initials: RC
authors.lastname: Davidson
authors.forename: Min
authors.initials: M
authors.lastname: Du
authors.forename: Daniel
authors.initials: D
authors.lastname: Hopkins
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Muralidhar R
authors.initials: MR
authors.lastname: Mallem
authors.forename: Adam
authors.initials: A
authors.lastname: Nylen
authors.forename: Seemab S
authors.initials: SS
authors.lastname: Shaikh
authors.forename: Nathan
authors.initials: N
authors.lastname: Sharkey
authors.forename: Gavin C
authors.initials: GC
authors.lastname: Barnard
authors.forename: Victoria
authors.initials: V
authors.lastname: Copeland
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
authors.forename: Raymond
authors.initials: R
authors.lastname: Evers
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Peter M
authors.initials: PM
authors.lastname: Gray
authors.forename: Russell B
authors.initials: RB
authors.lastname: Lingham
authors.forename: Denise
authors.initials: D
authors.lastname: Visco
authors.forename: Gail
authors.initials: G
authors.lastname: Forrest
authors.forename: Julie
authors.initials: J
authors.lastname: DeMartino
authors.forename: Thomas
authors.initials: T
authors.lastname: Linden
authors.forename: Thomas I
authors.initials: TI
authors.lastname: Potgieter
authors.forename: Stefan
authors.initials: S
authors.lastname: Wildt
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
authors.forename: Marc
authors.initials: M
authors.lastname: d'Anjou
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Natarajan
authors.initials: N
authors.lastname: Sethuraman
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D000068256:Darbepoetin alfa; D004921:Erythropoietin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000097:blood / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology; D005260:Female; D006031:Glycosylation; D006801:Humans; D008297:Male; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols; D011134:Polysaccharides / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C554270:EPO protein, human; D011134:Polysaccharides; D011994:Recombinant Proteins; C508174:pegylated erythropoietin; D004921:Erythropoietin; D000068256:Darbepoetin alfa; D011092:Polyethylene Glycols