title: Evaluation of bioactivity and pharmacokinetic characteristics of PEGylated P.pastoris-expressed erythropoietin.
abstract: High costs of production and relatively short serum half-life of mammalian cell-derived recombinant human erythropoietin (rHuEpo) necessitate finding and developing superior hosts/technologies for more efficient production of longer-acting erythropoietic agents. With these aims, we provide the first report on reductive alkylation of low-cost P.pastoris-expressed rHuEpo (PPEpo) with PEG-aldehyde. The PCR-amplified cDNA of native rHuEpo was cloned into the pPICZαA vector and transformed into the yeast Pichia pastoris. The best expressing transformant was selected and employed for secreted-expression of PPEpo using the standard protocols. Purified PPEpo was N-terminally PEGylated with 20-kDa mPEG-propionaldehyde in a low pH (5) condition. The in vitro and in vivo biological activities of purified mono-PEGylated PPEpo was evaluated by the UT-7 cells proliferation assay and normocythaemic mice assay, respectively. Pharmacokinetic parameters were determined following intravenous administration of Epo proteins in rabbits. While PPEpo showed a higher in vitro bioactivity compared to rHuEpo, no in vivo efficiency was determined for PPEpo. However, the in vivo activity of PEG-PPEpo conjugate was comparable to that of rHuEpo. Pharmacokinetic studies showed that the terminal half-life and mean residence time of PEG-PPEpo were increased approximately 4-fold and 6.5-fold respectively, compared with those of PPEpo. The results indicate that N-terminal PEGylation of Pichia-expressed Epo could be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.
authors: Faculty of Pharmacy, Tehran University of Medical Science, Postal Code: 1417653761, Tehran, Iran.
authors: Ahmad
authors: A
authors: Maleki
authors: Abdolhossein Rouholamini
authors: AR
authors: Najafabadi
authors: Farzin
authors: F
authors: Roohvand
authors: Abbas
authors: A
authors: Shafiee
authors: Hossein
authors: H
authors: Khanahmad
authors: Homa
authors: H
authors: Faghihi
authors: Mohammad H
authors: MH
authors: Hedayati
authors: Hosnieh
authors: H
authors: Tajerzadeh
mesh_terms: D000818:Animals; D019540:Area Under Curve; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology*; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D010843:Pichia / Q000378:metabolism; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000493:pharmacokinetics* / Q000494:pharmacology*; D017701:Reticulocyte Count
chemical_list: C554270:EPO protein, human; D011994:Recombinant Proteins; C508174:pegylated erythropoietin; D004921:Erythropoietin; D011092:Polyethylene Glycols