Abstract
Erythropoietin (EPO) is the principal hormone which, has somewhat short half-life involved in the differentiation and regulation of circulating red blood cells. The present study was carried out to evaluate the capability of a polyethylene glycol mimetic technology as a biological alternative to improve pharmaceutical properties of human recombinant EPO. In silico models of EPO fused to 200 amino acids of proline, alanine, and serine (PAS) were initially generated and assessed by molecular dynamic (MD) simulation. The fluctuations of the modeled structure reached a plateau after 6000 ps of MD simulation. The Phi and psi analysis showed >99.2% of residues were located in the allowed regions. An expression vector consisting of EPO cDNA tagged to PAS coding sequences was synthesized and expressed in CHO-K1 Cells. The produced PASylated molecule was purified and characterized by standard analytical methods. The molecular weight of fusion protein was expanded to 70 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Analytical size exclusion chromatography revealed an approximately sevenfold increase in apparent size of produced protein. Although the in vitro potency of the fusion protein was significantly reduced (1.26 ± 0.05 vs. 0.24 ± 0.03 ng/ml) but, the in vivo activity was considerably increased up to 1.58 × 105 IU/ml in normocythemic mice assay. Pharmacokinetic animal studies revealed strongly 15.6-fold plasma half-life extension for the PASylated EPO (83.16 ± 13.28 h) in comparison to epoetin α (8.5 ± 2.4 h) and darbepoetin α (25.3 ± 2.2h).
Similar content being viewed by others
Abbreviations
- PAS:
-
Proline, alanine, serine
- rHuEPO:
-
Recombinant human erythropoietin
- PEG:
-
Poly ethylene glycol
- CD:
-
Circular dichroism
- MTT:
-
[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
- DMSO:
-
Dimethyl sulfoxide
- t1/2 :
-
Terminal half-life
- RMSD:
-
Root-mean-square deviation
- CZE:
-
Capillary zone electrophoresis
References
Jelkmann W (1992) Erythropoietin: structure, control of production, and function. Physiol Rev 72(2):449–489
Inoue N, Takeuchi M, Ohashi H, Suzuki T (1995) The production of recombinant human erythropoietin. Biotechnol Annu Rev 1:297–313
Jelkmann W (2011) Regulation of erythropoietin production. J Physiol 589:1251–1258
Sasaki H, Bothner B, Dell A, Fukuda M (1987) Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J Biol Chem 262(25):12059–12076
Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, Kobata A (1988) Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem 263(8):3657–3663
Takeuchi M, Takasaki S, Shimada M, Kobata A (1990) Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J Biol Chem 265(21):12127–12130
Tsuda E, Kawanishi G, Ueda M, Masuda S, Sasaki R (1990) The role of carbohydrate in recombinant human erythropoietin. Eur J Biochem 188(2):405–411
Joanne TM (2011) Erythropoietin-measurement and clinical applications. Ann Clin Biochem 43:93–104
Joung CH, Shin JY, Koo JK, Lim JJ, Wang JS, Lee SJ, Tan HK, Kim SL, Lim SM (2009) Production and characterization of long-acting recombinant human albumin-EPO fusion protein expressed in CHO cell. Protein Expr Purif 68(2):137–145
Egrie JC, Browne JK (2001) Development and characterization of novel erythropoiesis stimulating protein (NESP). Nephrol Dial Transp 16:3–13
Veronese FM, Pasut G (2005) PEGylation, successful approach to drug delivery. Drug Discov Today 10(21):1451–1458
Skerra A, Theobald I, Schlapschy M (2008) Biological active proteins having increased in vivo and/or in vitro stability, WO 155134 A1
Kontermann RE (2011) Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol 22:868–876
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Meyer TS, Lamberts BL (1965) Use of coomassie brilliant blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide-gel strips. Biochim Biophys Acta 107(1):144–145
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63
Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89(2):271–277
Davis BH, DiCorato M, Bigelow NC, Langweiler MH (1993) Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements. Cytometry 14(3):318–326
Gibaldi M, Perrier D (1982) Pharmacokinetics, 2nd edn. Marcel Dekker, New York
Fiser A, Sali A (2003) ModLoop: automated modeling of loops in protein structures. Bioinformatics 19(18):2500–2501
Kontermann RE (2009) Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 23(2):93–109
Sytkowski AJ, Lunn ED, Davis KL, Feldman L, Siekman S (1998) Human erythropoietin dimers with markedly enhanced in vivo activity. Proc Natl Acad Sci USA 95(3):1184–1188
Dalle B, Henri A, Rouyer-Fessard P, Bettan M, Scherman D, Beuzard Y, Payen E (2001) Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in vivo. Blood 97(12):3776–3782
Egrie JC, Dwyer E, Browne JK, Hitz A, Lykos MA (2003) Darbepoetin alfa has a longer circulating half-life and greater in vivo potency than recombinant human erythropoietin. Exp Hematol 31(4):290–299
Su D, Zhao H, Xia H (2010) Glycosylation-modified erythropoietin with improved half-life and biological activity. Int J Hematol 91(2):238–244
Schriebl K, Trummer E, Lattenmayer C, Weik R, Kunert R, Muller D, Katinger H, Vorauer-Uhl K (2006) Biochemical characterization of rhEpo-Fc fusion protein expressed in CHO cells. Protein Expr Purif 49(2):265–275
Shi X, Yang J, Zhu H, Ye L, Feng M, Li J, Huang H, Tao Q, Ye D, Sun LH, Sun BN, Sun CR, Han G, Liu Y, Yao M, Zhou P, Ju D (2013) Pharmacokinetics and pharmacodynamics of recombinant human EPO-Fc fusion protein in vivo. PLoS ONE 8(8):e72673
Fares F, Havron A, Fima E (2011) Designing a long acting erythropoietin by fusing three carboxyl-terminal peptides of human chorionic gonadotropin beta subunit to the N-terminal and C-terminal coding sequence. Int J Cell Biol 2011:275063
Wang YJ, Hao SJ, Liu YD, Hu T, Zhang GF, Zhang X, Qi QS, Ma GH, Su ZG (2010) PEGylation markedly enhances the in vivo potency of recombinant human non-glycosylated erythropoietin: a comparison with glycosylated erythropoietin. J Control Release 145(3):306–313
Cohan RA, Madadkar-Sobhani A, Khanahmad H, Roohvand F, Aghasadeghi MR, Hedayati MH, Barghi Z, Ardestani MS, Inanlou DN, Norouzian D (2011) Design, modeling, expression, and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin. Int J Nanomed 6:1217–1227
Maleki A, Madadkar-Sobhani A, Roohvand F, Najafabadi AR, Shafiee A, Khanahmad H, Cohan RA, Namvar N, Tajerzadeh H (2012) Design, modeling, and expression of erythropoietin cysteine analogs in Pichia pastoris: improvement of mean residence times and in vivo activities through cysteine-specific PEGylation. Eur J Pharm Biopharm 80(3):499–507
Binder U, Skerra A (2012) Half-life extension of therapeutic proteins via genetic fusion to recombinant PEG mimetics. In: Kontermann RE (ed) Therapeutic proteins. Wiley, New York
Lee DE, Son W, Ha BJ, Oh MS, Yoo OJ (2006) The prolonged half-lives of new erythropoietin derivatives via peptide addition. Biochem Biophys Res Commun 339(1):380–385
Schlapschy M, Theobald I, Mack H, Schottelius M, Wester HJ, Skerra A (2007) Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel 20(6):273–284
Jolling K, Ruixo JJ, Hemeryck A, Piotrovskij V, Greway T (2004) Population pharmacokinetic analysis of pegylated human erythropoietin in rats. J Pharm Sci 93(12):3027–3038
Armstrong JK, Hempel G, Koling S, Chan LS, Fisher T, Meiselman HJ, Garratty G (2007) Antibody against poly(ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients. Cancer 110(1):103–111
Fishburn CS (2008) The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics. J Pharm Sci 97(10):4167–4183
Bendele A, Seely J, Richey C, Sennello G, Shopp G (1998) Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins. Toxicol Sci 42(2):152–157
Schlapschy M, Binder U, Borger C, Theobald I, Wachinger K, Kisling S, Haller D, Skerra A (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins. Protein Eng Des Sel 26(8):489–501
Mendler CT, Friedrich L, Laitinen I, Schlapschy M, Schwaiger M, Wester HJ, Skerra A (2015) High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation. MAbs 7(1):96–109
Acknowledgements
This research has been supported by Tehran University of Medical Sciences & Health Services Grant 21960. The authors wish to express their deep gratitude to Pasteur Institute of Iran for scientific collaboration during the course of this research.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
There are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. All welfare of animals was conformed to the regulations and guidelines of the National Ethics Committee of the Ministry of Health and Medical Education of Iran.
Rights and permissions
About this article
Cite this article
Hedayati, M.H., Norouzian, D., Aminian, M. et al. Molecular Design, Expression and Evaluation of PASylated Human Recombinant Erythropoietin with Enhanced Functional Properties. Protein J 36, 36–48 (2017). https://doi.org/10.1007/s10930-017-9699-9
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-017-9699-9