, Volume 32, Issue 2, pp 153–168 | Cite as

Comprehensive Physicochemical and Biological Characterization of the Proposed Biosimilar Darbepoetin Alfa, LBDE, and Its Originator Darbepoetin Alfa, NESP®

  • Yeong Ran Jeong
  • Rae Ung Jeong
  • Jeong Hyun Son
  • Joon Cheol Kwon
  • Saem Jung
  • Mi A Song
  • Jin Ah Hwang
  • Gyun Min Lee
Original Research Article



For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization.


To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and NESP®, its originator, we performed a comprehensive physicochemical and biological characterization study.


Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography–mass spectrometry (LC–MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography–high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) were evaluated using rats through two injection routes.


Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and NESP® have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with NESP®. For both LBDE and NESP®, a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with NESP® in the in vitro and in vivo functional analyses. PK parameters of LBDE were in good agreement with those of NESP®.


LBDE shows high similarity to NESP® with regard to structure and function.


Author contributions

All authors were involved in drafting the article and revising it critically for important intellectual content. All authors read and approved the final manuscript submitted for publication.

Compliance with Ethical Standards

The protocol of the in vivo potency assay and rat PK study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).


 This research was supported in part by a grant from the Bio & Medical Technology Development Program of the NRF funded by the Korean government (2013M3A9B6075931).

Conflicts of interest

Yeong Ran Jeong, Rae Ung Jeong, Jeong Hyun Son, Joon Cheol Kwon, Saem Jung, Mi A. Song, Jin Ah Hwang, and Gyun Min Lee declare that they have no conflicts of interest.

Supplementary material

40259_2018_272_MOESM1_ESM.pdf (97 kb)
Supplementary material 1 (PDF 98 kb)


  1. 1.
    Overbay DK, Manley HJ. Darbepoetin-alpha: a review of the literature. Pharmacotherapy. 2002;22:889–97.CrossRefPubMedGoogle Scholar
  2. 2.
    Egrie JC, Browne JK. Development and characterization of novel erythropoiesis stimulating protein (NESP). Br J Cancer. 2001;84:3–10.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Maxwell AP. Novel erythropoiesis-stimulating protein in the management of the anemia of chronic renal failure. Kidney Int. 2002;62:720–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Biosimilars of darbepoetin alfa. Generics and Biosimilars Initiative. 2016. Accessed 28 Oct 2017.
  5. 5.
    Phase I study shows darbepoetin alfa biosimilar to be well tolerated. Generics and Biosimilars Initiative. 2015. Accessed 28 Oct 2017.
  6. 6.
    US$67 billion worth of biosimilar patents expiring before 2020. Generics and Biosimilars Initiative. 2012. Accessed 28 Oct 2017.
  7. 7.
    Committee for Medicinal Products for Human Use (CHMP), European Medicines Agency. Guideline on similar biological medicinal products. CHMP/437/04 Rev 1. 2014. Accessed 28 Oct 2017.
  8. 8.
    Shahrokh Z, Royle L, Saldova R, Bones J, Abrahams JL, Artemenko NV, et al. Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines. Mol Pharm. 2011. Scholar
  9. 9.
    Harazono A, Hashii N, Kuribayashi R, Nakazawa S, Kawasaki N. Mass spectrometric glycoform profiling of the innovator and biosimilar erythropoietin and darbepoetin by LC/ESI-MS. J Pharm Biomed Anal. 2013. Scholar
  10. 10.
    Boucher S, Kane A, Girard M. Qualitative and quantitative assessment of marketed erythropoiesis-stimulating agents by capillary electrophoresis. J Pharm Biomed Anal. 2012. Scholar
  11. 11.
    Okano M, Sato M, Kageyama S. Mass spectrometric characterisation of darbepoetin alfa biosimilars with C-terminal arginine residues. Drug Test Anal. 2016. Scholar
  12. 12.
    Anumula KR, Dhume ST. High resolution and high sensitivity methods for oligosaccharide mapping and characterization by normal phase high performance liquid chromatography following derivatization with highly fluorescent anthranilic acid. Glycobiology. 1998;8:685–94.CrossRefPubMedGoogle Scholar
  13. 13.
    European Directorate for the Quality of Medicines (EDQM). European pharmacopoeia, 9th ed. Erythropoietin concentrated solution, pp 2391–5; 2017.Google Scholar
  14. 14.
    DePaolis AM, Advani JV, Sharma BG. Characterization of erythropoietin dimerization. J Pharm Sci. 1995;84:1280–4.CrossRefPubMedGoogle Scholar
  15. 15.
    Sinclair AM, Elliott S. Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci. 2005;94:1626–35.CrossRefPubMedGoogle Scholar
  16. 16.
    Byrne B, Donohoe GG, O’Kennedy R. Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discov Today. 2007;12:319–26.CrossRefPubMedGoogle Scholar
  17. 17.
    Schauer R. Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol. 2009. Scholar
  18. 18.
    Ghaderi D, Taylor RE, Padler-Karavani V, Diaz S, Varki A. Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol. 2010. Scholar
  19. 19.
    Lai PH, Everett R, Wang FF, Arakawa T, Goldwasser E. Structural characterization of human erythropoietin. J Biol Chem. 1986;261:3116–21.PubMedGoogle Scholar
  20. 20.
    Bunn HF. Erythropoietin. Cold Spring Harb Perspect Med. 2013. Scholar
  21. 21.
    Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin. J Biol Chem. 1987;262:17156–63.PubMedGoogle Scholar
  22. 22.
    Lappin TR, Winter PC, Elder GE, McHale CM, Hodges VH, Bridges JM. Structure-function relationships of the erythropoietin molecule. Ann N Y Acad Sci. 1994;718:191–201.CrossRefPubMedGoogle Scholar
  23. 23.
    Jiang G, Yu C, Yadav DB, Hu Z, Amurao A, Duenas E, et al. Evaluation of heavy-chain C-terminal deletion on product quality and pharmacokinetics of monoclonal antibodies. J Pharm Sci. 2016. Scholar
  24. 24.
    Bennett CL, Silver SM, Djulbegovic B, Samaras AT, Blau CA, Gleason KJ, et al. Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. JAMA. 2008. Scholar
  25. 25.
    Thomson RI, Gardner RA, Strohfeldt K, Fernandes DL, Stafford GP, Spencer DIR, et al. Analysis of three epoetin alpha products by LC and LC-MS indicates differences in glycosylation critical quality attributes, including sialic acid content. Anal Chem. 2017. Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Yeong Ran Jeong
    • 1
    • 2
  • Rae Ung Jeong
    • 2
  • Jeong Hyun Son
    • 2
  • Joon Cheol Kwon
    • 2
  • Saem Jung
    • 2
  • Mi A Song
    • 2
  • Jin Ah Hwang
    • 3
  • Gyun Min Lee
    • 1
  1. 1.Department of Biological SciencesKAISTDaejeonRepublic of Korea
  2. 2.Biopharmaceuticals R&DDaejeonRepublic of Korea
  3. 3.Department of Medical BioscienceKonyang UniversityNonsanRepublic of Korea

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