Analytical and Bioanalytical Chemistry

, Volume 406, Issue 26, pp 6559–6567 | Cite as

Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers

  • Michaella J. Levy
  • Ashley C. Gucinski
  • Cynthia D. Sommers
  • Houman Ghasriani
  • Bo Wang
  • David A. Keire
  • Michael T. BoyneII
Research Paper
Part of the following topical collections:
  1. Analysis of Biological Therapeutic Agents and Biosimilars

Abstract

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733–737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310–312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D 1H,15N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.

Keywords

Top–down mass spectrometry Granulocyte-colony stimulating factor Bioassay Cell proliferation Circular dichroism NMR 

Supplementary material

216_2013_7469_MOESM1_ESM.pdf (207 kb)
ESM 1(PDF 206 kb)

References

  1. 1.
    Lanthier M, Behrman R, Nardinelli C (2008) Economic issues with follow-on protein products. Nat Rev Drug Discov 7(9):733–737CrossRefGoogle Scholar
  2. 2.
    Schiestl M, Stangler T, Torella C, Cepeljnik T, Toll H, Grau R (2011) Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat Biotechnol 29(4):310–312CrossRefGoogle Scholar
  3. 3.
    Kaushansky K, O’Hara PJ, Berkner K, Segal GM, Hagen FS, Adamson JW (1986) Genomic cloning, characterization, and multilineage growth-promoting activity of human granulocyte-macrophage colony-stimulating factor. Proc Natl Acad Sci U S A 83(10):3101–3105CrossRefGoogle Scholar
  4. 4.
    Nemunaitis J, Rabinowe SN, Singer JW, Bierman PJ, Vose JM, Freedman AS, Onetto N, Gillis S, Oette D, Gold M et al (1991) Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 324(25):1773–1778CrossRefGoogle Scholar
  5. 5.
    Souza LM, Boone TC, Gabrilove J, Lai PH, Zsebo KM, Murdock DC, Chazin VR, Bruszewski J, Lu H, Chen KK et al (1986) Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells. Science 232(4746):61–65CrossRefGoogle Scholar
  6. 6.
    Hill CP, Osslund TD, Eisenberg D (1993) The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci U S A 90(11):5167–5171CrossRefGoogle Scholar
  7. 7.
    Chirino AJ, Mire-Sluis A (2004) Characterizing biological products and assessing comparability following manufacturing changes. Nat Biotechnol 22(11):1383–1391CrossRefGoogle Scholar
  8. 8.
    Watson C, Sharp JS (2012) Conformational analysis of therapeutic proteins by hydroxyl radical protein footprinting. AAPS J 14(2):206–217CrossRefGoogle Scholar
  9. 9.
    Yamazaki K, Iwura T, Ishikawa R, Ozaki Y (2006) Effects of ionic strength on the thermal unfolding process of granulocyte-colony stimulating factor. J Biochem 139(1):41–49CrossRefGoogle Scholar
  10. 10.
    Yamazaki K, Iwura T, Ishikawa R, Ozaki Y (2006) Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor. J Biochem 140(1):49–56CrossRefGoogle Scholar
  11. 11.
    Bristow AF, Bird C, Bolgiano B, Thorpe R (2012) Regulatory requirements for therapeutic proteins: the relationship between the conformation and biological activity of filgrastim. Pharmeur Bio Sci Notes 2012:103–117Google Scholar
  12. 12.
    Zink T, Ross A, Ambrosius D, Rudolph R, Holak TA (1992) Secondary structure of human granulocyte colony-stimulating factor derived from NMR spectroscopy. FEBS Lett 314(3):435–439CrossRefGoogle Scholar
  13. 13.
    Zink T, Ross A, Luers K, Cieslar C, Rudolph R, Holak TA (1994) Structure and dynamics of the human granulocyte colony-stimulating factor determined by NMR spectroscopy. Loop mobility in a four-helix-bundle protein. Biochemistry 33(28):8453–8463CrossRefGoogle Scholar
  14. 14.
    Werner JM, Breeze AL, Kara B, Rosenbrock G, Boyd J, Soffe N, Campbell ID (1994) Secondary structure and backbone dynamics of human granulocyte colony-stimulating factor in solution. Biochemistry 33(23):7184–7192CrossRefGoogle Scholar
  15. 15.
    Aubin Y, Gingras G, Sauve S (2008) Assessment of the three-dimensional structure of recombinant protein therapeutics by NMR fingerprinting: demonstration on recombinant human granulocyte macrophage-colony stimulation factor. Anal Chem 80(7):2623–2627CrossRefGoogle Scholar
  16. 16.
    Sauve S, Gingras G, Aubin Y (2008) NMR assignment of human granulocyte-macrophage colony-stimulating factor. Biomol NMR Assign 2(1):5–7CrossRefGoogle Scholar
  17. 17.
    Klick S, Muijselaar PG, Waterval J, Eichinger T, Kom C, Gerding TK, Debets AJ, Sanger-van de Griend C, van den Beld C, Somsen GW, De Jong GJ (2005) Toward a generic approach for stress testing of drug substances and drug products. Pharm Tech 29:48–66Google Scholar
  18. 18.
    Quali EED, EDQM (2008) European Pharmacopoeia supplement 6.3: supplement 6.3 W/6.4 & 6.5 when available. Worldwide Book ServiceGoogle Scholar
  19. 19.
    Aritomi M, Kunishima N, Okamoto T, Kuroki R, Ota Y, Morikawa K (1999) Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme. Nature 401(6754):713–717CrossRefGoogle Scholar
  20. 20.
    Brahms S, Brahms J (1980) Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J Mol Biol 138(2):149–178CrossRefGoogle Scholar
  21. 21.
    Holzmann J, Hausberger A, Rupprechter A, Toll H (2013) Top-down MS for rapid methionine oxidation site assignment in filgrastim. Anal Bioanal Chem 405(21):6667–6674CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg (outside the USA) 2013

Authors and Affiliations

  • Michaella J. Levy
    • 2
  • Ashley C. Gucinski
    • 2
  • Cynthia D. Sommers
    • 2
  • Houman Ghasriani
    • 2
  • Bo Wang
    • 2
  • David A. Keire
    • 2
  • Michael T. BoyneII
    • 1
  1. 1.Division of Pharmaceutical Analysis, Office of Testing and Research, Center for Drug Evaluation and ResearchU.S. Food and Drug AdministrationSilver SpringUSA
  2. 2.Division of Pharmaceutical Analysis, Office of Testing and Research, Center for Drug Evaluation and ResearchU.S. Food and Drug AdministrationSt. LouisUSA

Personalised recommendations