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Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR

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Abstract

Purpose

Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®.

Methods

Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with 13C and 15 N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation.

Results

The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions

The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.

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Abbreviations

ANC:

Absolute neutrophil count

CMC:

Critical micelle concentration

CRS:

Chemical Reference Standard

EP:

European Pharmacopeia

HSQC:

Heteronuclear single quantum coherence

IMAC:

Immobilized metal affinity chromatography

Met-G-CSF:

Recombinant methionyl granulocyte-colony stimulating factor

PS-80:

Polysorbate-80

SEB:

Subsequent entry biologics

References

  1. Parham P. The Immune System 2nd Ed. New York: Garland Science; 2005. p. 227–76.

    Google Scholar 

  2. Zink T, Ross A, Lüers K, Cieslar C, Rudolph R, Holak TA. Structure and dynamics of the human granulocyte colony-stimulating factor determined by NMR spectroscopy. Loop mobility in a four-helix-bundle protein. Biochemistry. 1994;33:8453–63.

    Article  CAS  PubMed  Google Scholar 

  3. Hill CP, Osslund TD, Eisenberg D. The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc Natl Acad Sci U S A. 1993;90:5167–71.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Arakawa T, Prestrelski SJ, Narhi LO, Boone TC, Kenney WC. Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed. J Protein Chem. 1993;12:525–31.

    Article  CAS  PubMed  Google Scholar 

  5. EMEA Comparability of biotechnological/biological products subject to changes in their manufacturing process. CPMP/ICH/5721/03, 13 (2006). Canada, H. GUIDANCE FOR SPONSORS: Information and Submission Requirements for Subsequent Entry Biologics (SEBs). 2010/03/05. Health Canada - Publications, 17 (2010). FDA, U. Guidance for Industry: Quality Considerations in Demonstrating Biosimilarity to a Reference Protein Product. Office of Communications US FDA UCM291134, 1-20 (2012). FDA, U. Guidance for Industry: Scientific Considerations in Demonstrating Biosimilarity to a Reference Product. Office of Communications US FDA UCM291128, 1-25 (2012).

  6. Aubin Y, Gingras G, Sauvé S. Assessment of the three-dimensional structure of recombinant protein therapeutics by nmr fingerprinting: demonstration on rhGM-CSF. Anal Chem. 2008;80:2623–7.

    Article  CAS  PubMed  Google Scholar 

  7. Bishop B, Koay DC, Sartorelli AC, Reagan L. Reengineering granulocyte colony-stimulating factor for enhanced stability. J Biol Chem. 2001;276(36):33465–70.

    Article  CAS  PubMed  Google Scholar 

  8. Panjwani N, Hodgson DJ, Sauvé S, Aubin Y. Assessment of the effects of pH, formulation and deformulation on the conformation of interferon alpha-2 by NMR. J Pharm Sci. 2010;99:3334–42.

    Article  CAS  PubMed  Google Scholar 

  9. Delaglio F, Grzesiek S, Vuiter GW, Zhu G, Pfeifer J, Bax A. NMRpipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995;6:277–93.

    Article  CAS  PubMed  Google Scholar 

  10. Zink T, Ross A, Ambrosius D, Rudolph R, Holak TA. Secondary structure of human granulocyte colony-stimulating factor derived from NMR spectroscopy. FEBS Lett. 1992;314:435–9.

    Article  CAS  PubMed  Google Scholar 

  11. Johnson BA, Blevins RA. NMRView: a computer program for the visualization and analysis of NMR data. J Biomol NMR. 1994;4:603–14.

    Article  CAS  PubMed  Google Scholar 

  12. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12.

    Article  CAS  PubMed  Google Scholar 

  13. Baxter NJ, Williamson MP. Temperature dependence of 1H chemical shifts in proteins. J Biomol NMR. 1997;9:359–69.

    Article  CAS  PubMed  Google Scholar 

  14. Kolvenbach CG, Narhi LO, Philo JS, Li T, Zhang M, Arakawa T. Granulocyte-colony stimulating factor maintains a thermally stable, compact, partially folded structure at pH 2. J Pept Res. 1997;50:310–8.

    Article  CAS  PubMed  Google Scholar 

  15. Ricci MS, Sarkar CA, Fallon EM, Lauffenburger DA, Brems DN. pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor. Protein Sci. 2003;12:1030–8.

    Article  PubMed Central  PubMed  Google Scholar 

  16. Herman AC, Boone TC, Lu HS. Characterization, formulation and stability of Neupogen (Filgrastim), a recombinant human granulocyte-colony stimulating factor. In: Pearlman R, Wang J, editors. Formulation, characterization and stability of protein drugs. New York: Plenum Press; 1996. p. 303–28.

    Google Scholar 

  17. Narhi LO, Kenney WC, Arakawa T. Conformational changes of recombinant human granulocyte- colony stimulating factor induced by pH and guanidine hydrochloride. J Protein Chem. 1991;10:359–67.

    Article  CAS  PubMed  Google Scholar 

  18. Tamada T, Honjo E, Maeda Y, Okamoto T, Ishibashi M, Tokunaga M, et al. Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex. Proc Natl Acad Sci U S A. 2006;103:3135–40.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. Mecozzi S, West Jr AP, Dougherty DA. Cation-π interactions in aromatics of biological and medicinal interest: Electrostatic potential surfaces as a useful qualitative guide. Proc Natl Acad Sci U S A. 1996;93:10566–71.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Okada A, Miura T, Takeuchi H. Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 Ion channel from influenza a virus. Biochemistry. 2001;40:6053–60.

    Article  CAS  PubMed  Google Scholar 

  21. Loewenthal R, Sancho J, Fersht AR. Histidine-aromatic interactions in barnase. Elevation of histidine pKa and contribution to protein stability. J Mol Biol. 1992;224:759–70.

    Article  CAS  PubMed  Google Scholar 

  22. Van Gilst M, Hudson BS. Histidine-tryptophan interactions in T4 lysozyme: anomalous pH dependence of fluorescence. Biophys Chem. 1996;63:17–25. references cited therein.

    Article  PubMed  Google Scholar 

  23. Chou DK, Krishnamurthy R, Randolph TW, Carpenter JF, Manning MC. Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation. J Pharm Sci. 2005;94(6):1368–81.

    Article  CAS  PubMed  Google Scholar 

  24. Bristow AF, Bird C, Bolgiano B, Thorpe R. Regulatory requirements for therapeutic proteins: the relationship between the conformation and biological activity of filgrastim. Pharmeur Bio Sci Notes. 2012;2012:103–17.

    CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank Dr. Adrian Bristow (NIBSC) for stimulating discussions and for sharing his results on the mutants prior to publication, and Drs. Michel Girard and Michael Johnston for critical reading of the manuscript. The mass spectrometry support by Drs Daryl Smith and Terry Cyr is also acknowledged.

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Authors and Affiliations

  1. Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney’s Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada

    Yves Aubin, Derek J. Hodgson, William B. Thach, Geneviève Gingras & Simon Sauvé

Authors
  1. Yves Aubin
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  2. Derek J. Hodgson
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  3. William B. Thach
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  4. Geneviève Gingras
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  5. Simon Sauvé
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Correspondence to Yves Aubin.

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Aubin, Y., Hodgson, D.J., Thach, W.B. et al. Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR. Pharm Res 32, 3365–3375 (2015). https://doi.org/10.1007/s11095-015-1713-3

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  • DOI: https://doi.org/10.1007/s11095-015-1713-3

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