Pharmaceutical Research

, 35:137 | Cite as

Denaturation and Aggregation of Interferon-τ in Aqueous Solution

  • Ryan R. Manning
  • Glenn A. Wilson
  • Ryan E. Holcomb
  • Nathaniel J. Zbacnik
  • Auria A. Tellechea
  • Chelsey L. Gilley-Dunn
  • Ryan J. Krammes
  • Nathan S. Krammes
  • Gabriel J. Evans
  • Charles S. Henry
  • Mark Cornell Manning
  • Brian M. Murphy
  • Robert W. Payne
  • Derrick S. KatayamaEmail author
Research Paper



To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.


Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system.


The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1.


Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Key words

AF4 aggregation chaotropes denaturation flow microscopy guanidinium hydrochloride interferon-tau protein conformational stability urea 



Gibbs free energy of unfolding


Asymmetrical flow field-flow fractionation


Dynamic light scattering


Guanidinium hydrochloride




Multi-angle laser light scattering


Size exclusion chromatography




  1. 1.
    Chon TW, Bixler S. Interferon-t: current applications and potential in antiviral therapy. J Interferon Cytokine Res. 2010;30:477–85.CrossRefPubMedGoogle Scholar
  2. 2.
    Radhakrishnan R, Walter LJ, Subramaniam PS, Johnson HM, Walter MR. Crystal structure of ovine interferon-tau at 2.1 a resolution. J Mol Biol. 1999;286:151–62.CrossRefPubMedGoogle Scholar
  3. 3.
    Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqeuous solutions: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003;20:1325–32.CrossRefPubMedGoogle Scholar
  4. 4.
    Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27:544–75.CrossRefPubMedGoogle Scholar
  5. 5.
    Freire E, Schön A, Hutchins BM, Brown RK. Chemical denaturation as a tool in the formulation optimization of biologics. Drug Discov Today. 2013;18:1007–13.CrossRefPubMedGoogle Scholar
  6. 6.
    Katayama DS, Nayar R, Chou DK, Campos J, Cooper J, Vander Velde DG, et al. Solution of a novel type 1 interferon, interferon-t. J Pharm Sci. 2005;94:2703–15.CrossRefPubMedGoogle Scholar
  7. 7.
    Katayama DS, Nayar R, Chou DK, Valente JJ, Cooper J, Henry CS, et al. Effect of buffer species on the thermally induced aggregation of interferon-tau. J Pharm Sci. 2006;95:1212–26.CrossRefPubMedGoogle Scholar
  8. 8.
    Monera OD, Kay CM, Hodges RS. Protien denaturation with guanidine hydrochloride or urea provides a different estimate of stability depanding on the contributions of electrostatic interactions. Protein Sci. 1994;3:1984–91.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Povarova OI, Kuznetsova IM, Turoverov KK. Differences in the pathways of proteins unfolding induced by urea and guanidine hydrochloride: molten globule state and aggregates. PLos ONE. 2010;5. Article # e15035.Google Scholar
  10. 10.
    Smith JS, Scholtz JM. Guanidine hydrochloride unfolding of peptide helices: separation of denaturant and salt effects. Biochemistry. 1996;35:7292–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Greene RF Jr, Pace CN. Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, a-chymotrypsin, and b-lactoglobulin. J Biol Chem. 1974;249:5388–93.PubMedGoogle Scholar
  12. 12.
    Lim WK, Roesgen J, Englandder SW. Urea, but not guanidinium, destabilizes protein by forming hydrogen bonds to the peptide group. Proc Natl Acad Sci U S A. 2009;106:2595–600.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Stumpe MC, Grubmüller H. Urea impedes the hydrophobic collapse of partially unfolded proteins. Biophys J. 2009;96:3744–52.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ragone R, Colonna G, Balestrieri C, Servillo L, Irace G. Determination of tyrosine exposure in proteins by second -derivative spectroscopy. Biochemistry. 1984;23:1871–5.CrossRefPubMedGoogle Scholar
  15. 15.
    Kueltzo LA, Ersoy B, Ralston JP, Middaugh CR. Derivative absorbance spectroscopy and protein phase diagrams as tools for comprehensive protein characterization: a bGCSF case study. J Pharm Sci. 2003;92:1805–20.CrossRefPubMedGoogle Scholar
  16. 16.
    Pace CN. Linear extapolation method of analyzing solvent denaturation curves. Proteins Struct Funct Genet 2000; Suppl 4 1–7.Google Scholar
  17. 17.
    Vaz DC, Rodrigues JR, Sebald W, Dobson CM, Brito RMM. Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4. Protein Sci. 2006;15:33–44.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Bishop B, Koay DC, Sartorelli AC, Regan L. Reengineering granulocye-stimulating factor for enhanced stability. J Biol Chem. 2001;276:33465–70.CrossRefPubMedGoogle Scholar
  19. 19.
    Brems DN, Brown PL, Becker GW. Equilibrium denaturation of human growth hormone and its cysteine modified forms. J Biol Chem. 1990;265:5504–11.PubMedGoogle Scholar
  20. 20.
    Monera OD, Kay CM, Hodges RM. Protein denaturation with guanidinium hydrochloride or urea provides a different estimate of stability depending on electrostatic interactions. Protein Sci. 1994;3:1984–91.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Pace CN, Huyghues-Despointes BMP, Fu HL, Takano K, Scholtz JM, Grimsley GR. Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins. Protein Sci. 2010;19:929–43.CrossRefGoogle Scholar
  22. 22.
    Robertson AD, Murphy KP. Protein structure and the energetics of protein stability. Chem Rev. 1997;97:1251–67.CrossRefPubMedGoogle Scholar
  23. 23.
    Younvanich SS, Britt BM. The stability curve of hen egg white lysozyme. Protein Pept Lett. 2006;13:769–72.CrossRefPubMedGoogle Scholar
  24. 24.
    Myers JK, Pace CN, Scholtz JM. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995;4:2138–48.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Scholtz JM, Grimsley GR, Pace CN. Solvent denaturation of proteins and interpretations of the m value, methods in enzymology, Vol 466: Biothermodynamics, Pt B, Elsevier Academic Press Inc, San Diego; 2009 p. 549–565Google Scholar
  26. 26.
    Ye MQ, Yi TY, Li HP, Guo LL, Zou GL. Study on thermal and thermal chemcial denaturation of bovine immunoglobulin G. Acta Chim Sin. 2005;63:2047–54.Google Scholar
  27. 27.
    Jiao M, Liang Y, Li HT, Wang X. Studies on the unfolding of catalase induced by urea and guanidine hydrochloride. Acta Chim Sin. 2003;61:1362–8.Google Scholar
  28. 28.
    Wong HJ, Stathopulos PB, Bonner JM, Sawyer M, Meiering EM. Non-linear effects of temperature and urea on the thermodynamics and kinetics of folding and unfolding of hisactophilin. J Mol Biol. 2004;344:1089–107.CrossRefPubMedGoogle Scholar
  29. 29.
    Makhatadze GI, Privalov PL. Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol. 1992;226:491–505.CrossRefPubMedGoogle Scholar
  30. 30.
    Zweifel ME, Barrick D. Relationships between the temperature dependence of solvent denaturation and the denaturant dependence of protein stability curves. Biophys Chem. 2002;101-102:221–37.CrossRefPubMedGoogle Scholar
  31. 31.
    Radhakrishnan R, Walter LJ, Subramanian PS, Johnson HM, Walter MR. Crystal structure of ovine interferon-tau at 2.1 Å resolution. J Mol Biol. 1999;286:151–62.CrossRefPubMedGoogle Scholar
  32. 32.
    Street TO, Bolen DW, Rose GD. A molecular mechanism for osmolyte-induced protein stability. P Natl Acad Sci USA. 2006;103:13997–4002.CrossRefGoogle Scholar
  33. 33.
    Manning RR, Holcomb RE, Wilson GA, Manning MC. Review of orthogonal methods to SEC for quantitation and characterization of protein aggregates. Biopharm Int. 2014;27:32+.Google Scholar
  34. 34.
    Bria CRM, Jones J, Charlesworth A, Williams SKR. Probign submicron aggregation kinetics of an IgG protein using asymmetrical flow field-flow fractionation. J Pharm Sci. 2016;105:31–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Davis JM, Zhang N, Payne RW, Murphy BM, Abdul-Fattah AM, Matsuura JE, et al. Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation. Pharm Dev Technol. 2013;18:883–96.CrossRefPubMedGoogle Scholar
  36. 36.
    Simler RB, Hui G, Dahl JE, Perez-Ramirez B. Mechanistic complexity of subvisible particle formation: links to protein aggregation are highly specific. J Pharm Sci. 2012;101:4140–54.CrossRefPubMedGoogle Scholar
  37. 37.
    Barnard JG, Singh S, Randolph TW, Carpenter JF. Subvisible particle counting provides a sensitive method of detecting and quantifying aggregation of monoclonal antibody caused by freeze-thawing: insights into the roles of particles in the protein aggregation pathway. J Pharm Sci. 2011;100:492–503.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ryan R. Manning
    • 1
  • Glenn A. Wilson
    • 2
  • Ryan E. Holcomb
    • 3
    • 4
  • Nathaniel J. Zbacnik
    • 3
  • Auria A. Tellechea
    • 3
    • 4
  • Chelsey L. Gilley-Dunn
    • 3
    • 4
  • Ryan J. Krammes
    • 3
  • Nathan S. Krammes
    • 3
  • Gabriel J. Evans
    • 3
  • Charles S. Henry
    • 4
  • Mark Cornell Manning
    • 3
    • 4
  • Brian M. Murphy
    • 5
  • Robert W. Payne
    • 3
    • 4
  • Derrick S. Katayama
    • 3
    • 4
    Email author
  1. 1.Great Lakes Bio DesignCharlotteUSA
  2. 2.West Coast BioDesignSanta BarbaraUSA
  3. 3.Legacy BioDesign LLCJohnstownUSA
  4. 4.Department of ChemistryColorado State UniversityFort CollinsUSA
  5. 5.Alder BiopharmaceuticalsSeattleUSA

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