Advertisement

Differential Scanning Calorimetry to Quantify Heat-Induced Aggregation in Concentrated Protein Solutions

  • Matthew R. Jacobs
  • Mark Grace
  • Alice Blumlein
  • Jennifer J. McManusEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2039)

Abstract

Differential scanning calorimetry (DSC) is an important technique to measure the thermodynamics of protein unfolding (or folding). Information including the temperature for the onset of unfolding, the melt transition temperature (Tm), enthalpy of unfolding (ΔH), and refolding index (RI) are useful for evaluating the heat stability of proteins for a range of biochemical, structural biology, industrial, and pharmaceutical applications. We describe a procedure for careful sample preparation of proteins for DSC measurements and data analysis to determine a range of thermodynamic parameters. In particular, we highlight a measure of protein refolding following complete thermal denaturation (RI), which quantifies the proportion of protein lost to irreversible aggregation after thermal denaturation.

Key words

Differential scanning calorimetry Melt transition temperature Enthalpy Refolding index Protein aggregation Thermal denaturation 

Notes

Acknowledgments

The research work presented in this chapter was made possible through the financial support of Enterprise Ireland under grant number [IP/2015 0358] and the Synthesis and Solid State Pharmaceutical Centre, cofunded under the European Regional Development Fund, and the Department of Agriculture Food and Marine, FIRM grant number [11/F/037], SFI Stokes Lectureship to J.J.M., and Maynooth University Teaching Fellowship to A.B.

References

  1. 1.
    Chi EY, Krishnan S, Randolph TW, Carpenter JF (2003) Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res 20:12.  https://doi.org/10.1023/A:1025771421906CrossRefGoogle Scholar
  2. 2.
    Kamerzell TJ, Esfandiary R, Joshi SB, Middaugh CR, Volkin DB (2011) Protein–excipient interactions: mechanisms and biophysical characterization applied to protein formulation development. Adv Drug Deliv Rev 63:1118–1159.  https://doi.org/10.1016/j.addr.2011.07.006CrossRefPubMedGoogle Scholar
  3. 3.
    Roberts CJ (2014) Protein aggregation and its impact on product quality. Curr Opin Biotechnol 30:211–217.  https://doi.org/10.1016/j.copbio.2014.08.001CrossRefPubMedGoogle Scholar
  4. 4.
    Wu H, Kroe-Barrett R, Singh S, Robinson AS, Roberts CJ (2014) Competing aggregation pathways for monoclonal antibodies. FEBS Lett 588:936–941.  https://doi.org/10.1016/j.febslet.2014.01.051CrossRefPubMedGoogle Scholar
  5. 5.
    Barnett GV, Drenski M, Razinkov V, Reed WF, Roberts CJ (2016) Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering. Anal Biochem 511:80–91.  https://doi.org/10.1016/j.ab.2016.08.002CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Alam P, Siddiqi K, Chturvedi SK, Khan RH (2017) Protein aggregation: from background to inhibition strategies. Int J Biol Macromol 103:208–219.  https://doi.org/10.1016/j.ijbiomac.2017.05.048CrossRefPubMedGoogle Scholar
  7. 7.
    Wang W, Roberts CJ (2018) Protein aggregation – mechanisms, detection, and control. Int J Pharm 550:251–268.  https://doi.org/10.1016/j.ijpharm.2018.08.043CrossRefPubMedGoogle Scholar
  8. 8.
    Privalov PL, Khechinashvili NN (1974) A thermodynamic approach to the problem of stabilization of globular protein structure: a calorimetric study. J Mol Biol 86:665–684.  https://doi.org/10.1016/0022-2836(74)90188-0CrossRefPubMedGoogle Scholar
  9. 9.
    Cooper A (1999) Thermodynamic analysis of biomolecular interactions. Curr Opin Chem Biol 3:557–563.  https://doi.org/10.1016/S1367-5931(99)00008-3CrossRefPubMedGoogle Scholar
  10. 10.
    Johnson CM (2013) Differential scanning calorimetry as a tool for protein folding and stability. Arch Biochem Biophys 531:100–109.  https://doi.org/10.1016/j.abb.2012.09.008CrossRefPubMedGoogle Scholar
  11. 11.
    Bruylants G, Wouters J, Michaux C (2011) Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Curr Med Chem 12:2011–2020.  https://doi.org/10.2174/0929867054546564CrossRefGoogle Scholar
  12. 12.
    James S, McManus JJ (2012) Thermal and solution stability of lysozyme in the presence of sucrose, glucose, and trehalose. J Phys Chem B 116:10182–10188.  https://doi.org/10.1021/jp303898gCrossRefPubMedGoogle Scholar
  13. 13.
    Rosa M, Roberts CJ, Rodrigues MA (2017) Connecting high-temperature and low-temperature protein stability and aggregation. PLoS One 12:e0176748.  https://doi.org/10.1371/journal.pone.0176748CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Blumlein A, McManus JJ (2013) Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength. Biochim Biophys Acta 1834:2064–2070.  https://doi.org/10.1016/j.bbapap.2013.06.001CrossRefPubMedGoogle Scholar
  15. 15.
    Stavropoulos P, Thanassoulas A, Nounesis G (2018) The effect of cations on reversibility and thermodynamic stability during thermal denaturation of lysozyme. J Chem Thermodyn 118:331–337.  https://doi.org/10.1016/j.jct.2017.10.006CrossRefGoogle Scholar
  16. 16.
    Zbacnik TJ, Holcomb RE, Katayama DS, Murphy BM, Payne RW, Coccaro RC, Evans GJ, Matsuura JE, Henry CS, Manning MC (2017) Role of buffers in protein formulations. J Pharm Sci 106:713–733.  https://doi.org/10.1016/j.xphs.2016.11.014CrossRefPubMedGoogle Scholar
  17. 17.
    Mahler H-C, Friess W, Grauschopf U, Kiese S (2009) Protein aggregation: pathways, induction factors and analysis. J Pharm Sci 98:2909–2934.  https://doi.org/10.1002/jps.21566CrossRefPubMedGoogle Scholar
  18. 18.
    Schön A, Clarkson BR, Siles R, Ross P, Brown RK, Freire E (2015) Denatured state aggregation parameters derived from concentration dependence of protein stability. Anal Biochem 488:45–50.  https://doi.org/10.1016/j.ab.2015.07.013CrossRefPubMedGoogle Scholar
  19. 19.
    Wu S, Ding Y, Zhang G (2015) Mechanic insight into aggregation of lysozyme by ultrasensitive differential scanning calorimetry and sedimentation velocity. J Phys Chem B 119:15789–15795.  https://doi.org/10.1021/acs.jpcb.5b08190CrossRefPubMedGoogle Scholar
  20. 20.
    Peng J, Tang J, Barrett DM, Sablani SS, Anderson N, Powers JR (2017) Thermal pasteurization of ready-to-eat foods and vegetables: critical factors for process design and effects on quality. Crit Rev Food Sci Nutr 57:2970–2995.  https://doi.org/10.1080/10408398.2015.1082126CrossRefPubMedGoogle Scholar
  21. 21.
    Ling B, Tang J, Kong F, Mitcham EJ, Wang S (2015) Kinetics of food quality changes during thermal processing: a review. Food Bioprocess Technol 8:343–358.  https://doi.org/10.1007/s11947-014-1398-3CrossRefGoogle Scholar
  22. 22.
    Niedziela-Majka A, Kan E, Weissburg P, Mehra U, Sellers S, Sakowicz R (2015) High-throughput screening of formulations to optimize the thermal stability of a therapeutic monoclonal antibody. J Biomol Screen 20:552–559.  https://doi.org/10.1177/1087057114557781CrossRefPubMedGoogle Scholar
  23. 23.
    He F, Woods CE, Trilisky E, Bower KM, Litowski JR, Kerwin BA, Becker GW, Narhi LO, Razinkov VI (2011) Screening of monoclonal antibody formulations based on high-throughput thermostability and viscosity measurements: design of experiment and statistical analysis. J Pharm Sci 100:1330–1340.  https://doi.org/10.1002/jps.22384CrossRefPubMedGoogle Scholar
  24. 24.
    He F, Hogan S, Latypov RF, Narhi LO, Razinkov VI (2010) High throughput thermostability screening of monoclonal antibody formulations. J Pharm Sci 99:1707–1720.  https://doi.org/10.1002/jps.21955CrossRefPubMedGoogle Scholar
  25. 25.
    Harn N, Allan C, Oliver C, Middaugh CR (2007) Highly concentrated monoclonal antibody solutions: direct analysis of physical structure and thermal stability. J Pharm Sci 96:532–546.  https://doi.org/10.1002/jps.20753CrossRefPubMedGoogle Scholar
  26. 26.
    Moussa EM, Panchal JP, Moorthy BS, Blum JS, Joubert MK, Narhi LO, Topp EM (2016) Immunogenicity of therapeutic protein aggregates. J Pharm Sci 105:417–430.  https://doi.org/10.1016/j.xphs.2015.11.002CrossRefPubMedGoogle Scholar
  27. 27.
    Dobson CM, Evans PA, Radford SE (1994) Understanding how proteins fold: the lysozyme story so far. Trends Biochem Sci 19:31–37.  https://doi.org/10.1016/0968-0004(94)90171-6CrossRefPubMedGoogle Scholar
  28. 28.
    Esposito A, Comez L, Cinelli S, Scarponi F, Onori G (2009) Influence of glycerol on the structure and thermal stability of lysozyme: a dynamic light scattering and circular dichroism study. J Phys Chem B 113:16420–16424.  https://doi.org/10.1021/jp906739vCrossRefPubMedGoogle Scholar
  29. 29.
    Meng-Lund H, Friis N, van de Weert M, Rantanen J, Poso A, Grohganz H, Jorgensen L (2017) Correlation between calculated molecular descriptors of excipient amino acids and experimentally observed thermal stability of lysozyme. Int J Pharm 523:238–245.  https://doi.org/10.1016/j.ijpharm.2017.03.043CrossRefPubMedGoogle Scholar
  30. 30.
    Alam MT, Rizvi A, Rauf MA, Owais M, Naeem A (2018) Thermal unfolding of human lysozyme induces aggregation: recognition of the aggregates by antisera against the native protein. Int J Biol Macromol 113:976–982.  https://doi.org/10.1016/j.ijbiomac.2018.02.095CrossRefPubMedGoogle Scholar
  31. 31.
    Sophianopoulos AJ, Rhodes CK, Holcomb DN, van Holde KE (1962) Physical studies of lysozyme: I. Characerization. J Biol Chem 237:1107–1112PubMedGoogle Scholar
  32. 32.
    Harding SE, Chowdhry B (2000) Protein-ligand interactions: hydrodynamics and calorimetry: a practical approach. In: Oxford University Press. Oxford, New York, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Matthew R. Jacobs
    • 1
  • Mark Grace
    • 1
  • Alice Blumlein
    • 1
  • Jennifer J. McManus
    • 2
    Email author
  1. 1.Department of ChemistryMaynooth UniversityMaynoothIreland
  2. 2.Department of ChemistryMaynooth UniversityMaynoothIreland

Personalised recommendations