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Fluorescence Correlation Spectroscopy for Particle Sizing in Highly Concentrated Protein Solutions

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

Abstract

Highly concentrated solutions of biomolecules play an increasingly important role in biopharmaceutical drug development. In these systems, the formation of reversible aggregates by self-association creates a significant analytical challenge, since dilution is often required for techniques such as HPLC/liquid chromatography and analytical ultracentrifugation. There is a growing demand for methods capable of analyzing these assemblies, ideally under formulation conditions (i.e., in the presence of excipients). One approach that addresses this need is based on fluorescence correlation spectroscopy (FCS), which is a flexible and powerful technique to measure the diffusion of fluorescently labeled particles. It is particularly suited to measuring the size distribution of reversible aggregates of proteins or peptides in highly concentrated formulations, since it overcomes some of the challenges associated with other methods. In this protocol, we describe state-of-the-art measurement and analysis of protein self-assembly by determination of particle size distributions in highly concentrated protein solutions using FCS.

Key words

Fluorescence correlation spectroscopy Protein self-assembly Size distribution High concentration Polydispersity Gaussian distribution model Formulation 

Notes

Acknowledgments

This work has emanated from research supported by the Synthesis and Solid State Pharmaceutical Centre, the National University of Ireland Maynooth and funded by a research grant from Enterprise Ireland (EI) under Grant Number IP 2015 0358. We thank J.O. Rädler for use of his laboratory and the FCS setup at LMU Munich (Germany).

References

  1. 1.
    Shire SJ, Shahrokh Z, Liu J (2014) Challenges in the development of high protein concentration formulations. J Pharm Sci 93(6):1390–1402CrossRefGoogle Scholar
  2. 2.
    Harris RJ, Shire SJ, Winter C (2004) Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies. Drug Dev Res 61(3):137–154CrossRefGoogle Scholar
  3. 3.
    Daugherty AL, Mrsny RJ (2006) Formulation and delivery issues for monoclonal antibody therapeutics. Adv Drug Deliv Rev 58(5–6):686–706CrossRefGoogle Scholar
  4. 4.
    Lowe D, Dudgeon K, Rouet R, Schofield P, Jermutus L, Christ D (2011) Aggregation, stability, and formulation of human antibody therapeutics. Adv Protein Chem Struct Biol 84:41–61CrossRefGoogle Scholar
  5. 5.
    Staub A, Guillarme D, Schappler J, Veuthey J-L, Rudaz S (2011) Intact protein analysis in the biopharmaceutical field. J Pharm Biomed Anal 55(4):810–822CrossRefGoogle Scholar
  6. 6.
    Muneeruddin K, Thomas JJ, Salinas PA, Kaltashov IA (2014) Characterization of small protein aggregates and oligomers using size exclusion chromatography with online detection by native electrospray ionization mass spectrometry. Anal Chem 86(21):10962–10999CrossRefGoogle Scholar
  7. 7.
    Mittag JJ, Milani S, Walsh DM, Rädler JO, McManus JJ (2014) Simultaneous measurement of a range of particle sizes during Aβ1-42 fibrillogenesis quantified using fluorescence correlation spectroscopy. Biochem Biophys Res Comm 448(2):195–199CrossRefGoogle Scholar
  8. 8.
    Mittag JJ, Rädler JO, McManus JJ (2018) Peptide self-assembly measured using fluorescence correlation spectroscopy. Methods Mol Biol 1777:159–171CrossRefGoogle Scholar
  9. 9.
    Wolff M, Mittag JJ, Herling T, de Genst E, Dobson CM, Knowles TPJ, Braun D, Buell AK (2016) Quantitative thermophoretic study of disease-related protein aggregates. Sci Rep 6:22829CrossRefGoogle Scholar
  10. 10.
    Elson EL (2011) Fluorescence correlation spectroscopy: past, present and future. Biophys J 101:2855–2870CrossRefGoogle Scholar
  11. 11.
    Kim SA, Heinze KG, Schwille P (2007) Fluorescence correlation spectroscopy in living cells. Nat Methods 4:963–973CrossRefGoogle Scholar
  12. 12.
    Engelke H, Heinrich D, Rädler JO (2010) Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy. Phys Biol 7(4):046014CrossRefGoogle Scholar
  13. 13.
    Krieger JW, Langowski J (2015) QuickFit 3.0 (status: beta, compiled: 2015-03-18, SVN: 3891): a data evaluation application for biophysics. http://www.dkfz.de/Macromol/quickfit/. Accessed 2 Jan 2018Google Scholar
  14. 14.
    Provencher SW (1982) Contin: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27:229–242CrossRefGoogle Scholar
  15. 15.
    Nyeo SL, Chu B (1989) Maximum-entropy analysis of photon correlation spectroscopy data. Macromolecules 22(10):3998–4009CrossRefGoogle Scholar
  16. 16.
    Garai K, Sahoo B, Sengupta P, Maiti S (2008) Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation. J Chem Phys 128(4):045102-1-7CrossRefGoogle Scholar
  17. 17.
    Pal N, Verma SD, Singh MK, Singh MK, Sobhan S (2011) Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution. Anal Chem 83(20):7736–7744CrossRefGoogle Scholar
  18. 18.
    Sengupta P, Garai K, Balaji J, Periasamy N, Maiti S (2003) Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. Biophys J 84(3):1977–1984CrossRefGoogle Scholar
  19. 19.
    Banachowicz E, Patkowski A, Meier G, Klamecka K, Gapiński J (2014) Successful FCS experiment in nonstandard conditions. Langmuir 30(29):8945–8955CrossRefGoogle Scholar
  20. 20.
    Vira S, Mekhedov E, Humphrey G, Blank PS (2010) Fluorescent labeled antibodies – balancing functionality and degree of labeling. Anal Biochem 402(2):146–150CrossRefGoogle Scholar
  21. 21.
    Cilliers C, Nessler I, Christodolu N, Thurber GM (2017) Tracking antibody distribution with near-infrared fluorescent dyes: impact of dye structure and degree of labeling on plasma clearance. Mol Pharm 14:1623–1633CrossRefGoogle Scholar
  22. 22.
    Brinkley (1992) A brief survey of methods for preparing protein conjugate with dyes, haptens, and cross linking reagents. Bioconjug Chem 3:2CrossRefGoogle Scholar
  23. 23.
    Müller C, Eckert T, Loman A, Enderlein J, Richtering W (2008) Dual-focus fluorescence correlation spectroscopy: a robust tool for studying molecular crowding. Soft Matter 5:1358–1366CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Judith J. Mittag
    • 1
  • Matthew R. Jacobs
    • 1
  • Jennifer J. McManus
    • 2
    Email author
  1. 1.Department of ChemistryMaynooth UniversityMaynoothIreland
  2. 2.Department of ChemistryMaynooth UniversityMaynoothIreland

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