Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products


Particle size distribution, a measurable physicochemical quantity, is a critical quality attribute of drug products that needs to be controlled in drug manufacturing. The non-invasive methods of dynamic light scattering (DLS) and Diffusion Ordered SpectroscopY (DOSY) NMR can be used to measure diffusion coefficient and derive the corresponding hydrodynamic radius. However, little is known about their use and sensitivity as analytical tools for particle size measurement of formulated protein therapeutics. Here, DLS and DOSY-NMR methods are shown to be orthogonal and yield identical diffusion coefficient results for a homogenous monomeric protein standard, ribonuclease A. However, different diffusion coefficients were observed for five insulin drug products measured using the two methods. DOSY-NMR yielded an averaged diffusion coefficient among fast exchanging insulin oligomers, ranging between dimer and hexamer in size. By contrast, DLS showed several distinct species, including dimer, hexamer, dodecamer and other aggregates. The heterogeneity or polydisperse nature of insulin oligomers in formulation caused DOSY-NMR and DLS results to differ from each other. DLS measurements provided more quality attributes and higher sensitivity to larger aggregates than DOSY-NMR. Nevertheless, each method was sensitive to a different range of particle sizes and complemented each other. The application of both methods increases the assurance of complex drug quality in this similarity comparison.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3



Dynamic light scattering


Diffusion Ordered SpectroscopY


Nuclear magnetic resonance


  1. 1.

    Woodcock J, Griffin J, Behrman R, Cherney B, Crescenzi T, Fraser B, et al. The FDA’s assessment of follow-on protein products: a historical perspective. Nat Rev Drug Discov. 2007;6(6):437–42.

    Article  PubMed  Google Scholar 

  2. 2.

    Berkowitz SA, Engen JR, Mazzeo JR, Jones GB. Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nat Rev Drug Discov. 2012;11(7):527–40.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Ahmadi M, Bryson CJ, Cloake EA, Welch K, Filipe V, Romeijn S, et al. Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics. Pharm Res. 2015;32(4):1383–94.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8(3):E501–7.

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Hjorth CF, Norrman M, Wahlund PO, Benie AJ, Petersen BO, Jessen CM, et al. Structure, aggregation, and activity of a covalent insulin dimer formed during storage of neutral formulation of human insulin. J Pharm Sci. 2016;105(4):1376–86.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Philo JS. Is any measurement method optimal for all aggregate sizes and types? AAPS J. 2006;8(3):E564–71.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Gilard V, Trefi S, Balayssac S, Delsuc MA, Gostan T, Malet-Martino M, et al. Chapter 6—DOSY NMR for drug analysis A2—Holzgrabe, Ulrike. In: Wawer I, Diehl B, editors. NMR Spectroscopy in pharmaceutical analysis. Amsterdam: Elsevier; 2008. p. 269–89.

    Google Scholar 

  8. 8.

    Arakawa T, Philo JS, Ejima D, Tsumoto K, Arisaka F. Aggregation analysis of therapeutic proteins, part 2. Bioprocess Int. 2007;5(4):36–47.

    CAS  Google Scholar 

  9. 9.

    Clark TD, Bartolotti L, Hicks RP. The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids. Biopolymers. 2013;99(8):548–61.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Li X, Shantz DF. PFG NMR investigations of tetraalkylammonium-silica mixtures. J Phys Chem C. 2010;114(18):8449–58.

    CAS  Article  Google Scholar 

  11. 11.

    Li CG, Pielak GJ. Using NMR to distinguish viscosity effects from nonspecific protein binding under crowded conditions. J Am Chem Soc. 2009;131(4):1368–9.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Bocian W, Sitkowski J, Tarnowska A, Bednarek E, Kawecki R, Kozminski W, et al. Direct insight into insulin aggregation by 2D NMR complemented by PFGSE NMR. Proteins. 2008;71(3):1057–65.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Berne BJ, Pecora R. Dynamic light scattering: with applications to chemistry, biology, and physics. New York: Dover Publications; 2000.

    Google Scholar 

  14. 14.

    Panchal J, Kotarek J, Marszal E, Topp EM. Analyzing subvisible particles in protein drug products: a comparison of dynamic light scattering (DLS) and resonant mass measurement (RMM). AAPS J. 2014;16(3):440–51.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Hinton DPJ, C.S. Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions. J Phys Chem. 1993;97:9064–72.

    CAS  Article  Google Scholar 

  16. 16.

    Hawe A, Hulse WL, Jiskoot W, Forbes RT. Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates. Pharm Res. 2011;28(9):2302–10.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Demeester JDSS, Sanders N, Haustraete J. Methods for structural analysis of protein pharmaceuticals. Arlington: AAPS; 2005.

    Google Scholar 

  18. 18.

    Chang X, Jorgensen AM, Bardrum P, Led JJ. Solution structures of the R6 human insulin hexamer. Biochemistry. 1997;36(31):9409–22.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Xu Y, Yan Y, Seeman D, Sun L, Dubin PL. Multimerization and aggregation of native-state insulin: effect of zinc. Langmuir. 2012;28(1):579–86.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Derewenda U, Derewenda Z, Dodson EJ, Dodson GG, Reynolds CD, Smith GD, et al. Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer. Nature. 1989;338(6216):594–6.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Teska BM, Alarcon J, Pettis RJ, Randolph TW, Carpenter JF. Effects of phenol and meta-cresol depletion on insulin analog stability at physiological temperature. J Pharm Sci. 2014;103(8):2255–67.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Lin MF, Larive CK. Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy. Anal Biochem. 1995;229(2):214–20.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Hassiepen U, Federwisch M, Mulders T, Wollmer A. The lifetime of insulin hexamers. Biophys J. 1999;77(3):1638–54.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Whittingham JL, Edwards DJ, Antson AA, Clarkson JM, Dodson GG. Interactions of phenol and m-cresol in the insulin hexamer, and their effect on the association properties of B28 pro --> Asp insulin analogues. Biochemistry. 1998;37(33):11516–23.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Gualandi-Signorini AM, Giorgi G. Insulin formulations—a review. Eur Rev Med Pharmacol Sci. 2001;5(3):73–83.

    CAS  PubMed  Google Scholar 

  26. 26.

    Smith GD, Swenson DC, Dodson EJ, Dodson GG, Reynolds CD. Structural stability in the 4-zinc human insulin hexamer. Proc Natl Acad Sci U S A. 1984;81(22):7093–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Ciszak E, Beals JM, Frank BH, Baker JC, Carter ND, Smith GD. Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin. Structure. 1995;3(6):615–22.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Palmieri LC, Favero-Retto MP, Lourenco D, Lima LM. A T3R3 hexamer of the human insulin variant B28Asp. Biophys Chem. 2013;173-174:1–7.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Ortega A, Amoros D. Garcia de la Torre J. Prediction of hydrodynamic and other solution properties of rigid proteins from atomic- and residue-level models. Biophys J. 2011;101(4):892–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Garcia de la Torre J, Huertas ML, Carrasco B. HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations. J Magn Reson. 2000;147(1):138–46.

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Nauman JV, Campbell PG, Lanni F, Anderson JL. Diffusion of insulin-like growth factor-I and ribonuclease through fibrin gels. Biophys J. 2007;92(12):4444–50.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    van Beers MM, Bardor M. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins. Biotechnol J. 2012;7(12):1473–84.

    Article  PubMed  Google Scholar 

  33. 33.

    Wang W, Singh SK, Li N, Toler MR, King KR, Nema S. Immunogenicity of protein aggregates—concerns and realities. Int J Pharm. 2012;431(1–2):1–11.

    CAS  PubMed  Google Scholar 

Download references


We thank Darón Freedberg, Marcos Battistel, and Hugo Azurmendi for the assistance in setting up the DOSY-NMR experiments and for their helpful discussions. Support for this work comes from the US FDA CDER Critical Path funds and is gratefully acknowledged.

Author information



Corresponding author

Correspondence to Kang Chen.

Ethics declarations


This article reflects the views of the author and should not be construed to represent US FDA’s views or policies.

Electronic Supplementary Material


Bruker 2D DOSY-NMR pulse sequence employed for data acquisition. Individual diffusion coefficients obtained from NMR and DLS. (DOCX 66 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Patil, S.M., Keire, D.A. & Chen, K. Comparison of NMR and Dynamic Light Scattering for Measuring Diffusion Coefficients of Formulated Insulin: Implications for Particle Size Distribution Measurements in Drug Products. AAPS J 19, 1760–1766 (2017).

Download citation


  • diffusion
  • particle size distribution
  • physicochemical equivalence
  • protein drug product
  • similarity