Abstract
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.
Similar content being viewed by others
Abbreviations
- DLS:
-
Dynamic light scattering
- DOSY:
-
Diffusion Ordered SpectroscopY
- NMR:
-
Nuclear magnetic resonance
References
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.
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.
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.
Rosenberg AS. Effects of protein aggregates: an immunologic perspective. AAPS J. 2006;8(3):E501–7.
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.
Philo JS. Is any measurement method optimal for all aggregate sizes and types? AAPS J. 2006;8(3):E564–71.
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.
Arakawa T, Philo JS, Ejima D, Tsumoto K, Arisaka F. Aggregation analysis of therapeutic proteins, part 2. Bioprocess Int. 2007;5(4):36–47.
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.
Li X, Shantz DF. PFG NMR investigations of tetraalkylammonium-silica mixtures. J Phys Chem C. 2010;114(18):8449–58.
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.
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.
Berne BJ, Pecora R. Dynamic light scattering: with applications to chemistry, biology, and physics. New York: Dover Publications; 2000.
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.
Hinton DPJ, C.S. Diffusion ordered 2D NMR spectroscopy of phospholipid vesicles: determination of vesicle size distributions. J Phys Chem. 1993;97:9064–72.
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.
Demeester JDSS, Sanders N, Haustraete J. Methods for structural analysis of protein pharmaceuticals. Arlington: AAPS; 2005.
Chang X, Jorgensen AM, Bardrum P, Led JJ. Solution structures of the R6 human insulin hexamer. Biochemistry. 1997;36(31):9409–22.
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.
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.
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.
Lin MF, Larive CK. Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy. Anal Biochem. 1995;229(2):214–20.
Hassiepen U, Federwisch M, Mulders T, Wollmer A. The lifetime of insulin hexamers. Biophys J. 1999;77(3):1638–54.
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.
Gualandi-Signorini AM, Giorgi G. Insulin formulations—a review. Eur Rev Med Pharmacol Sci. 2001;5(3):73–83.
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.
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.
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.
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.
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.
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.
van Beers MM, Bardor M. Minimizing immunogenicity of biopharmaceuticals by controlling critical quality attributes of proteins. Biotechnol J. 2012;7(12):1473–84.
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.
Acknowledgements
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
Authors and Affiliations
Corresponding author
Ethics declarations
Disclaimer
This article reflects the views of the author and should not be construed to represent US FDA’s views or policies.
Electronic Supplementary Material
ESM 1
Bruker 2D DOSY-NMR pulse sequence employed for data acquisition. Individual diffusion coefficients obtained from NMR and DLS. (DOCX 66 kb)
Rights and permissions
About this article
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). https://doi.org/10.1208/s12248-017-0127-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1208/s12248-017-0127-z