Pharmaceutical Research

, Volume 28, Issue 5, pp 1194–1210 | Cite as

The Degradation of Polysorbates 20 and 80 and its Potential Impact on the Stability of Biotherapeutics

  • Ravuri S. K. Kishore
  • Sylvia Kiese
  • Stefan Fischer
  • Astrid Pappenberger
  • Ulla Grauschopf
  • Hanns-Christian MahlerEmail author
Research Paper



To study the potential impact of the degradation of Polysorbates (PS) 20 and 80 on the stability of therapeutic proteins in parenteral formulations.


First, degradation products of PS20 and 80 were identified. Subsequently, the effect of degraded polysorbate on physical characteristics and long-term stability of protein formulations was assessed. Further, the impact of polysorbate degradation on protein stability was evaluated via shaking stress studies on formulations spiked with artificially degraded polysorbate or degradants like fatty acids. Additionally, aged formulations with reduced polysorbate content were shaken.


The degradation of polysorbate leads to a buildup of various molecules, some of which are poorly soluble, including fatty acids and polyoxyethylene (POE) esters of fatty acids. Spiking studies showed that the insoluble degradants could potentially impact protein stability and that the presence of sufficient intact polysorbate was crucial to prevent this. End-of-shelf-life shaking of protein formulations showed that the stability of various monoclonal antibodies was, however, not affected.


Although some degradants can potentially influence the stability of the protein (as discerned from spiking studies), degradation of polysorbates did not impact the stability of the different proteins tested in pharmaceutically relevant temperature and storage conditions.


auto-oxidation degradation hydrolysis polysorbate protein formulations surfactant 



The authors wish to thank Dr. Balz Fischer and B. Gessier for SBSE-GC-MS measurements, Dr. Heribert Dolt and Dr. Siegfred Stolz for FT-MS measurements, Dr. Alfred Ross for NMR measurements, Dr. Monira Siam for FT-IR measurements, Dr. Andreas Staempfli for GC-MS measurements, and Christian Lehrmayer, Thomas Steffen and Martin Weiss for their help in the experimental work.


  1. 1.
    Hillgren A, Lindgren J, Alden M. Protection mechanism of Tween 80 during freeze-thawing of a model protein, LDH. Int J Pharm. 2002;237:57–69.PubMedCrossRefGoogle Scholar
  2. 2.
    Kiese S, Pappenberger A, Friess W, Mahler HC. Shaken, not stirred: mechanical stress testing of an IgG1 antibody. J Pharm Sci. 2008;97:4347–66.PubMedCrossRefGoogle Scholar
  3. 3.
    Jones LS, Bam NB, Randolph TW. Surfactant-stabilized protein formulations: a review of protein-surfactant interactions and novel analytical methodologies. In: Shahrokh Z, Cleland JL, Shire SJ, editors. Therapeutic proteins and peptide formulation and delivery. Washington: American Chemical Society; 1997. p. 206–22.CrossRefGoogle Scholar
  4. 4.
    Kreilgaard L, Jones LS, Randolph TW, Frokjaer S, Flink JM, Manning MC, et al. Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII. J Pharm Sci. 1998;87:1597–603.PubMedGoogle Scholar
  5. 5.
    Wang W. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm. 2005;289:1–30.PubMedCrossRefGoogle Scholar
  6. 6.
    Mahler HC, Friess W, Grauschopf U, Kiese S. Protein aggregation: pathways, induction factors and analysis. J Pharm Sci. 2009;98:2909–34.PubMedCrossRefGoogle Scholar
  7. 7.
    Mahler H-C, Mueller R, Friess W, Delille A, Matheus S. Induction and analysis of aggregates in a liquid IgG1-antibody formulation. Eur J Pharm Biopharm. 2005;59:407–17.PubMedCrossRefGoogle Scholar
  8. 8.
    Maaand Y-F, Hsu CC. Protein denaturation by combined effect of shear and air-liquid interface. Biotechnol Bioeng. 1997;54:503–12.CrossRefGoogle Scholar
  9. 9.
    Cromwell MEM, Hilario E, Jacobson F. Protein aggregation and bioprocessing. Aaps J. 2006;8:E572–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Carpenter JF, Chang BS, Garzon-Rodriguez W, Randolph TW. Rational design of stable lyophilized protein formulations: theory and practice. Pharm Biotechnol. 2002;13:109–33.PubMedGoogle Scholar
  11. 11.
    Carpenter JF, Pikal MJ, Chang BS, Randolph TW. Rational design of stable lyophilized protein formulations: some practical advice. Pharm Res. 1997;14:969–75.PubMedCrossRefGoogle Scholar
  12. 12.
    Ayorinde FO, Gelain SV, Johnson Jr JH, Wan LW. Analysis of some commercial polysorbate formulations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 2000;14:2116–24.PubMedCrossRefGoogle Scholar
  13. 13.
    Brandner JD. The composition of NF-defined emulsifiers: sorbitan monolaurate, monopalmitate, monostearate, monooleate, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Drug Dev Ind Pharm. 1998;24:1049–54.PubMedCrossRefGoogle Scholar
  14. 14.
    Frison-Norrieand S, Sporns P. Investigating the molecular heterogeneity of polysorbate emulsifiers by MALDI-TOF MS. J Agric Food Chem. 2001;49:3335–40.CrossRefGoogle Scholar
  15. 15.
    Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton AC, Xu H, et al. Evaluation of hydroperoxides in common pharmaceutical excipients. J Pharm Sci. 2007;96:106–16.PubMedCrossRefGoogle Scholar
  16. 16.
    Ha E, Wang W, Wang YJ. Peroxide formation in polysorbate 80 and protein stability. J Pharm Sci. 2002;91:2252–64.PubMedCrossRefGoogle Scholar
  17. 17.
    Harmon PA, Kosuda K, Nelson E, Mowery M, Reed RA. A novel peroxy radical based oxidative stressing system for ranking the oxidizability of drug substances. J Pharm Sci. 2006;95:2014–28.PubMedCrossRefGoogle Scholar
  18. 18.
    Kerwin BA. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J Pharm Sci. 2008;97:2924–35.PubMedCrossRefGoogle Scholar
  19. 19.
    Donbrow M, Azaz E, Pillersdorf A. Autoxidation of polysorbates. J Pharm Sci. 1978;67:1676–81.PubMedCrossRefGoogle Scholar
  20. 20.
    Donbrow M, Hamburger R, Azaz E. Surface tension and cloud point changes of polyoxyethylenic nonionic surfactants during autoxidation. J Pharm Pharmacol. 1975;27:160–6.PubMedGoogle Scholar
  21. 21.
    Donbrow M, Hamburger R, Azaz E, Pillersdorf A. Development of acidity in nonionic surfactants: formic and acetic acid. Analyst (London). 1978;103:400–2.CrossRefGoogle Scholar
  22. 22.
    Kishore RSK, Pappenberger A, Dauphin IB, Ross A, Buergi B, Staempfli A, Mahler HC. Degradation of polysorbates 20 and 80: Studies on thermal auto-oxidation in bulk and hydrolysis in formulations. J Pharm Sci. 2011;100:721–31.Google Scholar
  23. 23.
    Mahler H-C, Senner F, Maeder K, Mueller R. Surface activity of a monoclonal antibody. J Pharm Sci. 2009;98:4525–33.PubMedCrossRefGoogle Scholar
  24. 24.
    Mahler HC, Huber F, Kishore RSK, Reindl J, Rückert P, Müller R. Adsorption behavior of a surfactant and a monoclonal antibody to sterilizing-grade filters. J Pharm Sci. 2010;99:2620–7.PubMedGoogle Scholar
  25. 25.
    Kiese S. Protein aggregation: induction, analytical methods and inhibition in biopharmaceutical formulations. Faculty of Chemistry und Pharmacy Vol. Doctorate, Ludwig-Maximillians-Universität München Munich, Germany, 2009, p. 298.Google Scholar
  26. 26.
    Carey FA, Sundberg RJ. Advanced organic chemistry, Springer Verlag, 2007.Google Scholar
  27. 27.
    Bates TR, Nightingale CH, Dixon E. Kinetics of hydrolysis of poly(oxyethylene) (20) sorbitan fatty acid ester surfactants. J Pharm Pharmacol. 1973;25:470–7.PubMedGoogle Scholar
  28. 28.
    Decker C, Marchal J. Autoxydation radio-induite du poly (oxyéthylène) en solution aqueuse, 7. Cinétique de la consommation d’oxygène. Die Makromolekulare Chemie. 1974;175:3531–40.CrossRefGoogle Scholar
  29. 29.
    Dulogand VL, Storck G. Die oxydation von polyepoxiden mit molekularem sauerstoff. Die Makromolekulare Chemie. 1966;91:50–73.CrossRefGoogle Scholar
  30. 30.
    Donbrow M. Stability of polyoxyethylene chain in non ionic surfactants. In: Schick MJ, editor. Nonionic surfactants: physical chemistry, vol. 23. new york: CRC; 1987. p. 1135.Google Scholar
  31. 31.
    Yao J, Dokuru DK, Noestheden M, Park SS, Kerwin BA, Jona J, et al. A quantitative kinetic study of polysorbate autoxidation: the role of unsaturated fatty acid ester substituents. Pharm Res-Dord. 2009;26:2303–13.CrossRefGoogle Scholar
  32. 32.
    Zhou Y, Woo LK, Angelici RJ. Solid acid catalysis of tandem isomerization-lactonization of olefinic acids. Applied Catalysis A. 2007.Google Scholar
  33. 33.
    Shepherdand IS, Showell JS. The mechanism of the aqueous perchloric acid isomerization of oleic acid to -stearolactone. Journal of the American Oil Chemists’ Society. 1969;46:479–81.CrossRefGoogle Scholar
  34. 34.
    Arudi RL, Sutherland MW, Bielski BH. Purification of oleic acid and linoleic acid. J Lipid Res. 1983;24:485.PubMedGoogle Scholar
  35. 35.
    Li S, Schöneich C, Borchardt RT. Chemical instability of protein pharmaceuticals: mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng. 2004;48:490–500.CrossRefGoogle Scholar
  36. 36.
    Tomita M, Irie M, Ukita T. Sensitized photooxidation of histidine and its derivatives. Products and mechanism of the reaction. Biochemistry. 1969;8:5149–60.PubMedCrossRefGoogle Scholar
  37. 37.
    Müller R, Karle A, Vogt A, Kropshofer H, Ross A, Maeder K, et al. Evaluation of the immuno-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout. J Pharm Sci. 2009;98:3548–61.CrossRefGoogle Scholar
  38. 38.
    Porter NA, Cladwell SE, Mills KA. Mechanisms of free-radical oxidation of unsaturated lipids. Amer Oil Chemists Soc. 1995;30:277–90.Google Scholar
  39. 39.
    O’Brien EP, Dima RI, Brooks B, Thirumalai D. Interactions between hydrophobic and ionic solutes in aqueous guanidinium chloride and urea solutions: lessons for protein denaturation mechanism. J Am Chem Soc. 2007;129:7346–53.PubMedCrossRefGoogle Scholar
  40. 40.
    Mirgorodskaya AB, Yatskevich EI, Zakharova LY. The solubilization of fatty acids in systems based on block copolymers and nonionic surfactants. Russ J Phys Chem A. 2010;84:2066–70.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ravuri S. K. Kishore
    • 1
  • Sylvia Kiese
    • 2
  • Stefan Fischer
    • 2
  • Astrid Pappenberger
    • 2
  • Ulla Grauschopf
    • 1
  • Hanns-Christian Mahler
    • 1
    Email author
  1. 1.Pharmaceutical & Device Development, Pharma Technical DevelopmentF. Hoffmann-La Roche LtdBaselSwitzerland
  2. 2.Formulation Research, Pharma Research and Early DevelopmentF. Hoffmann-La Roche LtdBaselSwitzerland

Personalised recommendations