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Considerations for the Use of Polysorbates in Biopharmaceuticals

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Abstract

Purpose

Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations.

Methods

A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations.

Results

Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients.

Conclusions

An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

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Abbreviations

CHO:

Chinese hamster ovary

CMC:

Critical micelle concentrations

DOE:

Design of experiments

EP:

European pharmacopeia

FFA:

Free fatty acid

HCCF:

Harvested cell culture fluid

HCP:

Host cell protein

HLB:

High hydrophile-lipophile balance

JP:

Japanese pharmacopeia

LC:

Liquid chromatography

LPLA2 :

Lysosomal phospholipase A2

PLBL2:

Phospholipase B-like 2

POE:

Polyoxyethylene

PS20:

Polysorbate 20

PS80:

Polysorbate 80

USP:

United States pharmacopeia

References

  1. 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.

    Article  CAS  Google Scholar 

  2. Charman SA, Mason KL, Charman WN. Techniques for assessing the effects of pharmaceutical excipients on the aggregation of porcine growth hormone. Pharm Res. 1993;10:954–62.

    Article  CAS  Google Scholar 

  3. Chi EY, Krishnan S, Randolph TW, Carpenter JF. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. Pharm Res. 2003;20:1325–36.

    Article  CAS  Google Scholar 

  4. Katakam M, Bell LN, Banga AK. Effect of surfactants on the physical stability of recombinant human growth hormone. J Pharm Sci. 1995;84:713–6.

    Article  CAS  Google Scholar 

  5. Mahler HC, Muller 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.

    Article  CAS  Google Scholar 

  6. Bam NB, Cleland JL, Yang J, Manning MC, Carpenter JF, Kelley RF, et al. Tween protects recombinant human growth hormone against agitation-induced damage via hydrophobic interactions. J Pharm Sci. 1998;87:1554–9.

    Article  CAS  Google Scholar 

  7. Chang BS, Kendrick BS, Carpenter JF. Surface-induced denaturation of proteins during freezing and its inhibition by surfactants. J Pharm Sci. 1996;85:1325–30.

    Article  CAS  Google Scholar 

  8. Kerwin BA, Heller MC, Levin SH, Randolph TW. Effects of tween 80 and sucrose on acute short-term stability and long-term storage at −20 degrees C of a recombinant hemoglobin. J Pharm Sci. 1998;87:1062–8.

    Article  CAS  Google Scholar 

  9. 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.

    Article  CAS  Google Scholar 

  10. Carpenter JF, Arakawa T, Crowe JH. Interactions of stabilizing additives with proteins during freeze-thawing and freeze-drying. Dev Biol Stand. 1992;74:225–38. discussion 238-229

    CAS  PubMed  Google Scholar 

  11. Katakamand M., Banga AK. Use of poloxamer polymers to stabilize recombinant human growth hormone against various processing stresses. Pharm Dev Technol 1997;2:143–149.

    Article  CAS  Google Scholar 

  12. Levine HL, Ransohoff TC, Kawahata RT, Mcgregor WC. The use of surface-tension measurements in the design of antibody-based product formulations. J Parenter Sci Technol 1991;45:160–165.

  13. Bam NB, Randolph TW, Cleland JL. Stability of protein formulations: investigation of surfactant effects by a novel EPR spectroscopic technique. Pharm Res. 1995;12:2–11.

    Article  CAS  Google Scholar 

  14. Ian M. Tucker, Jordan T. Petkov, Jeffrey Penfold, Robert K. Thomas, Peixun Li, Andrew R. Cox, Nick Hedges, John R. P. Webster Spontaneous surface self-assembly in protein−surfactant mixtures: interactions between hydrophobin and ethoxylated polysorbate surfactants J Phys Chem B 2014;118(18), 4867–4875.

  15. Jones LS, Bam NB, Randolph TW. Surfactant-stabilized protein formulations: a review of protein-surfactants interactions and novel analytical methodologies. ACS Symp Ser. 1997;675:206–22.

    Article  CAS  Google Scholar 

  16. Ludwig DB, Carpenter JF, Hamel JB, Randolph TW. Protein adsorption and excipient effects on kinetic stability of silicone oil emulsions. J Pharm Sci. 2010;99:1721–33.

    Article  CAS  Google Scholar 

  17. Mahler HC, Huber F, Kishore RS, Reindl J, Ruckert P, Muller R. Adsorption behavior of a surfactant and a monoclonal antibody to sterilizing-grade filters. J Pharm Sci. 2010;99:2620–7.

    Article  CAS  Google Scholar 

  18. Randolphand TW, Jones LS. Surfactant-protein interactions. Pharm Biotech. 2002;13:159–75.

    Article  Google Scholar 

  19. Silha J, Bares M, Zeman I, Smidrkal J. The Hlb number determination of polyoxyethylene surfactants. Collect Czechoslov Chem Commun. 1989;54:945–52.

    Article  CAS  Google Scholar 

  20. Mittal KL. Determination of CMC of polysorbate 20 in aqueous solution by surface tension method. J Pharm Sci. 1972;61:1334–5.

    Article  CAS  Google Scholar 

  21. Borisov OV, Ji JA, Wang YJ, Vega F, Ling VT. Toward understanding molecular heterogeneity of polysorbates by application of liquid chromatography-mass spectrometry with computer-aided data analysis. Anal Chem. 2011;83:3934–42.

    Article  CAS  Google Scholar 

  22. 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.

    Article  CAS  Google Scholar 

  23. Hewitt D, Alvarez M, Robinson K, Ji J, Wang YJ, Kao YH, et al. Mixed-mode and reversed-phase liquid chromatography-tandem mass spectrometry methodologies to study composition and base hydrolysis of polysorbate 20 and 80. J Chromatogr A. 2011;1218:2138–45.

    Article  CAS  Google Scholar 

  24. Li Y, Hewitt D, Lentz YK, Ji JA, Zhang TY, Zhang K. Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry. Anal Chem. 2014;86:5150–7.

    Article  CAS  Google Scholar 

  25. C. USP. The United States Pharmacopeia. USP 40 NF 35, 2017; NF Monograph: Polysorbate 80.

  26. C.O. Europe. European pharmacopoeia (PhEur) European Medicines Agency 9th ed. 2017;2267–2271.

  27. Japanese Pharmacopeia 17th Edition, March 7, 2016, the MHLW Ministerial Notification No. 64.

  28. Polysorbate 80 for Injection [9005–65-6], Chinese Pharmacopeia Volume 4, 2015.

  29. Kerwin BA. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J Pharm Sci. 2008;97:2924–35.

    Article  CAS  Google Scholar 

  30. Bates, TR, Nightingale, CH, Dixon E. Kinetics of hydrolysis of polyoxyethylene (20) sorbitan fatty acid ester surfactantsJ Pharm Pharmacol 1973;25, 470–477.

    Article  CAS  Google Scholar 

  31. LaBrenz S. Ester hydrolysis of polysorbate 80 in mAb drug product: evidence in support of the hypothesized risk after the observation of visible particulate in mAb formulations. J Pharm Sci. 2014;103:2268–77.

    Article  CAS  Google Scholar 

  32. Hall T, Sandefur SL, Frye CC, Tuley TL, Huang L. Polysorbates 20 and 80 degradation by group XV lysosomal phospholipase A2 isomer X1 in monoclonal antibody formulations. J Pharm Sci 2016;105;1633–1642.

    Article  CAS  Google Scholar 

  33. Dixit N, Salamat-Miller N, Salinas PA, Taylor KD. Residual host cell protein promotes polysorbate 20 degradation in a sulfatase drug product leading to free fatty acid particles. J Pharm Sci. 2016;105:1657–66.

    Article  CAS  Google Scholar 

  34. Tomlinson A, Demeule B, Lin B, Yadav S. polysorbate 20 degradation in biopharmaceutical formulations: quantification of free fatty acids, characterization of particulates, and insights into the degradation mechanism. Mol Pharm. 2015;12(11):3805–15.

    Article  CAS  Google Scholar 

  35. Doshi N, Demeule B, Yadav S. Understanding particle formation: solubility of free fatty acids as polysorbate 20 degradation byproducts in therapeutic monoclonal antibody formulations. Mol Pharm. 2015;12(11):3792–804.

    Article  CAS  Google Scholar 

  36. Saggu M, Liu J, Patel A. Identification of subvisible particle in biopharmaceutical formulations using raman spectroscopy provides insight into polysorbate 20 degradation pathway. J Pharm Sci. 2015;104(4):1282–90.

    Article  Google Scholar 

  37. Chiu J, Valente KN, Levy NE, Min L, Lenhoff AM, Lee KH. Knockout of a difficult-to-remove CHO host cell protein, lipoprotein, lipase for improved polysorbate stability in monoclonal antibody formulations. Biotechnol Bioeng. 2017;114:1006–15.

    Article  CAS  Google Scholar 

  38. Levy NE, Valente KN, Lee KH, Lenhoff AM. Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification. Biotechnol Bioeng. 2016;113:1260–72.

    Article  CAS  Google Scholar 

  39. Valente KN, Lenhoff AM, Lee KH. Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification. Biotechnol Bioeng. 2016;113:1260–72.

    Article  Google Scholar 

  40. Siska Christine C, Pierini Christopher J, Lau Hollis R, Latypov Ramil F, Fesinmeyer RM, Litowski JR. Free fatty acid particles in protein formulations, part 2: contribution of polysorbate raw material. J Pharm Sci. 2015;104(2):447–56.

    Article  CAS  Google Scholar 

  41. Mueller R, Karle A, Vogt A, Kropshofer H, Ross A, Maeder K, et al. Evaluation of the immune-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout. J Pharm Sci. 2009;98:3548–61.

    Article  CAS  Google Scholar 

  42. Matthew FR. Free fatty acid components of Polysorbate-20 linked to particle formation in drug product. Polysorbate 80 Hydrolysis And Considerations For Control Strategy Development. AAPS NBS, June 9th. 2015.

  43. Corvari V. Polysorbate 80 hydrolysis and considerations for control strategy development. AAPS NBC Boston, MA. 2016.

  44. Kishore RS, Pappenberger A, Dauphin IB, Ross A, Buergi B, Staempfli A, et al. Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis. J Pharm Sci. 2011;100:721–31.

    Article  CAS  Google Scholar 

  45. Treuheit MJ, Kosky AA, Brems DN. Inverse relationship of protein concentration and aggregation. Pharm Res. 2002;19:511–6.

    Article  CAS  Google Scholar 

  46. Zhang L, Yadav S, Demeule B, Wang JY, Mozziconacci O, Schoneich C. Degradation mechanisms of polysorbate 20 differentiated by 18O-labeling and mass spectrometry. Pharm Res. 2017;34:84–100.

    Article  CAS  Google Scholar 

  47. Wang W, Wang JY, Wang DQ. Dual effects of tween 80 on protein stability. Int J Pharm. 2008;347:31–8.

    Article  CAS  Google Scholar 

  48. Kishore RS, Kiese S, Fisher S, Pappenberger A, Grauschopf U, Mahler HC. The degradation of polysorbate 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res. 2011;28:1194–210.

    Article  CAS  Google Scholar 

  49. Agarkhed M, O’Dell C, Hsich MC, Zhang J, Goldstein J, Srivastava A. Effect of polysorbate 80 concentration on thermal and photostability of a monoclonal antibody. AAPS PharmSciTech. 2013;14(1):1–9.

    Article  CAS  Google Scholar 

  50. Brito RMM, Vaz WLC. Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine. Anal Biochem. 1986;152:250–5.

    Article  CAS  Google Scholar 

  51. Wenger MD, Bowman AM, Thorsteinsson MV, Little KK, Wang L, Zhong J, et al. An automated homogeneous method for quantifying polysorbate using fluorescence polarization. Anal Biochem. 2005;337:48–54.

    Article  CAS  Google Scholar 

  52. Adamo M, Dick LW Jr, Qiu D, Lee AH, Devincentis J, Cheng KC. A simple reversed phase high-performance liquid chromatography method for polysorbate 80 quantitation in monoclonal antibody drug products. J Chromatogr B. 2010;878:1865–70.

    Article  CAS  Google Scholar 

  53. Tani TH, Moore JM, Patapoff TW. Single step method for the accurate concentration determination of polysorbate 80. J Chromatogr A. 1997;786:99–106.

    Article  CAS  Google Scholar 

  54. Takeda Y, Abe Y, Ishiwata H, Yamada T. Determination method of polysorbates in powdered soup by HPLC. J Food Hyg Soc Jpn. 2001;42:91–5.

    Article  CAS  Google Scholar 

  55. Brown EG, Hayes TJ. The absorptiometric determination of polyethyleneglycol mono-oleate. Analyst. 1955;80:755–67.

    Article  CAS  Google Scholar 

  56. Greff RA, Setzkorn EA, Leslie WD. A colorimetric method for the determination of parts/million of nonionic surfactants. J Am Oil Chem Soc 1965; 42:180–185.

    Article  CAS  Google Scholar 

  57. Kim J, Qiu J. Quantitation of low concentrations of polysorbates in high protein concentration formulations by solid phase extraction and cobalt-thiocyanate derivatization. Anal Chim Acta. 2014;806:144–51.

    Article  CAS  Google Scholar 

  58. Savjani N, Babcock E, Khor HK, Raghani A. Use of ferric thiocyanate derivatization for quantification of polysorbate 80 in high concentration protein formulations. Talanta. 2014;130:542–6.

    Article  CAS  Google Scholar 

  59. Nair LM, Stephens NV, Vincent S, Raghavan N, Sand PJ. Determination of polysorbate 80 in parenteral formulations by high-performance liquid chromatography and evaporative light scattering detection. J Chromatogr A. 2003;1012:81–6.

    Article  CAS  Google Scholar 

  60. Hewitt D, Zhang T, Kao YH. Quantitation of polysorbate 20 in protein solutions using mixed-mode chromatography and evaporative light scattering detection. J Chromatogr A. 2008;1215:156–60.

    Article  CAS  Google Scholar 

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Acknowledgments and Disclosures

The Authors would like to acknowledge Mark Bolgar and Rajesh Gandhi from Bristol Meyers Squibb for helpful discussions during manuscript preparation.

Author information

Authors and Affiliations

  1. Pfizer, Inc, New York, New York, USA

    Michael T. Jones

  2. Lonza, Basel, Switzerland

    Hanns-Christian Mahler, Atanas Koulov & Satish K. Singh

  3. Genentech, South San Francisco, California, USA

    Sandeep Yadav & John Wang

  4. Bristol Meyers Squibb, New York, New York, USA

    Dilbir Bindra

  5. Eli Lilly and Company, Indianapolis, Indiana, USA

    Vincent Corvari

  6. Amgen, Thousand Oaks, California, USA

    R. Matthew Fesinmeyer & Ping Y. Yeh

  7. Biogen Idec, Cambridge, Massachusetts, USA

    Kapil Gupta & Kevin Maloney

  8. Johnson & Johnson, New Brunswick, New Jersey, USA

    Alexander M. Harmon, Kenneth D. Hinds & Wei Liu

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  1. Michael T. Jones
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  2. Hanns-Christian Mahler
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Correspondence to Michael T. Jones.

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Jones, M.T., Mahler, HC., Yadav, S. et al. Considerations for the Use of Polysorbates in Biopharmaceuticals. Pharm Res 35, 148 (2018). https://doi.org/10.1007/s11095-018-2430-5

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  • DOI: https://doi.org/10.1007/s11095-018-2430-5

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