Reviews
Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways

https://doi.org/10.1002/jps.21190Get rights and content

ABSTRACT

Polysorbates 20 and 80 (Tween® 20 and Tween® 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97: 2924-2935, 2008

Section snippets

INTRODUCTION

The proper formulation of recombinant proteins for pharmaceutical products is vital to their long-term stability and reproducible activity. To maintain biological activity, proteins generally must be maintained in a specific, three-dimensional conformation. This conformation is only marginally stable, and thus relatively minor perturbing forces can disrupt protein structure causing loss of biological activity or an immunological response. Such perturbations are commonly encountered as proteins

POLYSORBATE STRUCTURE

The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan.21 Polysorbate 20 (polyoxyethylene sorbitan monolaurate) and Polysorbate 80 (polyoxyethylene sorbitan monooleate) are the most common polysorbates currently used in formulation of protein biopharmaceuticals.22 Their structures are shown in Figure 1. The structures shown here are those of the chemically homogenous polysorbates. Solutions of polysorbate sold by manufacturers and

SOLUTION AND SURFACE ACTIVE PROPERTIES OF POLYSORBATE

Because of their dual hydrophobic/hydrophilic nature, surfactants in solution tend to orient themselves so that the exposure of the hydrophobic portion of the surfactant to the aqueous solution is minimized.20 In systems containing air/water interfaces, surfactants will tend to accumulate at these interfaces, forming a surface layer of surfactant oriented in such a fashion that only their hydrophilic ends are exposed to water.20, 27, 28 Such orientation and hydrophobic surface adsorption can

CHEMICAL STABILITY OF POLYSORBATES

Chemical stability of the polysorbates is another important consideration for their use. The polysorbates are notorious for undergoing autooxidation34, 73., 74., 75., 76., 77., 78., 79. and cleavage at the ethylene oxide subunits34, 74 as well as hydrolysis of the fatty acid ester bond.34, 80 A generalized reaction scheme depicting these processes is shown in Figure 6. Autooxidation of the ethylene oxide results in hydroperoxide formation, side-chain cleavage and eventually formation of short

CONCLUDING REMARKS

Polysorbates are present in a large number of biopharmaceutical drugs listed in the 2006 Physicians Desk Reference. The concentrations used in the formulations range from 0.0003% (w/v) to 0.3% (w/v). Over the past decade the quality of polysorbate solutions has increased dramatically such that many manufacturers now offer highly purified, low peroxide, and low acid content solutions. While the solutions are initially low in reactive oxygen species care must still be used when storing drug

ACKNOWLEDGEMENTS

The author would like to thank Dr. Sungae Park for the information from the European Pharmacopoeia, Arnold McAuley, and Hyo Jin Le for the surface tension measurements and Dr. Darren Reid, Dr. David Brems, and Dr. Richard Remmele for many useful discussions and critical reading of the manuscript.

REFERENCES (91)

  • S. Paria et al.

    A review on experimental studies of surfactant adsorption at the hydrophilic solid-water interface

    Adv Colloid Interface Sci

    (2004)
  • H.C. Mahler et al.

    Induction and analysis of aggregates in a liquid IgG1-antibody formulation

    Eur J Pharm Biopharm

    (2005)
  • B.A. Kerwin et al.

    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)
  • D.K. Chou et al.

    Effects of Tween 20 and Tween 80 on the stability of Albutropin during agitation

    J Pharm Sci

    (2005)
  • B.S. Chang et al.

    Surface-induced denaturation of proteins during freezing and its inhibition by surfactants

    J Pharm Sci

    (1996)
  • S.Y. Patro et al.

    Protein formulation and fill-finish operations

    Biotechnol Annu Rev

    (2002)
  • P.S. Norman et al.

    Human serum albumin and Tween 80 as stabilizers of allergen solutions

    J Allergy Clin Immun

    (1978)
  • P.A. Gunning et al.

    The effect of surfactant type on protein displacement from the air-water interface

    Food Hydrocolloids

    (2004)
  • K.L. Mittal

    Determination of CMC of polysorbate 20 in aqueous solution by surface tension method

    J Pharm Sci

    (1972)
  • A.I. Rusanov

    Determination of micellar characteristics by measuring quantities related to the surfactant chemical potential

    Mendeleev Commun

    (1996)
  • R.M.M. Brito et al.

    Determination of the critical micelle concentration of surfactants using the fluorescent probe N phenyl-1-naphthylamine

    Anal Biochem

    (1986)
  • A. Chattopadhyay et al.

    Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge

    Anal Biochem

    (1984)
  • P.J. Tummino et al.

    Determination of the aggregation number of detergent micelles using steady-state fluorescence quenching

    Biophys J

    (1993)
  • X. Zhang et al.

    Determination of surfactant critical micelle concentration by a novel fluorescence depolarization technique

    J Biochem Biophys Methods

    (1996)
  • E. Fuguet et al.

    Critical micelle concentration of surfactants in aqueous buffered and unbuffered systems

    Anal Chim Acta

    (2005)
  • H. Heerklotz et al.

    Titration calorimetry of surfactant-membrane partitioning and membrane solubilization

    Biochim Biophys Acta

    (2000)
  • X. Li et al.

    Isothermal titration calorimetry and dynamic light scattering studies of interactions between gemini surfactants of different structure and Pluronic block copolymers

    J Colloid Interface Sci

    (2005)
  • S. Reis et al.

    Noninvasive methods to determine the critical micelle concentration of some bile acid salts

    Anal Biochem

    (2004)
  • A. Helenius et al.

    Properties of detergents

    Methods Enzymol

    (1979)
  • J. Jaeger et al.

    Peroxide accumulation in detergents

    J Biochem Biophys Methods

    (1994)
  • M. Lever

    Peroxides in detergents as interfering factors in biochemical analysis

    Anal Biochem

    (1977)
  • A. Magill et al.

    Spectrophotometric method for quantitation of peroxides in sorbitan monooleate and monostearate

    J Pharm Sci

    (1984)
  • M. Hu et al.

    High-performance liquid chromatographic determination of polysorbate 80 in pharmaceutical suspensions

    J Chromatogr A

    (2003)
  • P.A. Harmon et al.

    A novel peroxy radical based oxidative stressing system for ranking the oxidizability of drug substances

    J Pharm Sci

    (2006)
  • N.A. Porter et al.

    Phospholipid oxidation

    Adv Free Rad Bio

    (1986)
  • A.G. McLeod et al.

    Loss of factor VIII activity during storage in PVC containers due to adsorption

    Haemophilia

    (2000)
  • Y.-.F. Maa et al.

    Protein denaturation by combined effect of shear and air-liquid interface

    Biotechnol Bioeng

    (1997)
  • H. Thurow et al.

    Stabilisation of dissolved proteins against denaturation at hydrophobic interfaces

    Diabetologia

    (1984)
  • B.M. Eckhardt et al.

    Effect of freezing on aggregation of human growth hormone

    Pharm Res

    (1991)
  • K.-.I. Izutsu et al.

    Stabilizing effect of amphiphilic excipients on the freeze-thawing and freeze-drying of lactate dehydrogenase

    Biotechnol Bioeng

    (1994)
  • S. Nema et al.

    Freeze-thaw studies of a model protein, lactate dehydrogenase, in the presence of cryoprotectants

    J Parent Sci Technol

    (1993)
  • J.F. Carpenter et al.

    Rational design of stable lyophilized protein formulations: Theory and practice

    Pharm Biotechnol

    (2002)
  • J.F. Carpenter et al.

    Rational design of stable lyophilized protein formulations: Some practical advice

    Pharm Res

    (1997)
  • J. Broadhead et al.

    The effect of process and formulation variables on the properties of spray-dried beta-galactosidase

    J Pharm Pharmacol

    (1994)
  • Y.F. Maa et al.

    Effect of spray drying and subsequent processing conditions on residual moisture content and physical/biochemical stability of protein inhalation powders

    Pharm Res

    (1998)
  • Cited by (605)

    View all citing articles on Scopus

    Published online in Wiley InterScience (www.interscience.wiley.com).

    View full text