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Interaction of Polysorbate 80 with Erythropoietin: A Case Study in Protein–Surfactant Interactions

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Purpose

The cause of antibody positive pure red cell aplasia associated with the subcutaneous administration of EPREX® to patients with chronic kidney failure has been determined to be due to the leaching of weakly adjuvant compounds from the uncoated rubber stoppers that were formerly used in prefilled syringes. Other researchers have suggested that polysorbate 80 micelles containing erythropoietin may be a causative factor. The purpose of this work was to repeat previously published studies in a more controlled manner and to define the precise nature of the interactions between polysorbate 80 and erythropoietin.

Methods

The contents of EPREX® prefilled syringes and laboratory-prepared, well-characterized formulations of EPREX® were analyzed by size exclusion chromatography. Fractions were analyzed for the presence of erythropoietin by ELISA. EPREX® formulations prepared with increasing amounts of polysorbate 80 were analyzed by light scattering.

Results

Well-controlled chromatographic studies showed that when EPREX® formulations containing no aggregate were analyzed by high-performance liquid chromatography, erythropoietin monomer could not be detected under the polysorbate 80 peak. Dimer and oligomers of erythropoietin coeluted under the polysorbate 80 peak as the molecular weights overlapped on the size exclusion chromatogram. Solution light scattering indicated that polysorbate 80 associates with erythropoietin in a defined stoichiometric ratio of 1:12.

Conclusions

Based on controlled studies, previous results suggesting that EPREX® contains micelle-associated erythropoietin were incorrect. As with other surfactants and proteins, polysorbate 80 associates with erythropoietin in a defined stoichiometric ratio.

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Acknowledgments

The authors would like to acknowledge the excellent technical assistance provided by Patricia Brennan, Wise Lumax, Ken Bui, Alexandra Marin, and Samantha Ranaweera.

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Authors and Affiliations

  1. Biotech Development, Global Biological Supply Chain, 100 Route 202 South, Raritan, New Jersey, 08869, USA

    Annabelle P. Villalobos

  2. Pharmaceutical Sciences, Global Biological Supply Chain, 920 Route 202 South, Raritan, New Jersey, 08869, USA

    Srinivas R. Gunturi

  3. Biopharmaceutical Sciences, Protein Design, Characterization, Optimization and Analysis, Centocor, Inc., 145 King of Prussia Road, Radnor, Pennsylvania, 19087, USA

    George A. Heavner

Authors
  1. Annabelle P. Villalobos
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  2. Srinivas R. Gunturi
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  3. George A. Heavner
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Correspondence to George A. Heavner.

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Villalobos, A.P., Gunturi, S.R. & Heavner, G.A. Interaction of Polysorbate 80 with Erythropoietin: A Case Study in Protein–Surfactant Interactions. Pharm Res 22, 1186–1194 (2005). https://doi.org/10.1007/s11095-005-5356-7

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  • DOI: https://doi.org/10.1007/s11095-005-5356-7

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