Interaction of polysorbate 80 with erythropoietin: a case study in protein-surfactant interactions.
OBJECTIVE
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.
Villalobos
Annabelle P
AP
Biotech Development, Global Biological Supply Chain, 100 Route 202 South, Raritan, New Jersey 08869, USA.
Gunturi
Srinivas R
SR
Heavner
George A
GA
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D004797:Enzyme-Linked Immunosorbent Assay; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000032:analysis / Q000737:chemistry*; D005079:Excipients / Q000737:chemistry*; D008823:Micelles; D008970:Molecular Weight; D011136:Polysorbates / Q000737:chemistry*; D011994:Recombinant Proteins; D012010:Red-Cell Aplasia, Pure; D013501:Surface-Active Agents / Q000737:chemistry*; D013594:Syringes
D005079:Excipients; D008823:Micelles; D011136:Polysorbates; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D004921:Erythropoietin; D000068817:Epoetin Alfa