Evidence for aluminum-binding erythropoietin by size-exclusion chromatography coupled to electrothermal absorption atomic spectrometry

Erythropoietin (EPO) is a glycoprotein that stimulates erythropoiesis and is clinically used for treating anemia during chronic renal failure and for anemia in preterm infants. EPO formulations usually have elevated rates of contamination due to aluminum (Al), which is toxic to both types of patients. Size-exclusion chromatography (SEC) coupled with graphite furnace atomic absorption spectrometry (GF AAS) was employed to separate proteins and to quantify the amount of aluminum present in the elution volume corresponding to EPO and, therefore, to evaluate possible binding. Because EPO formulations contain human serum albumin (HSA), a chromatographic method was optimized for the separation of these proteins. Subsequent to the chromatographic separation, 1-mL fractions of the column effluent were collected, and the Al content in these aliquots was measured by GF AAS. EPO and HSA samples were incubated with Al for 4 h at 4 °C and 37 °C as well as for 16 h at 4 °C and 37 °C. Afterwards, they were injected into the chromatographic system. These samples were also submitted to ultrafiltration (10 and 50 kDa membranes), and Al was measured in the ultrafiltrates. The results showed that Al was present in the eluent volume corresponding to the EPO peak but not in the HSA peak in the chromatograms. Temperature strengthened the interaction because the Al present in the EPO fraction was 3 times higher at 37 °C compared to 4 °C. Thirty-eight percent of the Al present in a 2.4 μg/mL EPO standard solution, and approximately 50% of the Al in formulation samples containing approximately 11 μg/mL EPO and either citrate or phosphate, were non-ultrafiltrable, which suggests that EPO is an effective Al acceptor in vitro.