Recombinant human erythropoietin (rHuEPO) has been widely used for treatment of anemia associated with chronic renal failure. Cases of anti-rHuEPO-associated pure red cell aplasia (PRCA) have been found in patients undergone rHuEPO treatment. Patients become resistant to administered rHuEPO and develop antibodies against rHuEPO. Structures of proteins depend largely on their surrounding environment; any change in their environment may affect their conformation and stability.
This study aimed to investigate the possibilities of two unexplored conditions in destabilizing and causing immunogenicity of rHuEPO—the imbalanced electrolyte levels causing metabolic acidosis in end-stage renal failure patients and the presence of heavy metal such as cadmium in blood circulation. The distributions of rHuEPO and cadmium in blood fractions were examined. In the experimental conditions carried out, the supplemented rHuEPO and cadmium were exclusively found in the plasma fractions of the blood and no interference on each other’s distributions was observed. The effects of acidic pH stress and the presence of cadmium in the solution on changes in secondary and tertiary structures of rHuEPO and aggregate formation upon different treatments were monitored with far-UV circular dichroism spectroscopy, tryptophan fluorescence emission scan, and blue native (BN)-PAGE techniques, respectively. Observations from BN-PAGE demonstrated that conformational changes and aggregate formation of rHuEPO appeared after a few days of incubation at 37°C at the physiological pH 7.4; in mild acidic pH 6.4 and 5.4 similar aggregate species to that of pH 7.4 were observed, whereas in strong acidic pH 4.4 the aggregate formation was escalated.
Over the course of 25-day incubation at the physiological pH 7.4, the tertiary structure of rHuEPO gradually became more exposed to the surrounding environment. Acidic pH further accelerated the extent of conformational changes, especially at pH 4.4. No effect of acidic pH was observed on the secondary structure of rHuEPO except at pH 4.4 where its helical content decreased by 18%. Interestingly, at pH 7.4 and mild acidic pH 6.4, cadmium appeared to decelerate the conformational changes in tertiary structure of rHuEPO; the cadmium protective effect was no longer observed in more acidic pH 5.4 and 4.4. In all the pH’s tested, no effect of cadmium was observed on the secondary structure of rHuEPO. The observations in this study suggest that the presence of cadmium in rHuEPO solution is unlikely to cause destabilization of rHuEPO structure. On the other hand, pH of the solution plays a crucial role in stability of rHuEPO. Depending on the severity of electrolyte imbalance and acidosis in the blood system of chronic renal failure patients, acidic pH of the blood may have destabilizing effects and, among other factors, contribute to the immunogenicity of administered rHuEPO. More information from in vivo studies will be necessary to confirm the physiological significance of these findings.