Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2-8°C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products.

Renal ischemic and reperfusion injury resulting in acute renal failure is a multidisciplinary problem at the junction of pathophysiology, transplantology, urology, nephrology, cardiac surgery and pharmacology. One of renal protection strategies is using the phenomenon of preconditioning. Preconditioning is one of the ways to adopt a tissue to repeated short-term effects of damaging factors to induce an enhanced tolerance to the long period of hypoxia and/or ischemia. There are multiple cellular and molecular mechanisms of the renal protective effects of preconditioning stimuli, but the key effectors and signaling molecules are ATP-dependent potassium channels, nitric oxide synthase, nitric oxide, and mitochondrial pore. Contradictory data on the protective effect of ischemic preconditioning allow searching for approaches to pharmacological correction of ischemic and reperfusion injuries. The article provides data on possible ways of using erythropoietin, darbepoetin and phosphodiesterase 5 inhibitors.