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.

阿尔贝达泊汀（DA）; 高糖基化促红细胞生成素α（EPO）是慢性肾脏衰竭和癌症患者贫血的基本治疗方法。在这项研究中，DA和EPO受到在生产，运输和储存过程中可能遇到的物理化学胁迫因素（pH，温度，搅拌，反复冻融和氧化）。建立了由SE-HPLC，RP-HPLC，ELISA和SDS-PAGE组成的正交性指示试验方案，并进行了验证，以研究DA进一步糖基化对降解模式和动力学的影响。结果表明，与EPO相比，在等效应力条件下，与DA相比，DA相对较高的稳定性和较低的形成高分子量聚集体的趋势。两种药物在整个pH范围内以及在高于2–8°C的温度下孵育或反复冷冻/融化后均会形成二聚体和聚集体。在高速搅拌在配制缓冲液中制备的标准样品并用过氧化氢氧化时，也观察到相同的观察结果。SE-HPLC之间的协议（由光谱纯度数据支持）和ELISA证实了这两种技术对完整药物的特异性。RP-HPLC和SDS-PAGE的结果表明，在pH和氧化的情况下，二聚分别通过二硫键和双酪氨酸共价键发生。明显的是，随着聚糖大小的增加，聚集被显着抑制，并且在任何研究的应力因素下都没有观察到聚糖的损失。这些观察到的缓慢的降解动力学证实了糖工程优于化学聚乙二醇化以增强EPO的稳定性。在所有测试的压力因素下，此类潜在的与免疫原性产品相关的杂质的形成证实了需要正交测试方案来研究此类敏感产品降解的复杂模式。