Abed Heba S, Al-Ghobashy Medhat A, Fathalla Faten A, Salem Maissa Y
National Organization for Research and Control of Biologicals, Egypt.
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt; Bioanalysis Research Group, School of Pharmacy, New Giza University, Egypt.
Biologicals. 2017 Nov;50:129-136. doi: 10.1016/j.biologicals.2017.08.012. Epub 2017 Sep 27.
Recombinant human erythropoietin (rhEPO) is a commonly used biopharmaceutical for the treatment of anemia-associated disorders. Epogen; glycosylated erythropoietin (G-EPO) has short half-life and poor stability. Pegylated Epogen (Peg-G-EPO) was introduced to the market to overcome these limitations. The combined effects of pegylation and glycosylation on the stability of Peg-G-EPO was studied. Determination of Peg-G-EPO in the presence of its degradation products was achieved using SE-HPLC. The assay was validated according to ICH guidelines over concentration range of 50.00-320.00 μg/mL (r 0.9999). A mobile phase of 50 mM phosphate buffer (pH 6.5) with 300 mM sodium chloride and 20% ethanol was employed. Isocratic elution was carried out at 0.5 mL/min over run time of 30 min. Peg-G-EPO was found stable towards mechanical agitation only at low concentrations while it was stable towards repeated freeze/thaw; regardless of the concentration. Effect of temperature and pH were also investigated and Peg-G-EPO was found stable within narrow ranges. Results indicated formation of small molecular weight and very high molecular weight aggregates that have been filtered-off the column. Although Peg-G-EPO was found relatively more stable than its non-pegylated but glycosylated version, results indicated the need for careful stability-assessment of Peg-G-EPO.
重组人促红细胞生成素（rhEPO）是一种常用于治疗贫血相关疾病的生物制药。促红细胞生成素；糖基化促红细胞生成素（G-EPO）半衰期短且稳定性差。聚乙二醇化促红细胞生成素（Peg-G-EPO）被引入市场以克服这些局限性。研究了聚乙二醇化和糖基化对Peg-G-EPO稳定性的综合影响。使用尺寸排阻高效液相色谱法（SE-HPLC）在其降解产物存在的情况下测定Peg-G-EPO。该测定法在50.00 - 320.00μg/mL的浓度范围内根据国际人用药品注册技术协调会（ICH）指南进行了验证（r = 0.9999）。采用含有300 mM氯化钠和20%乙醇的50 mM磷酸盐缓冲液（pH 6.5）作为流动相。在30分钟的运行时间内以0.5 mL/分钟的流速进行等度洗脱。发现Peg-G-EPO仅在低浓度下对机械搅拌稳定，而对反复冻融稳定，无论浓度如何。还研究了温度和pH的影响，发现Peg-G-EPO在较窄的范围内稳定。结果表明形成了已从柱中滤出的小分子和非常高分子量的聚集体。尽管发现Peg-G-EPO比其未聚乙二醇化但糖基化的版本相对更稳定，但结果表明需要对Peg-G-EPO进行仔细的稳定性评估。
