National Institute for Biological Standards & Control (NIBSC), Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, United Kingdom.
National Institute for Food and Drug Control (NIFDC), Tiantan Xili No. 2, 100050, Dongcheng District, Beijing, China.
J Pharm Biomed Anal. 2018 May 10;153:214-220. doi: 10.1016/j.jpba.2018.02.043. Epub 2018 Feb 24.
Recombinant, human, erythropoietin (rhEPO) is a glycoprotein hormone which is prescribed throughout the world to treat anaemia caused by chronic kidney disease or chemotherapy. rhEPO is at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Due to its complex glycosylation profile, which has a crucial influence upon biological activity, therapeutic rhEPO preparations must be closely monitored to ensure consistency, safety and efficacy. Here, we have compared twelve rhEPO preparations from eleven manufacturers in China and one in Japan, measuring in vivo biological activity and exploring its relationship with glycosylation through sialic acid content determination, isoform distribution via capillary electrophoresis (CE), O-glycan profiling, and N-glycan mapping using a novel anion-exchange/hydrophilic interaction chromatography-mass spectrometry (AEX/HILIC-MS) approach. We observed differences between glycosylation profiles, including the varying occurrence of sialic acid O-acetylation, extension of N-glycan antennae with N-acetyllactosamine units, and the distribution of sialic acids across multi-antennary structures. The presence of unusually high levels of suspected penta- and hexa-anionic N-glycans in several samples is consistent with elevated rhEPO isoform acidity, which is reflected by slightly elevated in vivo bioactivities. This aside, the observed differences in glycosylation profile do not appear to have a significant influence upon biological activity in mice. Nonetheless, with the continued emergence of biosimilars, the study highlights the importance of monitoring glycosylation profiles in biological medicines, in order to detect and account for divergence between products, as well as the presence of unusual or unexpected glycans.
重组人促红细胞生成素（rhEPO）是一种糖蛋白激素，被广泛用于治疗慢性肾病或化疗引起的贫血。rhEPO 是最近生物类似药出现的前沿领域，全球有许多产品。由于其复杂的糖基化谱对生物活性有至关重要的影响，治疗用 rhEPO 制剂必须密切监测以确保一致性、安全性和疗效。在这里，我们比较了来自中国的十一家制造商和日本的一家制造商的十二种 rhEPO 制剂，通过体内生物活性测定，并通过唾液酸含量测定、毛细管电泳（CE）的同工型分布、O-聚糖分析以及使用新型阴离子交换/亲水相互作用色谱-质谱法（AEX/HILIC-MS）的 N-聚糖图谱，探索其与糖基化的关系。我们观察到糖基化谱之间的差异，包括唾液酸 O-乙酰化的不同发生、N-乙酰乳糖胺单元扩展 N-聚糖天线以及唾液酸在多天线结构中的分布。在几个样本中存在异常高水平的疑似五元和六元阴离子 N-聚糖与 rhEPO 同工型酸度升高一致，这反映在体内生物活性略有升高。除此之外，观察到的糖基化谱差异似乎对小鼠的生物活性没有显著影响。尽管如此，随着生物类似药的不断涌现，该研究强调了监测生物药物中糖基化谱的重要性，以便检测和解释产品之间的差异，以及异常或意外聚糖的存在。
