Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys
Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys
复制标题
DOI：
10.1093/glycob/cwm017
复制
发表时间：
2007-05-01
期刊：
影响因子：
4.3
通讯作者：
Battersby, John E.
中科院分区：
文献类型：
--
作者：
Jones, Andrew J. S.;Papac, Damon I.;Battersby, John E.
为了了解重组糖蛋白的碳水化合物部分如何影响其药代动力学(PK)特性，直接从食蟹猴和人类的PK研究期间采集的一系列血液样本中评估了糖链的分布。所研究的蛋白质是一种免疫粘附素(Lenercept)，含有人免疫球蛋白G(Ig G-1)的Fc结构域和肿瘤坏死因子受体p55广泛糖基化的胞外区的两个副本。采用双柱免疫亲和-反相高效液相色谱分离纯化蛋白。利用基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)对多糖进行释放和分析。另一种方法是用胰酶获得糖肽，并用MALDI-TOF进行分析。成分与时间的关系表明，在循环10天内，Fc结构域中的葡聚糖的分布没有改变，这与它们在蛋白质内部的隔离一致。然而，在最初的24小时内，受体结构域中的多糖组成发生了显著变化，然后保持相对恒定。对酸性多糖(仅来自受体结构域)的分析表明，在清除的快速初始阶段，携带末端N-乙酰氨基葡萄糖(TGlcNAc)的多糖被选择性地从循环中清除。这种现象同样发生在人类和食蟹猴身上。唾液酸含量和末端半乳糖变化不大。这些数据证实了tGlcNAc与分子半衰期的相关性，并支持了甘露糖受体(也可以与tGlcNAc结合)导致该分子的可变清除的假说。
To understand how the carbohydrate moieties of a recombinant glycoprotein affected its pharmacokinetic (PK) properties, the glycan distribution was directly assessed from serial blood samples taken during PK studies in cynomolgus monkeys and humans. The protein studied was an immunoadhesin (lenercept), containing an Fc domain from human immunoglobulin G (IgG-1) and two copies of the extensively glycosylated extra cellular domain of tumor necrosis factor receptor p55. The protein was recovered in pure form using a dual column, immunoaffinity-reversed-phase high-performance liquid chromatography method. The glycans were released and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Alternatively, trypsin was used to obtain glycopeptides, and these were analyzed by MALDI-TOF. The composition versus time profiles show that the distribution of glycans in the Fc domain was not altered over 10 days of circulation, consistent with their sequestration in the interior of the protein. However, the glycan composition in the receptor domain was changed dramatically in the first 24 h and then remained relatively constant. Analysis of the acidic glycans (derived exclusively from the receptor domain) showed that, in the rapid initial phase of clearance, glycans carrying terminal N-acetylglucosamine (tGlcNAc) were selectively cleared from the circulation. This phenomenon occurred similarly in humans and cynomolgus monkeys. Sialic acid content and terminal galactose showed only small changes. These data confirm the correlation of tGlcNAc and half-life of the molecule, and support the hypothesis that the mannose receptor (which can also bind tGlcNAc) causes the variable clearance of this molecule.