Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII
Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII
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DOI：
10.1016/j.thromres.2012.09.011
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发表时间：
2013-01-01
影响因子：
7.5
通讯作者：
Sandberg, Helena
中科院分区：
文献类型：
--
作者：
Kannicht, Christoph;Ramstrom, Margareta;Sandberg, Helena
简介：用于重组蛋白表达的宿主细胞系在执行翻译后修饰 (PTM) 的能力方面有所不同。目前可用的重组人 FVIII (rhFVIII) 产品是在哺乳动物、非人细胞系中产生的。对于 rhFVIII，糖基化和硫酸化对于功能性和冯维勒布兰德因子 (VWF) 结合亲和力至关重要。在这里，我们介绍了一种新型人细胞系衍生的重组人 FVIII (Human-cl rhFVIII) 的 PTM 特征。 rhFVIII 在人类细胞系中的表达可避免表达不需要的哺乳动物糖型，如 Gal α 1-3Gal beta 1-GlcNAc-R (α-Gal) 和 N-羟乙酰神经氨酸 (Neu5Gc)，这些糖型构成对人类具有抗原性的表位。 材料和方法：我们描述了使用液体的硫酸化分析、聚糖分析和表征 色谱-质谱法和带脉冲电流检测的高效阴离子交换色谱法。结果和结论：Human-cl rhFVIII 被证实与人血浆来源的 FVIII 相比被硫酸化和糖基化。最重要的是，人cl rhFVIII 缺乏在中国仓鼠卵巢和幼仓鼠肾脏衍生的rFVIII 产品中观察到的抗原性Neu5Gc 或α-Gal 表位。与现有的 rFVIII 产品相比，避免非人聚糖结构和实现完全硫酸化都被认为可以降低人-cl rhFVIII 的内在免疫原性。 (C) 2012 Elsevier Ltd. 保留所有权利。
Introduction: Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Gal alpha 1-3Gal beta 1-GlcNAc-R (alpha-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans.Materials and methods: We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection.Results and conclusions: Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or alpha-Gal epitopes observed in Chinese Hamster Ovary-and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products. (C) 2012 Elsevier Ltd. All rights reserved.