In-depth comparison of N-glycosylation of human plasmaderived factor VIII and different recombinant products: from structure to clinical implications
In-depth comparison of N-glycosylation of human plasmaderived factor VIII and different recombinant products: from structure to clinical implications
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DOI：
10.1111/jth.14204
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发表时间：
2018-08-01
影响因子：
10.4
通讯作者：
Malisauskas, M.
中科院分区：
文献类型：
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作者：
Canis, K.;Anzengruber, J.;Malisauskas, M.
背景/目的：人凝血因子VIII（FVIII）是一种血浆糖蛋白，其缺陷导致血友病A。目前的替代疗法使用从人血浆或从具有不同水平的B结构域缺失的各种细胞系（重组FVIII）纯化的FVIII产物。糖基化是FVIII中的一种翻译后蛋白质修饰，对其物理、功能和抗原特性具有实质性影响。糖基化的变化可能是FVIII产品在药代动力学、药效学和免疫原性方面不同的原因。然而，关于FVIII糖基化的文献是不一致的，阻止组装成一致的模型。为了更好地理解FVIII生物学基础的糖基化机制，我们研究了人血浆衍生（pd）FVIII和CHO、BHK或HEK细胞系中表达的6种rFVIII产物的N-糖基化。研究方法：使用MALDI-MS和MS/MS、GC-MS和UPLC-UV-MSE技术组合，对FVIII样品进行头对头详细的糖组学和糖蛋白质组学表征。结果/结论：我们的研究结果将pdFVIII的N-聚糖库详细描述到前所未有的水平，并首次提供了在pdFVIII上发现的N-羟乙酰神经氨酸（NeuGc）的证据。尽管rFVIII的位点特异性糖基化被证明与pdFVIII一致，无论表达系统如何，每个样品的整个N-聚糖含量似乎显著不同。尽管所有样品共有的生物学重要表位（即唾液酸化和高甘露糖）的比例在样品之间不同，但一些重组产物表达不同的免疫学相关表位，如LacdiNAc（LDN）、岩藻糖基化LacdiNAc（FucLDN）、NeuGc、刘易斯（X/Y）和Gal（α 1，3）Gal表位。HEK细胞中表达的rFVIII显示出与人pdFVIII最大的糖组学差异。
Background/Objective: Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods: FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion: The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of Nglycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, Lewis(X/Y) and Gal(alpha 1,3)Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.