Process Sciences Department, 100 Research Drive, Abbott Bioresearch Center, Worcester, Massachusetts 01605, USA.
Anal Chem. 2010 Dec 1;82(23):9871-7. doi: 10.1021/ac102332f. Epub 2010 Nov 8.
Glycosylation is a common posttranslational modification and a key quality attribute of recombinant monoclonal antibodies. In particular, the level of core-fucosylation is critical to several Fc mediated biological functions. However, it is challenging to accurately determine the level of afucosylation due to its low level and possible distribution in multiple oligosaccharide species. Methods that can accurately determine the level of afucosylation were developed in the current study. Digestion of monoclonal antibodies with Endoglycosidases F2 and H, which cleave the glycosidic bond between the two primary GlcNAc residues, reduced complicated oligosaccharide population into two groups with either only GlcNAc or GlcNAc and the core-fucose residue. Analysis of the digested antibodies by LC-MS provided a quick estimate of the level of afucosylation. Alternatively, PNGaseF released oligosaccharides were labeled with 2-aminobenzamide, digested with Endoglycosidases F2 and H followed by normal phase HPLC with fluorescence detection. The results demonstrated that the latter method can provide an accurate quantitation of the level of afucosylation.
糖基化是一种常见的翻译后修饰，也是重组单克隆抗体的关键质量属性。特别是核心岩藻糖基化水平对几种 Fc 介导的生物学功能至关重要。然而，由于其低水平和可能分布在多种寡糖种类中，因此难以准确确定去岩藻糖基化的水平。本研究开发了能够准确测定去岩藻糖基化水平的方法。用内切糖苷酶 F2 和 H 消化单克隆抗体，该酶可切断两个主要 GlcNAc 残基之间的糖苷键，将复杂的寡糖群体简化为仅含有 GlcNAc 或 GlcNAc 和核心岩藻糖残基的两组。通过 LC-MS 分析消化后的抗体可以快速估计去岩藻糖基化的水平。或者，用 2-氨基苯甲酰胺标记 PNGaseF 释放的寡糖，然后用内切糖苷酶 F2 和 H 消化，再用正相 HPLC 结合荧光检测。结果表明，后一种方法可以准确定量去岩藻糖基化的水平。
