Optimal Synthetic Glycosylation of a Therapeutic Antibody.
Optimal Synthetic Glycosylation of a Therapeutic Antibody.
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DOI：
10.1002/ange.201508723
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发表时间：
2016-02-12
期刊：
影响因子：
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通讯作者：
Davis BG
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作者：
Parsons TB;Struwe WB;Gault J;Yamamoto K;Taylor TA;Raj R;Wals K;Mohammed S;Robinson CV;Benesch JL;Davis BG
抗体中的糖基化模式决定着生物和物理特性，但其精确控制在生物学和生物技术中是一个重大挑战。我们在此描述了最畅销的生物治疗药物Herceptin的内糖苷酶催化的糖基化的优化，Herceptin是一种抗HER2抗体。对完整的四链抗体杂多聚体进行精确的MS分析，可以发现在标准变性条件下不能检测到的非特异性、非酶反应(糖基化)。到目前为止，这种竞争反应作为副产品的来源一直被低估，现在可以最小化了。优化后可获得迄今为止最纯天然的赫赛汀(≥90 %)。此外，通过使用包含非天然官能团的小糖库，可以容易地产生在特定位置(用于连接货物分子或“糖随机化”)包含限定数量的选择性可寻址化学标签(Sia C1处的反应手柄)的Ab变体。
Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase‐catalyzed glycosylation of the best‐selling biotherapeutic Herceptin, an anti‐HER2 antibody. Precise MS analysis of the intact four‐chain Ab heteromultimer reveals nonspecific, non‐enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non‐natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or “glycorandomization”) were readily generated.