NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana
NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana
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DOI：
10.1007/s00299-015-1757-1
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发表时间：
2015-02
影响因子：
6.2
通讯作者：
H. Yagi;Noriho Fukuzawa;Y. Tasaka;K. Matsuo;Ying Zhang;Takumi Yamaguchi;Sachiko Kondo;Shiori Nakazawa;N. Hashii;N. Kawasaki;T. Matsumura;Koichi Kato
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文献类型：
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作者：
H. Yagi;Noriho Fukuzawa;Y. Tasaka;K. Matsuo;Ying Zhang;Takumi Yamaguchi;Sachiko Kondo;Shiori Nakazawa;N. Hashii;N. Kawasaki;T. Matsumura;Koichi Kato
Keymessage我们成功地开发了一种用于转基因烟草中产生的重组蛋白的代谢同位素标记方法。这使得通过NMR analysis.AbstractA的植物来源的治疗性抗体的结构完整性的评估已经开发了各种各样的表达载体的生产有前途的生物制剂，包括植物，真菌，细菌，昆虫和哺乳动物。糖蛋白生物制剂通常经历以变异糖基化模式为代表的改变的折叠和翻译后修饰。这些差异可显著影响其功效，如治疗性抗体所例示。然而，通常难以验证使用不同表达载体生产的生物制剂的结构完整性。为了解决这一问题，我们开发并应用了稳定同位素辅助的核磁共振（NMR）光谱方法来表征植物中产生的重组抗体的构象。对由此制备的源自15 N标记抗体的Fc片段进行异频二维（2D）NMR测量。该方法能够通过将植物来源的治疗性抗体的NMR光谱性质与源自哺乳动物细胞的真实IgG-Fc的NMR光谱性质进行比较来评估植物来源的治疗性抗体的结构完整性。
KeymessageWe successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis.AbstractA variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants.Nicotiana benthamianaused as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a15N-enriched plant growth medium. The Fc fragment derived from the15N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.