中向下核磁共振用于治疗性单克隆抗体,糖剖析
Jiayi Li1, Grace Zhang1, Kang Chen1
1Division of Pharmaceutical Quality Research VI, Office of Pharmaceutical Quality Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland.
Current protocols
|November 3, 2025
概括
一种新的中向下核磁共振 (NMR) 方法描述了单克隆抗体 (mAb) 糖化,而不会损害甘氨酸. 这种技术为质量控制提供了全面的半定量单糖形状分析.
科学领域:
- 生物化学 生物化学
- 分析化学 分析化学
- 生物技术是生物技术.
背景情况:
- 再组合单克隆抗体 (mAbs) 是关键的治疗药物,糖化显著影响其质量,制造和生物相似性.
- 准确的糖甘分析对于确保mAb产品的一致性和治疗效果至关重要.
- 目前的甘氨酸分析方法通常涉及侵入性程序,可以改变甘氨酸的完整性.
研究的目的:
- 开发和验证一个中向下核磁共振 (NMR) 方法,用于全面的mAb糖甘造型.
- 为了在分析过程中保持连接到mAbs的Fc域的甘氨酸的完整性.
- 提供一种非侵入性,半定量方法,用于糖甘分布分析.
主要方法:
- 使用免疫球蛋白降解酶 (IdeS) 来产生Fc片段的mAbs的酶性消化.
- 在尿素中的Fc碎片的净化,变质和溶解.
- 高分辨率的二维1H-13C异质核单量子相干 (2D 1H-13C HSQC) 核磁共振光谱仪用于糖分析.
主要成果:
- 中向下的NMR方法成功地描述了mAb糖分布,包括主要和次要物种,如三诺西尔核和高曼诺斯变种.
- 在2D 1H-13C HSQC NMR光谱中的异构峰分布提供了半定量单糖形状.
- 该方法保留了甘氨酸和Fc域之间的共价键,保持了甘氨酸的完整性.
结论:
- 中下NMR为传统的糖甘地图绘制方法提供了一个非侵入性的替代方案,保留了糖甘的完整性.
- 这种精简的工作流程使得全面和半定量的甘氨酸分析成为可能,适合于受监管的环境.
- 该方法需要基本的生物化学和2DNMR技能,以促进其在开发和质量控制设置中的实施.
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