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通过icIEF-UV/MS和RP-HPLC-MS对化学酶结合ADC的多层次表征 EAD碎片化地图

Scott Mack¹, Haichuan Liu¹, Erica Andersson²

¹SCIEX, Redwood City, California, USA.

Electrophoresis

|January 13, 2026

概括

这项研究开发了一种高效的化学酶方法,以创建同质的抗体-药物联合体 (ADC),如Trastuzumab-MMAE. 先进的质谱技术证实了临床前应用的特定位点的结合和高药对抗体比率.

科学领域:

生物结合化学生物结合化学
分析化学分析化学
药理学药理学是指药理学的学科.

背景情况:

抗体-药物合物 (ADC) 是关键的治疗方法,它结合了有针对性的输送与强大的细胞毒性有效载荷.
开发具有可控药对抗体比率 (DAR) 的同质ADC对于疗效和安全至关重要.
酶介导的结合为抗体的特定位点修饰提供了一个有前途的途径.

研究的目的:

为了合成单甲基奥里斯丁E (MMAE) 的有效载荷,使用酶介导的甘氨酸重塑反应结合了Trastuzumab (TRA).
通过使用多层次分析技术,全面描述所产生的TRA-MMAE抗体-药物合物 (ADC).
建立一个精简的工作流程,以生产具有良好的特征和同质的ADC.

主要方法:

对TRA-MMAE结合物的化学酶合成.
基于微流体芯片的紫外线成像通道异电聚焦质谱法 (icIEF-UV/MS) 用于完整的蛋白形状分析.
逆相高性能液态染色学 (RP-HPLC) 基映射与电子激活解离 (EAD) 分片.
药物与抗体比率 (DAR) 的确定和特定部位的结合分析.

主要成果:

成功合成了具有高药对抗体比率 (DAR 2) 的TRA-MMAEADCs.
icIEF-UV/MS 可分离,定量和识别完整的ADC蛋白形体,揭示了暗示结合的质量转移.
使用EAD碎片化的RP-HPLC基映射证实了完整的质量发现,局部的翻译后修改,并验证了特定于位点的MMAE对Asn-300甘氨酸的结合.
化学酶反应表明TRA-MMAE ADC产品的高产量和同质性.

结论:

结合 icIEF-UV/MS 和 RP-HPLC 基映射与 EAD 碎片化,为 ADC 表征提供了一个强大的分析策略.
评估的化学酶结合反应有效地产生具有定义DAR的同质TRA-MMAEADC.
这种精简的工作流程有助于为临床前评估和扩展提供明确的ADC的生产和表征.

关键词:

抗体药物合物 (ADC) 的使用收费变种收费变种药物与抗体比率 (DAR) 是药物与抗体的比率.电子激活解离 (EAD) 是一种高分辨率质谱仪 (HRMS) 的使用图像化毛细血管同电聚焦 (icIEF)完整的质量分析.质谱学 (MS) 是一种质谱学.类映射绘制的类映射紫外线成像通道异电聚焦质谱法 (icIEF-UV/MS)

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