Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis
Site-specific antibody-drug conjugate heterogeneity characterization and heterogeneity root cause analysis
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DOI：
10.1080/19420862.2019.1624127
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发表时间：
2019-06-14
期刊：
影响因子：
5.3
通讯作者：
Liu, Dengfeng
中科院分区：
文献类型：
--
作者：
Cao, Mingyan;De Mel, Niluka;Liu, Dengfeng
位点特异性抗体-药物结合物(ADC)旨在克服第一代ADC的异质性，第一代ADC使用随机结合到表面暴露的赖氨酸残基或结合到链间二硫键。然而，尽管同质性显著增强，但位点特异性ADC的产生产生了一些与过程相关的物种异质性，包括立体异构体、未结合抗体、欠结合物种和过度结合物种。在最终的位点特异性ADC产物中，也观察到了较高水平的大小变体，如重链-轻链物种(半ADC)、重链-重链-轻链物种和轻链物种。为了了解在ADC接合过程中产生异质性的根本原因，我们为每个接合步骤设计了时间进程研究，包括还原、氧化、接合和猝灭。我们开发了非还原肽图和基于LabChip的毛细管电泳法，用于时程样品分析。根据我们的时间历程数据，在氧化过程中，由于替代的二硫键排列，产生了半ADC和未结合抗体。在氧化过程中，两个铰链半胱氨酸在半个ADC上形成一个链内二硫键，在未结合抗体中形成三个链间铰链二硫键。时间历程数据还表明，猝灭步骤导致尺寸变体水平增加，特别是重链-重链-轻链物种和轻链物种，在该步骤中，猝灭剂与ADC进行二硫键交换反应，并破坏连接重链和轻链的二硫键。欠共轭和超共轭物种产生于共轭反应过程中建立的平衡。
Site-specific antibody-drug conjugates (ADCs) are designed to overcome the heterogeneity observed with first-generation ADCs that use random conjugation to surface-exposed lysine residues or conjugation to interchain disulfide bonds. Despite significantly enhanced homogeneity, however, the production of site-specific ADCs yields some process-related species heterogeneity, including stereoisomers, unconjugated antibody, underconjugated species, and overconjugated species. An elevated level of size variants, such as heavy chain-light chain species (half ADC), heavy chain-heavy chain-light chain species, and light chain species, is also observed with the final site-specific ADC product. To understand the root cause of heterogeneity generated during the ADC conjugation process, we designed time-course studies for each conjugation step, including reduction, oxidation, conjugation, and quenching. We developed both non-reduced peptide map and LabChip-based capillary electrophoresis sodium dodecyl sulfate methods for time-course sample analysis. On the basis of our time-course data, the half ADC and unconjugated antibody were generated during oxidation as a result of alternative disulfide bond arrangements. During oxidation, two hinge cysteines formed an intra-chain disulfide bond in the half ADC, and three inter-chain hinge disulfide bonds were formed in the unconjugated antibody. Time-course data also showed that the elevated level of size variants, especially heavy chain-heavy chain-light chain species and light chain species, resulted from the quenching step, where the quenching reagent engaged in a disulfide bond exchange reaction with the ADC and broke the disulfide bonds connecting the heavy chain and light chain. Underconjugated and overconjugated species arose from the equilibrium established during the conjugation reaction.