Integrated Bioanalysis, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, South San Francisco, California, USA.
Rapid Commun Mass Spectrom. 2024 Dec 15;38(23):e9910. doi: 10.1002/rcm.9910.
Isomerism can be an important aspect in pharmaceutical drug development. Identification of isomers can provide insights into drug pharmacology and contribute to better design of drug molecules. The general approaches to differentiate isomers include Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and circular dichroism. Additionally, a commonly used method to differentiate isomers is liquid chromatography coupled with mass spectrometry (LC-MS). Notably, LC-MS is routinely applied to leucine and isoleucine differentiation to facilitate protein sequencing. This work focuses on isomer differentiation of widely employed thio-succinimide structure bridging the antibody backbone and linker-payload of antibody-drug conjugates (ADCs). Thio-succinimide hydrolysis stabilizes the payload-protein structure while generating a pair of constitutional isomers: thio-aspartyl and thio-isoaspartyl.
This paper introduces a hybrid method using ligand binding assay (LBA) and liquid chromatography coupled with tandem MS (LC-MS/MS) to reveal isomerization details of thio-succinimide hydrolysis over time in plasma samples incubated with ADC. Application of two orthogonal dissociation methods, collision-induced dissociation (CID) and electron-activated dissociation (EAD) revealed different MS/MS spectra for this pair of isomers. This observation enables a unique approach in distinguishing thio-succinimide hydrolysis isomers.
We observed signature [R + Thio + 57 + H], [R + Succ + HO - 57 + H], and [R + Succ + HO - 44 + 2H] product ions (Succ = succinimide) that differentiated thio-aspartyl and thio-isoaspartyl isomers using EAD. A newly discovered [R + ThioSucc + HO - 44 + 2H] ion also served as additional evidence that further supported our findings.
This study is a first-to-date identification of thio-succinimide hydrolysis isomers without using synthesized reference materials. This approach should be applicable to all thio-succinimide-linked molecules. Correct identification of thio-succinimide hydrolysis isomers may eventually benefit the development of ADCs in the future.
对映异构可能是药物开发中的一个重要方面。异构体的鉴定可以深入了解药物的药理学，并有助于更好地设计药物分子。区分异构体的一般方法包括傅里叶变换红外光谱（FTIR）、核磁共振（NMR）和圆二色性。此外，区分异构体的常用方法是液相色谱与质谱联用（LC-MS）。值得注意的是，LC-MS 通常用于区分亮氨酸和异亮氨酸，以促进蛋白质测序。这项工作专注于广泛使用的硫代琥珀酰亚胺结构的异构体区分，该结构桥接抗体骨架和抗体药物偶联物（ADC）的连接子-有效载荷。硫代琥珀酰亚胺水解稳定了有效载荷-蛋白质结构，同时产生一对结构异构体：硫代天冬氨酸和硫代异天冬氨酸。
本文介绍了一种混合方法，使用配体结合分析（LBA）和液相色谱与串联质谱（LC-MS/MS），揭示在含有 ADC 的血浆样品中孵育时，硫代琥珀酰亚胺水解随时间的变化细节。应用两种正交解离方法，碰撞诱导解离（CID）和电子激活解离（EAD），为这对异构体揭示了不同的 MS/MS 谱。这种观察为区分硫代琥珀酰亚胺水解异构体提供了一种独特的方法。
我们观察到特征[R + 硫代 + 57 + H]、[R + 琥珀酰 + HO - 57 + H]和[R + 琥珀酰 + HO - 44 + 2H]产物离子（琥珀酰 = 琥珀酰亚胺），使用 EAD 区分硫代天冬氨酸和硫代异天冬氨酸异构体。一个新发现的[R + 硫代琥珀酰 + HO - 44 + 2H]离子也作为额外的证据进一步支持了我们的发现。
这是首次在不使用合成参考物质的情况下鉴定硫代琥珀酰亚胺水解异构体。这种方法应该适用于所有硫代琥珀酰亚胺连接的分子。正确鉴定硫代琥珀酰亚胺水解异构体最终可能有益于未来 ADC 的开发。
