Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS UMR7178, IPHC , 67000 Strasbourg, France.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne , CMU - Rue Michel-Servet, 1, 1206 Geneva, Switzerland.
Anal Chem. 2018 Feb 6;90(3):1578-1586. doi: 10.1021/acs.analchem.7b02110. Epub 2018 Jan 5.
There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quality control laboratories. Native MS offers structural insights into multiple ADC critical quality attributes, thanks to accurate mass measurement. However, both techniques can lead to misinterpretations or incomplete characterization when used as standalone methods. Online coupling of both techniques can thus potentially be of great interest, but the presence of large amounts of nonvolatile salts in HIC mobile phases makes it not easily directly compatible with native MS. Here, we present an innovative multidimensional analytical approach combining comprehensive online two-dimensional (2D)-chromatography that consists of HIC and size-exclusion chromatography (SEC), to ion mobility and mass spectrometry (IM-MS) for performing analytical characterization of ADCs under nondenaturing conditions. This setup enabled comprehensive and streamlined characterization of both native and forced degraded ADC samples. The proposed 4D methodology might be more generally adapted for online all-in-one HIC×SEC-IM×MS analysis of single proteins or analysis of protein complexes in nondenaturing conditions.
目前有两种主要技术可以在天然条件下分析鉴定半胱氨酸连接的抗体药物偶联物（ADC）的链间结构，即疏水相互作用色谱（HIC）和天然质谱（MS）。HIC 是一种色谱技术，可用于评估药物负载谱并在质量控制实验室计算平均药物抗体比（DAR）。天然 MS 可以通过准确测量分子量，为多个 ADC 关键质量属性提供结构见解。然而，当单独使用这两种技术时，可能会导致误解或不完全的特征描述。因此，这两种技术的在线联用可能具有很大的吸引力，但 HIC 流动相中存在大量的不可挥发盐，使得它不易与天然 MS 直接兼容。在这里，我们提出了一种创新的多维分析方法，结合了全面的在线二维（2D）色谱，包括 HIC 和尺寸排阻色谱（SEC），离子迁移和质谱（IM-MS），用于在非变性条件下对 ADC 进行分析鉴定。该装置实现了对天然和强制降解 ADC 样品的全面简化的特征描述。该方法可更普遍地适用于非变性条件下单个蛋白质的在线一体式 HIC×SEC-IM×MS 分析或蛋白质复合物的分析。
