a Department of Chemistry , University of Massachusetts Amherst , Amherst , MA , USA.
b QuarryBio Inc ., Bloomington , IN , USA.
MAbs. 2019 Apr;11(3):463-476. doi: 10.1080/19420862.2019.1565748. Epub 2019 Feb 6.
Monoclonal antibodies are among the fastest growing therapeutics in the pharmaceutical industry. Detecting higher-order structure changes of antibodies upon storage or mishandling, however, is a challenging problem. In this study, we describe the use of diethylpyrocarbonate (DEPC)-based covalent labeling (CL) - mass spectrometry (MS) to detect conformational changes caused by heat stress, using rituximab as a model system. The structural resolution obtained from DEPC CL-MS is high enough to probe subtle conformation changes that are not detectable by common biophysical techniques. Results demonstrate that DEPC CL-MS can detect and identify sites of conformational changes at the temperatures below the antibody melting temperature (e.g., 55 ᴼC). The observed labeling changes at lower temperatures are validated by activity assays that indicate changes in the F region. At higher temperatures (e.g., 65 ᴼC), conformational changes and aggregation sites are identified from changes in CL levels, and these results are confirmed by complementary biophysical and activity measurements. Given the sensitivity and simplicity of DEPC CL-MS, this method should be amenable to the structural investigations of other antibody therapeutics.
单克隆抗体是制药行业中增长最快的治疗方法之一。然而，检测抗体在储存或处理不当时的高级结构变化是一个具有挑战性的问题。在这项研究中，我们描述了使用基于二乙基焦碳酸盐（DEPC）的共价标记（CL）-质谱（MS）来检测热应激引起的构象变化，以利妥昔单抗为模型系统。从 DEPC CL-MS 获得的结构分辨率足以探测到常见生物物理技术无法检测到的细微构象变化。结果表明，DEPC CL-MS 可以在低于抗体熔化温度（例如 55°C）的温度下检测和识别构象变化的部位。在较低温度下观察到的标记变化通过活性测定得到验证，该测定表明 F 区发生了变化。在较高温度（例如 65°C）下，通过 CL 水平的变化鉴定出构象变化和聚集部位，这些结果通过补充生物物理和活性测量得到证实。鉴于 DEPC CL-MS 的灵敏度和简单性，该方法应该适用于其他抗体治疗药物的结构研究。
