Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, Box 599, Husargatan 3, SE-75124 Uppsala, Sweden.
Recipharm OT Chemistry AB, Virdings Allé 18, SE-754 50 Uppsala, Sweden.
Anal Chem. 2020 Jul 7;92(13):9001-9007. doi: 10.1021/acs.analchem.0c01106. Epub 2020 Jun 8.
Characterization of antibody-drug conjugates (ADCs) using mass spectrometry (MS) is important in drug discovery and formulation development and as part of the quality control processes. To facilitate electrospray ionization (ESI) and produce high-quality mass spectra, common components of storage solutions for monoclonal antibodies (mAbs) and ADCs, such as nonvolatile phosphate-buffered saline (PBS), should be replaced before analysis. Centrifugal spin-type kits are extensively used for the desalting or buffer-exchange of mAbs and ADCs samples. The commercially available kits commonly require at least 100 μL of a sample at 1 mg/mL for optimal recovery. However, most ESI-MS based analyses require no more than 25 μg of protein for triplicate injection. In this study, we present a novel method for desalting of ADCs and mAbs building on the SP3 approach with nonfunctionalized carboxylate coated magnetic beads without affinity ligands. The analytes bind to the hydrophilic beads upon the addition of organic solvent, and various solutions of volatile salts or acids can be used in the elution step. The optimized protocol allowed for 88% recovery of ADC at a 25 μL sample volume and 90% recovery at 100 μL. More than 90% of the salts were removed using a process of 20 min. The intra- and interday precision showed little variation with an RSD of 1% and 2%, respectively. The compatibility of this new workflow with low sample volumes is highly valuable since a smaller fraction of the sample is wasted for analysis of the expensive analytes, without compromising recovery.
使用质谱（MS）对抗体药物偶联物（ADC）进行表征对于药物发现和制剂开发以及质量控制过程至关重要。为了促进电喷雾电离（ESI）并产生高质量的质谱，在分析之前，应将单克隆抗体（mAb）和 ADC 储存溶液的常见成分（如非挥发性磷酸盐缓冲盐水（PBS））替换掉。离心式旋转试剂盒广泛用于 mAb 和 ADC 样品的脱盐或缓冲液交换。市售试剂盒通常需要至少 100μL、浓度为 1mg/mL 的样品，以实现最佳回收率。然而，大多数基于 ESI-MS 的分析需要不超过 25μg 的蛋白质进行三次重复注射。在这项研究中，我们提出了一种基于 SP3 方法的新型脱盐方法，使用非功能化的羧酸涂层磁性珠，无需亲和配体。在加入有机溶剂后，分析物与亲水性珠结合，在洗脱步骤中可以使用各种挥发性盐或酸溶液。优化的方案允许在 25μL 样品体积下以 88%的回收率回收 ADC，在 100μL 样品体积下以 90%的回收率回收 ADC。使用 20min 的过程可以去除超过 90%的盐。日内和日间精密度变化很小，RSD 分别为 1%和 2%。由于分析昂贵分析物时浪费的样品量较小，因此这种新工作流程与小样品量的兼容性非常有价值，而不会影响回收率。
