Analytical Chemistry, Regeneron Pharmaceuticals Inc., Tarrytown, New York, 10591, USA.
Bioanalytical Sciences, Regeneron Pharmaceuticals Inc., Tarrytown, New York, 10591, USA.
AAPS J. 2021 Jan 6;23(1):16. doi: 10.1208/s12248-020-00538-w.
There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.
目前迫切需要开发新技术来评估生物疗法的免疫原性。在这里，我们开发了一种免疫捕获 LC-MS 分析方法，用于鉴定和半定量测定猴子针对全人源单克隆抗体 (mAb) 药物的抗体。使用全长 mAb 的 Fab 交联到磁珠上的免疫捕获步骤从血清样本中分离出抗体，以最大程度减少基质干扰。针对人免疫球蛋白 κ 轻链的阳性单克隆抗体对照被用作 ADA 定量的校准标准。最终的 LC-MS 方法包含 17 个多重反应监测 (MRM) 转换和优化的 15 分钟 LC 方法。结果表明，在 ADA 阳性样本中 IgG1 是最丰富的同种型。较低水平检测到 IgG2 和 IgG4，而 IgG3 和 IgA 水平仅在非常低的水平下观察到。此外，通过 LC-MS 分析测定的总 ADA 水平与使用传统配体结合分析 (LBA) 获得的结果相当。LC-MS ADA 分析方法能够快速评估免疫原性，并提供 LBA 无法提供的额外同种型信息。
