Abstract：
In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 μm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 μL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 μg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.
摘要：
近年来,由于单克隆抗体(mAb)在临床治疗中的广泛利用,人们对蓬勃发展的单克隆抗体(mAb)产业的兴趣与日俱增。需要稳健和准确的生物分析方法，以实现生物基质中单克隆抗体的快速定量。特别是在药代动力学（PKs）/药效学（PDs）和治疗药物监测（TDM）研究的背景下。在这次调查中,我们提出了一种新型的免疫磁捕获与液相色谱-串联质谱（LC-MS/MS）方法，旨在定量生物流体中基于免疫球蛋白G-κ的mAb。用于mAb药物纯化的免疫亲和吸收剂是通过将蛋白L固定到monossize上而精心制作的，磁性聚（甲基丙烯酸缩水甘油酯）（m-pGMA）珠，通过分散聚合合成。获得的微球的平均尺寸为1.6μm，并且从mAb水溶液中的mAb的最佳结合被确定为45.82mgg-1。通过使用m-pGMA@蛋白L珠（使用利妥昔单抗作为内标（IS））的亲和纯化，实现了10μL血清样品中mAb的定量。珠上减少，和快速的胰蛋白酶消化。值得注意的是,整个过程,花费不到2.5小时，具有简化预处理程序和最大限度地减少分析时间的巨大潜力。此外，所开发的方法按照欧洲药品管理局(EMA)指南进行了验证.该测定显示达雷图单抗和派姆单抗在2-400μgmL-1范围内的良好线性。测定内和测定间的变异系数落在0.7%至13.4%的范围内，符合既定的验收标准。其他验证参数也符合监管标准。最终,在对小鼠单剂量静脉给药后的药代动力学研究中证实了该方法的有效性，强调其在现实场景中的适用性和可靠性。
