ABSTRACT

Given the increasing use of combination therapy with multiple monoclonal antibodies (mAbs), there is a clinical need for multiplexing assays. For the frequently co-administered anti-human epidermal growth factor receptor 2 (HER2) mAbs trastuzumab and pertuzumab, we developed a high-throughput and robust hybrid ligand-binding liquid chromatography-mass spectrometry (LC–MS)/MS quantitative assay. Nanomolar concentrations of trastuzumab and pertuzumab were determined in 10 µL serum samples after extraction by affinity purification through protein A beads, followed by on-bead reduction, alkylation, and trypsin digestion. After electrospray ionization, quantification was obtained by multiple reaction monitoring LC-MS/MS using SILuMab as an internal standard. The method was validated according to the current guidelines from the US Food and Drug Administration and the European Medicines Agency. Assay linearity was established in the ranges 0.250–250 μg/mL for trastuzumab and 0.500–500 μg/mL for pertuzumab. The method was accurate and selective for the simultaneous determination of trastuzumab and pertuzumab in clinical samples, thereby overcoming the limitation of ligand binding assays that cannot quantify mAbs targeting the same receptor. Furthermore, this method requires a small blood volume, which reduces blood collection time and stress for patients. The assay robustness was verified in a clinical trial where trastuzumab and pertuzumab concentrations were determined in 670 serum samples. As we used commercially available reagents and standards, the described generic bioanalytical strategy can easily be adapted to multiplex quantifications of other mAb combinations in non-clinical and clinical samples.

摘要

鉴于多种单克隆抗体 (mAb) 联合疗法的使用越来越多，临床需要进行多重检测。对于经常联合给药的抗人表皮生长因子受体 2 (HER2) mAb 曲妥珠单抗和帕妥珠单抗，我们开发了一种高通量、稳健的混合配体结合液相色谱-质谱 (LC-MS)/MS 定量测定法。通过蛋白 A 珠进行亲和纯化提取，然后进行珠上还原、烷基化和胰蛋白酶消化，然后在 10 µL 血清样品中测定曲妥珠单抗和帕妥珠单抗的纳摩尔浓度。电喷雾电离后，使用 SILuMab 作为内标，通过多反应监测 LC-MS/MS 进行定量。该方法根据美国食品和药物管理局和欧洲药品管理局的现行指南进行了验证。曲妥珠单抗的测定线性范围为 0.250–250 μg/mL，帕妥珠单抗的测定线性范围为 0.500–500 μg/mL。该方法准确且具有选择性，可同时测定临床样品中的曲妥珠单抗和帕妥珠单抗，从而克服了配体结合测定法无法量化针对同一受体的单克隆抗体的局限性。此外，该方法需要的血量较小，减少了采血时间和患者的压力。该测定的稳健性在一项临床试验中得到了验证，其中测定了 670 个血清样本中的曲妥珠单抗和帕妥珠单抗浓度。由于我们使用市售试剂和标准品，所描述的通用生物分析策略可以轻松适应非临床和临床样品中其他 mAb 组合的多重定量。