Department of Medical Oncology, Cancer Center Amsterdam (CCA), Amsterdam University Medical Centers, University of Amsterdam , Amsterdam, The Netherlands.
Department of Business Development Bioanalysis Europe, QPS Netherlands BV , Groningen, The Netherlands.
MAbs. 2020 Jan-Dec;12(1):1795492. doi: 10.1080/19420862.2020.1795492.
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.
鉴于越来越多地使用多种单克隆抗体 (mAb) 的联合治疗，因此临床需要多重检测。对于经常联合使用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且稳健的混合配体结合液相色谱-质谱 (LC-MS)/MS 定量检测方法。在经过蛋白 A 珠亲和纯化提取后，纳摩尔浓度的曲妥珠单抗和帕妥珠单抗可在 10 µL 血清样本中被检测到，随后进行珠上还原、烷基化和胰蛋白酶消化。经电喷雾电离后，通过 SILuMab 作为内标进行多重反应监测 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。检测方法在曲妥珠单抗的 0.250-250 μg/mL 和帕妥珠单抗的 0.500-500 μg/mL 范围内具有线性。该方法可准确且选择性地用于临床样本中曲妥珠单抗和帕妥珠单抗的同时测定，从而克服了配体结合测定法无法定量针对同一受体的 mAb 的局限性。此外，该方法需要的血液量较少，从而减少了患者的采血时间和压力。该检测方法的稳健性在一项临床试验中得到了验证，该试验中对 670 个血清样本中的曲妥珠单抗和帕妥珠单抗浓度进行了测定。由于我们使用了市售的试剂和标准品，因此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本中其他 mAb 组合的多重定量。
