Quantitative LC-MS/MS method for nivolumab in human serum using IgG purification and immobilized tryptic digestion
Quantitative LC-MS/MS method for nivolumab in human serum using IgG purification and immobilized tryptic digestion
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DOI：
10.1039/c9ay02087j
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发表时间：
2020-01-07
影响因子：
3.1
通讯作者：
Kawakami, Junichi
中科院分区：
文献类型：
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作者：
Abe, Kazuki;Shibata, Kaito;Kawakami, Junichi
液相色谱-串联质谱（LC-MS/MS）法是一种新的用于治疗性单克隆抗体定量分析的方法。然而，该方法具有严重的电离抑制和耗时的样品制备。在本研究中，我们开发了一种简单快速的LC-MS/MS方法，用于定量血清纳武利尤单抗，使用免疫球蛋白G（IgG）纯化和固定化胰蛋白酶消化，具有良好的分析性能。通过混合四极杆-Orbitrap质谱仪鉴定源自纳武单抗的替代肽。IgG纯化和固定化胰蛋白酶消化在25分钟内完成。色谱分离在10分钟内完成，逐步梯度洗脱。干扰替代肽及其稳定同位素标记肽作为内标的色谱峰未从血清中观察到。校准曲线的动态范围为2-200 μ g mL（-1）。批内和批间准确度和不精密度分别为92.2-104.5%和小于10.0%。在14名癌症患者中，血清nivolumab浓度范围为12-112 μ g/mL（-1）。LC-MS/MS法与ELISA法测得的血药浓度呈显著正相关（r = 0.92，P < 0.01）。总之，发现使用固定化胰蛋白酶消化结合IgG纯化来定量血清nivolumab的简单且快速的LC-MS/MS方法对于临床环境是可接受的。
A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method is a novel approach for the quantification of therapeutic monoclonal antibody. However, this method has severe ionization suppression and time-consuming sample preparation. In the present study, we developed a simple and rapid LC-MS/MS method for quantifying serum nivolumab with good analytical performance using immunoglobulin G (IgG) purification and immobilized tryptic digestion. Surrogate peptide derived from nivolumab was identified by a hybrid quadrupole-Orbitrap mass spectrometer. IgG purification and immobilized tryptic digestion were completed within 25 minutes. The chromatographic separation was completed in 10 minutes with stepwise-gradient elution. Chromatographic peaks interfering with the surrogate peptide and its stable isotope-labeled peptide as an internal standard were not observed from serum digests. The dynamic range of the calibration curve was 2-200 mu g mL(-1). The intra- and inter-assay accuracies and imprecisions were 92.2-104.5% and less than 10.0%, respectively. Serum nivolumab concentrations ranged from 12-112 mu g mL(-1) in 14 cancer patients. The measured concentrations in the LC-MS/MS method were strongly correlated with those in the ELISA method (r = 0.92, P < 0.01). In conclusion, a simple and rapid LC-MS/MS method for quantifying serum nivolumab using immobilized tryptic digestion coupled to IgG purification was found to be acceptable for clinical settings.