Investigating the utility of minimized sample preparation and high‐resolution mass spectrometry for quantification of monoclonal antibody drugs
Investigating the utility of minimized sample preparation and high‐resolution mass spectrometry for quantification of monoclonal antibody drugs
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研究最小化样品制备和高分辨率质谱在单克隆抗体药物定量中的效用
DOI：
10.1016/j.jpba.2018.07.012
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发表时间：
2018
影响因子：
3.4
通讯作者：
K. Rand
中科院分区：
文献类型：
--
作者：
T. T. Nguyen;Ulrik H. Mistarz;N. Costa;A. Herbet;D. Boquet;F. Becher;K. Rand
确定治疗性单克隆抗体 (mAb) 的药代动力学 (PK) 特性对于其成功开发为药物至关重要。为此，除了传统的配体结合测定（LBA）之外，使用低分辨率质谱仪（例如三重四极杆（QqQ））的LC-MS/MS方法已成为常规使用，然而，为了获得足够的灵敏度和选择性，通常需要复杂而冗长的样品预处理（采用免疫亲和力）。在本研究中，我们研究了高分辨率 MS 仪器的能力，以规避目前基于 LC-MS/MS 的单克隆抗体定量的敏感 LC-MS/MS 所需的复杂样品制备过程。采用简单的一步样品预处理工作流程，我们比较了三种不同 LC-MS 平台对血清和血浆中代表性单克隆抗体 Rendomab-B1 进行绝对定量的能力。样品用甲醇进行蛋白质沉淀，然后用胰蛋白酶消化沉淀，然后进行 LC-MS 分析。 AQUA 肽基于从广泛的计算机和实验筛选中选出的两种替代单克隆抗体肽，用作内标。 MS/MS 采集的开发和系统检查包括：1) 在选择反应监测 (SRM) 采集模式下运行的低分辨率 QqQ，2) 在并行反应监测 (PRM) 采集模式下运行的高分辨率混合四极杆轨道阱 (Q-Orbitrap)，以及 3) 在具有增强占空比 (EDC) 功能的 SRM 采集模式下运行的高分辨率混合四极杆飞行时间 (Q-TOF)。血清/血浆样品中高分辨率 Q-Orbitrap 和 Q-TOF 方法的灵敏度显着高于低分辨率 QqQ 方法（LOD 为 80ng/mL）。最后，通过为期 15 天的 10 点体内研究确定 Rendomab-B1 在小鼠体内的 PK 曲线，证明并验证了所开发的高分辨率 MS 方法在最小化样品处理的现实世界中的实用性。
Determination of the pharmacokinetic (PK) properties of therapeutic monoclonal antibodies (mAbs) is essential for their successful development as drugs. For this purpose, besides the traditional ligand binding assay (LBA), LC–MS/MS method using low resolution mass spectrometers (e.g.triple quadrupole (QqQ)) has become routinely used, however, complicated and lengthy sample pre-treatment (employing immuno-affinity) is often necessary for obtaining sufficient sensitivity and selectivity. In this study, we investigate the capabilities of high-resolution MS instruments for circumventing the complex sample preparation currently needed for sensitive LC–MS/MS-based quantification of mAbs. Employing a simple one-step sample pre-treatment workflow, we compare the ability of three different LC–MS platforms for absolute quantification of a representative monoclonal antibody Rendomab-B1 in serum and plasma. The samples are subjected to protein precipitation with methanol, followed by pellet digestion with trypsin prior to LC–MS analysis. AQUA peptides based on two surrogate mAb peptides selected from an extensivein-silicoand experimental screening are used as internal standards. MS/MS acquisitions are developed and systematically examined for 1) a low-resolution QqQ operated in selected reaction monitoring (SRM) acquisition mode, 2) a high-resolution hybrid Quadrupole-Orbitrap (Q-Orbitrap) operated in parallel reaction monitoring (PRM) acquisition mode and 3) a high-resolution hybrid Quadrupole-Time-of-flight (Q-TOF) operated in SRM acquisition mode with enhanced duty cycle (EDC) function. The sensitivity of the high-resolution Q-Orbitrap and Q-TOF methods was significantly higher (LOD of 80 ng/mL) in serum/plasma samples than the low-resolution QqQ method. Finally, the real-world utility of the developed high-resolution MS method with minimized sample handling was demonstrated and validated by determining the PK profile of Rendomab-B1 in mice by a 10-pointin vivostudy over 15 days.
通过 LC-MS/MS 证明人体皮肤中存在胆固醇酯氢过氧化物
DOI：
--
发表时间：
2020
期刊：
影响因子：
--
作者：
石川絢野;伊藤隼哉;小林エリ;坂田修;成英次;仲川清隆
通讯作者：
仲川清隆