Toward Accurate and Robust Liquid Chromatography-Mass Spectrometry-Based Quantification of Antibody Biotherapeutics in Tissues
Toward Accurate and Robust Liquid Chromatography-Mass Spectrometry-Based Quantification of Antibody Biotherapeutics in Tissues
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DOI：
10.1021/acs.analchem.0c03620
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发表时间：
2020-11-17
影响因子：
7.4
通讯作者：
Qu, Jun
中科院分区：
文献类型：
--
作者：
An, Bo;Zhang, Ming;Qu, Jun
液相色谱-质谱法（LC-MS）为组织中生物治疗剂/靶标的绝对定量提供了非常有前途的解决方案，这对于药物开发至关重要。尽管如此，准确/稳健的组织定量仍然具有挑战性，这主要是由于缺乏解决以下基本先决条件的最佳方法：（i）有效去除残留血液而不损失组织相关生物治疗剂;（ii）从组织中彻底/定量回收靶蛋白的最佳方法;以及（iii）制备校准/质量控制样品以确保准确组织分析的适当策略。在这里，我们设计了新的分析程序，使上述问题的广泛和系统的调查，从而发展的最佳策略，准确的组织分析。主要发现包括：首先，使用一种新的顺序给予未标记的单克隆抗体（mAb）的程序，然后使用稳定同位素标记的mAb;确定用三个血容量的肝素化盐水灌注是最佳的，实现了有效的血液清除（95 - 99%）和低定量偏倚（0.5 - 13%）;第二，通过质量平衡、极限浸提建立的参比样品组允许准确测量给药动物组织的绝对蛋白质回收率;用这种方法，我们发现mAb生物治疗剂存在于游离的-（49.3 - 75.4%）和结合型（24.6 - 50.7%），即使没有目标;因此，变性洗涤剂缓冲液是极限浸提所必需的（回收率> 90%）;第三，发现将mAb掺入组织后过夜孵育校准样品可提高定量准确度，特别是对于非变性缓冲液提取。这些研究建立了关键参数和最佳方案，可普遍应用于实现组织中生物治疗药物/靶标的准确和稳健定量。作为概念验证，我们采用基于LC-MS的方法首次对mAb在主要组织中进行了广泛的药代动力学测量，观察到mAb组织分布的有趣特征。
Liquid chromatography-mass spectrometry (LC-MS) affords a highly promising solution for absolute quantification of biotherapeutics/targets in tissues, which is critical for drug development. Nonetheless, accurate/robust tissue quantification remains challenging largely owing to the lack of optimal approaches to address the following fundamental prerequisites: (i) efficient removal of residual blood without losing tissue-associated biotherapeutics; (ii) an optimal method to exhaustively/quantitatively recover target proteins from tissues; and (iii) an appropriate strategy to prepare calibration/quality-control samples to ensure accurate tissue analysis. Here, we devised novel analytical procedures enabling extensive and systematic investigation of the above issues and thereby development of optimal strategies for accurate tissue analysis. Key discoveries include: first, using a novel procedure of sequential administration of nonlabeled and then stable-isotope-labeled monoclonal antibody (mAb); it was determined that perfusion with three blood volumes of heparinized saline is optimal, achieving efficient blood removal (95-99%) and low quantitative bias (0.5-13%); second, a reference sample set established by mass-balanced, exhaustive extraction, permitted accurate measurement of absolute protein recovery from tissues of dosed animals; with this method, we found mAb biotherapeutics present in free-(49.3-75.4%) and bound-forms (24.6-50.7%) in tissues, even without a target; therefore, a denaturing detergent buffer is necessary for exhaustive extraction (recovery>90%); third, overnight-incubation of calibration samples after spiking mAb to tissue was found to improve quantitative accuracy, especially for nondenaturing buffer extraction. These investigations established the critical parameters and optimal protocols that can be universally applied to achieve accurate and robust quantification of biotherapeutics/targets in tissues. As a proof of concept, we conducted the first-ever extensive pharmacokinetics measurement of mAb in major tissues with a LC-MS-based method, where interesting features of mAb tissue disposition were observed.