The Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, United States.
New York State Center of Excellence in Bioinformatics and Life Science, Buffalo, New York 14203, United States.
Anal Chem. 2020 Nov 17;92(22):15152-15161. doi: 10.1021/acs.analchem.0c03620. Epub 2020 Nov 6.
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 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.
液相色谱-质谱联用 (LC-MS) 为生物治疗药物/靶点在组织中的绝对定量提供了极具前景的解决方案，这对药物开发至关重要。尽管如此，由于缺乏解决以下基本前提的最佳方法，准确/稳健的组织定量仍然具有挑战性：(i) 有效地去除残留血液而不丢失与组织相关的生物治疗药物；(ii) 一种从组织中彻底/定量回收目标蛋白的最佳方法；和 (iii) 一种制备校准/质控样品的适当策略，以确保准确的组织分析。在这里，我们设计了新的分析程序，能够广泛和系统地研究上述问题，并因此开发了用于准确组织分析的最佳策略。主要发现包括：首先，使用顺序给予非标记和稳定同位素标记的单克隆抗体 (mAb) 的新程序；确定肝素化盐水灌注三个血液体积是最佳的，可实现高效的血液去除（95-99%）和低定量偏差（0.5-13%）；其次，通过质量平衡、彻底提取建立的参考样本集，允许准确测量给药动物组织中蛋白质的回收率；使用这种方法，我们发现 mAb 生物治疗药物以游离形式（49.3-75.4%）和结合形式（24.6-50.7%）存在于组织中，即使没有靶标；因此，需要变性去污剂缓冲液进行彻底提取（回收率>90%）；第三，发现在向组织中添加 mAb 后过夜孵育校准样品可提高定量准确性，特别是对于非变性缓冲液提取。这些研究确定了关键参数和最佳方案，可以普遍应用于实现生物治疗药物/靶点在组织中的准确和稳健定量。作为概念验证，我们使用基于 LC-MS 的方法首次对 mAb 在主要组织中的药代动力学进行了广泛测量，观察到了 mAb 组织处置的有趣特征。
