The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC–MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500 μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%.

This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC–MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies.

定量生物液中的单克隆抗体（mAb），例如贝伐单抗，一种重组人源化免疫球蛋白G1（hIgG1），是任何药代动力学临床前和临床研究的必要前提。迄今为止，用于定量mAb的参考技术依赖于缺乏特异性的酶联免疫吸附测定（ELISA）。此外，用于定量人血浆中贝伐单抗的市售ELISA试剂盒仅评估药物的游离级分。但是，血浆样品的储存和分析条件可能会改变贝伐单抗的游离，结合和部分结合形式之间的生理平衡，这可能导致药物浓度过高或过低。我们开发了一种通过液相色谱串联质谱法（LC-MS / MS）对贝伐单抗总含量进行绝对定量的新方法，该方法基于贝伐单抗的两种特定肽的鉴定和定量。在本报告中，我们将检测方法与两种内标（IS）校准方法进行了比较：一种使用不同的人类单克隆抗体（曲妥珠单抗），另一种使用稳定同位素标记的特异性肽。通过亲和层析富集蛋白A并通过超滤浓缩后，人血浆样品通过胰蛋白酶进行蛋白水解。线性建立在12.5至500μg/ mL之间，日间精度介于101.7至110.6％之间，精度介于7.0％至9.9％之间。在本报告中，我们将检测方法与两种内标（IS）校准方法进行了比较：一种使用不同的人类单克隆抗体（曲妥珠单抗），另一种使用稳定同位素标记的特异性肽。通过亲和层析富集蛋白A并通过超滤浓缩后，人血浆样品通过胰蛋白酶进行蛋白水解。线性建立在12.5至500μg/ mL之间，日间精度介于101.7至110.6％之间，精度介于7.0％至9.9％之间。在本报告中，我们将检测方法与两种内标（IS）校准方法进行了比较：一种使用不同的人类单克隆抗体（曲妥珠单抗），另一种使用稳定同位素标记的特异性肽。通过亲和层析富集蛋白A并通过超滤浓缩后，人血浆样品通过胰蛋白酶进行蛋白水解。线性建立在12.5至500μg/ mL之间，日间精度介于101.7至110.6％之间，精度介于7.0％至9.9％之间。

这项研究证明了选择IS在定量人血浆中贝伐单抗中的重要性，并强调了难以获得具有足够回收率的可靠蛋白水解的困难。我们开发了一种可靠且经济高效的LC-MS / MS方法，用于定量测定人血浆中贝伐单抗的总血浆含量。通过我们的开发，我们提出了一种通用的方法，可以轻松地进行转座，以量化对临床药代动力学研究非常有用的所有IgG1亚类。