Legeron Rachel, Xuereb Fabien, Chaignepain Stephane, Gadeau Alain-Pierre, Claverol Stephane, Dupuy Jean-William, Djabarouti Sarah, Couffinhal Thierry, Schmitter Jean-Marie, Breilh Dominique
Department of Pharmacy, Groupe hospitalier Sud, CHU de Bordeaux, Avenue de Magellan, 33604, Pessac, France; Univ. Bordeaux, EPST, Biology of Cardiovascular Diseases, U1034, 33604, Pessac, France.
Univ. Bordeaux, EPST, CBMN, CGFB, UMR 5248, CNRS, 33076, Bordeaux Cedex, France.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Dec 1;1070:43-53. doi: 10.1016/j.jchromb.2017.10.042. Epub 2017 Oct 23.
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 500μg/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.
对生物流体中的单克隆抗体（mAb）进行定量分析，如贝伐单抗（一种重组人源化免疫球蛋白G1（hIgG1）），是任何临床前和临床药代动力学研究的重要前提。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部分。然而，血浆样本的储存和分析条件可能会改变贝伐单抗游离、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的高估或低估。我们开发了一种新的测定方法，通过液相色谱串联质谱（LC-MS/MS）对贝伐单抗的总部分进行绝对定量，该方法基于两种特定肽段对贝伐单抗进行鉴定和定量。在本报告中，我们将我们的测定方法与两种内标（IS）校准方法进行了比较：一种使用不同的人源mAb（曲妥珠单抗），另一种使用稳定同位素标记的特定肽段。通过蛋白A亲和色谱富集和超滤浓缩后，用人胰蛋白酶对人血浆样本进行蛋白水解。线性范围为12.5至500μg/mL，日间准确度范围为101.7%至110.6%，精密度范围为7.0%至9.9%。本研究证明了在定量人血浆中贝伐单抗时选择内标的重要性，并突出了实现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方法来定量人血浆中贝伐单抗的总血浆部分。通过我们的开发，我们提出了一种通用方法，可轻松转换用于定量所有IgG1亚类，这对临床药代动力学研究非常有用。
