Heudi Olivier, Barteau Samuel, Zimmer Dieter, Schmidt Joerg, Bill Kurt, Lehmann Natalie, Bauer Christian, Kretz Olivier
DMPK/Bioanalytics, and Biotechnology Development, Novartis Pharma AG, CH-4002 Basel, Switzerland.
Anal Chem. 2008 Jun 1;80(11):4200-7. doi: 10.1021/ac800205s. Epub 2008 May 9.
Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.
尽管液相色谱 - 质谱（LC-MS）方法越来越多地用于蛋白质的绝对定量，但缺乏合适的内标（IS）阻碍了用于体外和体内研究的快速且标准化分析方法的发展。在此，我们开发了一种用于治疗性蛋白质（即单克隆抗体（mAb））绝对定量的新方法。该方法将液相色谱串联质谱（LC-MS/MS）与蛋白质裂解同位素稀释质谱相结合，使用同位素标记的mAb作为内标。后者与分析的mAb相同，只是每个苏氨酸含有四个（13）C原子和一个（15）N原子。血清样品在过夜胰蛋白酶消化和随后的样品净化之前加入内标。使用11分钟的LC梯度时间在C18 ACE柱（150 mm×4.6 mm）上分析样品提取物。通过计算其特征肽与相应同位素标记肽的峰高比来确定内源性mAb浓度。线性动态范围在5.00至1000μg/mL mAb之间建立，所有浓度下的准确度和精密度在±15%以内，定量下限（LLOQ）处低于±20%。就mAb而言，整体方法回收率为14%。样品制备（消化和纯化）导致的损失为72%，其中约32%归因于该方法的第一步，即样品消化。样品制备过程中的巨大损失强烈强调了从一开始就使用内标的必要性。我们的方法成功应用于狨猴血清研究样品中的mAb定量，并且在大多数情况下，重复样品获得的精密度低于20%。与酶联免疫吸附测定（ELISA）的比较表明，LC-MS/MS方法在AUC和Cmax方面显示出更高的暴露量。本研究讨论了这种差异的可能原因。本研究结果表明，我们的LC-MS/MS方法是一种简单、快速且精确的治疗性mAb定量方法，可支持临床前和临床研究。
