Donners Anouk A M T, Gerencsér László, van der Elst Kim C M, Egberts Toine C G, de Maat Moniek P M, Huisman Albert, Urbanus Rolf T, El Amrani Mohsin
Department of Clinical Pharmacy University Medical Center Utrecht Utrecht University Utrecht The Netherlands.
Department of Pharmacoepidemiology and Clinical Pharmacology Utrecht Institute for Pharmaceutical Sciences Faculty of Science Utrecht University Utrecht The Netherlands.
Res Pract Thromb Haemost. 2022 Jun 8;6(4):e12725. doi: 10.1002/rth2.12725. eCollection 2022 May.
Emicizumab is a new treatment option for people with hemophilia A. Emicizumab was approved with a body-weight-based dosage regimen, without laboratory monitoring requirements. Guidelines, however, recommend measuring emicizumab concentrations when the presence of antidrug antibodies is suspected. Furthermore, drug monitoring can be useful in clinical decision making, in adherence checking, and for research purposes. Therefore, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying emicizumab. We performed a validation study on this LC-MS/MS method quantifying emicizumab in the plasma of people with hemophilia A.
Sample preparation for LC-MS/MS analysis included ammonium sulfate protein precipitation and trypsin digestion. A signature peptide of emicizumab and a matching stable isotope-labeled internal standard were used to quantify emicizumab by LC-MS/MS analysis. Validation was performed in accordance with the "Guideline on Bioanalytical Method Validation" of the European Medicines Agency (EMA). The LC-MS/MS method was cross validated against a modified and calibrated ( Diagnostics) one-stage clotting assay (OSA).
The LC-MS/MS method demonstrated linearity over a wide range of emicizumab concentrations, far exceeding the concentrations observed in people with hemophilia A. Precision and accuracy were excellent, and all other validation parameters were also within the acceptance EMA criteria. Cross validation showed that the LC-MS/MS method and the OSA-based method can be used interchangeably for drug monitoring of emicizumab without the application of a correction factor.
艾美赛珠单抗是治疗甲型血友病患者的一种新的治疗选择。艾美赛珠单抗获批采用基于体重的给药方案，无需实验室监测。然而，指南建议在怀疑存在抗药抗体时检测艾美赛珠单抗浓度。此外，药物监测在临床决策、依从性检查及研究目的方面可能有用。因此，我们开发了一种用于定量艾美赛珠单抗的液相色谱 - 串联质谱（LC-MS/MS）方法。我们对这种在甲型血友病患者血浆中定量艾美赛珠单抗的LC-MS/MS方法进行了验证研究。
LC-MS/MS分析的样品制备包括硫酸铵蛋白沉淀和胰蛋白酶消化。使用艾美赛珠单抗的一个特征肽段和一个匹配的稳定同位素标记内标，通过LC-MS/MS分析定量艾美赛珠单抗。根据欧洲药品管理局（EMA）的《生物分析方法验证指南》进行验证。将LC-MS/MS方法与一种经过改良和校准的（Diagnostics）一步凝血试验（OSA）进行交叉验证。
LC-MS/MS方法在很宽的艾美赛珠单抗浓度范围内均表现出线性关系，远远超过甲型血友病患者中观察到的浓度。精密度和准确度极佳，所有其他验证参数也均在EMA认可标准范围内。交叉验证表明，LC-MS/MS方法和基于OSA的方法可在不应用校正因子的情况下互换用于艾美赛珠单抗的药物监测。
