Scheffe Nina, Schreiner Rupert, Thomann Anne, Findeisen Peter
MVZ Laboratory Dr. Limbach & Colleagues, Heidelberg, Germany.
Department of Medicine II, Medical Faculty Mannheim of the University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany; and.
Ther Drug Monit. 2020 Aug;42(4):572-577. doi: 10.1097/FTD.0000000000000734.
Ustekinumab (UST) is a human monoclonal antibody used to treat moderate-to-severe Crohn disease by blocking the interleukin-12/23 pathway. Although an optimized therapeutic concentration of UST is associated with clinical response and improved prognosis, the availability of clinical laboratory methods for UST monitoring is limited. Furthermore, the commercially available methods are immunoassays that are prone to interference of antidrug antibodies. This study aimed to develop a liquid chromatography-tandem mass spectrometry method for quantification of UST in human serum specimens.
A tryptic peptide that is specific to the heavy chain variable region of UST was selected. Quantification of UST was performed by selective reaction monitoring on a quadrupole TQ-XS with an internal standard. After digestion with trypsin, peptides were separated by reverse-phase C18 liquid chromatography; peptides were detected by MS/MS, and analyte to internal standard peak area ratios were used for the quantification. Finally, serum samples from patients treated with UST were collected at trough levels (n = 66).
The assay showed a broad dynamic range with linearity between 0.4 and 20 mg/L (R = 0.995). The lower limit of quantification was found to be 0.4 mg/L. The reproducibility was tested with 3 different UST concentrations (2, 8, and 16 mg/L). The coefficients of intra-assay and interassay variations were 2.2%-4.0% and 2.7%-5.3%, respectively. UST serum concentrations of 2-16 mg/L were stable for up to 14 days when specimens were left at room temperature (20°C).
The newly developed LC/MS-based method was shown to be feasible for UST quantification. This analytical approach may lead to individualized dosing and improved patient care.
乌司奴单抗（UST）是一种人源单克隆抗体，通过阻断白细胞介素-12/23通路用于治疗中重度克罗恩病。尽管乌司奴单抗的优化治疗浓度与临床反应及预后改善相关，但用于监测乌司奴单抗的临床实验室方法有限。此外，市售方法为免疫测定法，容易受到抗药抗体的干扰。本研究旨在开发一种液相色谱-串联质谱法，用于定量测定人血清样本中的乌司奴单抗。
选择了一种对乌司奴单抗重链可变区特异的胰蛋白酶肽段。采用内标在四极杆TQ-XS上通过选择性反应监测进行乌司奴单抗的定量分析。用胰蛋白酶消化后，肽段通过反相C18液相色谱分离；通过串联质谱检测肽段，分析物与内标峰面积比用于定量。最后，收集了接受乌司奴单抗治疗患者的血清样本谷浓度（n = 66）。
该测定法显示出较宽的动态范围，线性范围为0.4至20 mg/L（R = 0.995）。定量下限为0.4 mg/L。用3种不同的乌司奴单抗浓度（2、8和16 mg/L）测试了重现性。批内和批间变异系数分别为2.2%-4.0%和2.7%-5.3%。当样本在室温（20°C）下放置时，2-16 mg/L的乌司奴单抗血清浓度在长达14天内保持稳定。
新开发的基于液相色谱/质谱的方法被证明可用于乌司奴单抗的定量分析。这种分析方法可能会实现个体化给药并改善患者护理。
