Presently, antibody concentration measurements for patients undergoing treatment are predominantly determined by ELISA, which still comes with known disadvantages. Therefore, our aim was to establish a targeted mass-spectrometric assay enabling the reproducible absolute quantification of peptides from the hypervariable and interaction regions of infliximab. Peptides of infliximab were measured post-trypsin digestion and subsequent separation on a Vanquish Horizon UHPLC coupled to a TSQ Altis Triple-Quad mass spectrometer. Normalization and absolute quantification were conducted using stable isotope-synthesized peptides. Calibration curves covering a range of 0.25-50 µg/ml were employed for quantitation. We demonstrated the substantial influence of peptide selection, choice of hydrolase for digestion, and digestion time on absolute peptide yield (28–44% for peptide 1 and 64–97% for peptide 2). Furthermore, we showed that the generated calibration curves for absolute quantification were highly reproducible and robust (LLOQ1 0.72 µg/ml and LLOQ2 1.00 µg/ml) over several months. In comparison to ELISA values, the absolute values obtained by mass spectrometry often yielded lower results for both targeted peptides. In this study, a semi-automated workflow was employed and tested with 8 patients and corresponding replicates (n = 3–4). We demonstrated the robust implementation of calibration curves for the absolute quantification of infliximab in patient samples, with coefficients of variation ranging from 0.5 to 9%. Taken together, we have developed a platform enabling the rapid (2 days of sample preparation and 30 min of measurement time per sample) and robust quantification of Infliximab antibody concentration in patients. The use of mass spectrometry also facilitates the straightforward expansion of the method to include additional antibody peptides.

中文翻译：

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与 ELISA 数据相比，通过靶向质谱法对血清中的英夫利昔单抗进行高灵敏度治疗药物监测

目前，接受治疗的患者的抗体浓度测量主要通过 ELISA 来确定，但它仍然存在已知的缺点。因此，我们的目标是建立一种靶向质谱分析方法，能够对英夫利昔单抗高变区和相互作用区域的肽进行可重复的绝对定量。英夫利昔单抗的肽在胰蛋白酶消化后进行测量，随后在与 TSQ Altis Triple-Quad 质谱仪联用的 Vanquish Horizo​​n UHPLC 上进行分离。使用稳定同位素合成的肽进行归一化和绝对定量。采用涵盖 0.25-50 µg/ml 范围的校准曲线进行定量。我们证明了肽选择、消化水解酶的选择以及消化时间对绝对肽产率的重大影响（肽 1 为 28-44%，肽 2 为 64-97%）。此外，我们还表明，生成的绝对定量校准曲线在几个月内具有高度可重复性和稳健性（LLOQ1 0.72 µg/ml 和 LLOQ2 1.00 µg/ml）。与 ELISA 值相比，通过质谱法获得的绝对值通常对两种目标肽产生较低的结果。在本研究中，采用了半自动化工作流程，并在 8 名患者和相应的重复实验中进行了测试 (n = 3-4)。我们展示了用于患者样本中英夫利昔单抗绝对定量的校准曲线的稳健实施，变异系数范围为 0.5% 至 9%。总之，我们开发了一个平台，能够快速（2 天的样品制备和每个样品 30 分钟的测量时间）和稳健地定量患者体内的英夫利昔单抗抗体浓度。质谱法的使用还有助于直接扩展该方法以包括额外的抗体肽。