Hentschel Andreas, Piontek Gina, Dahlmann Rob, Findeisen Peter, Sakson Roman, Carbow Phil, Renné Thomas, Reinders Yvonne, Sickmann Albert
Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., Dortmund, Germany.
MVZ Labor Dr. Limbach & Kollegen, Heidelberg, Germany.
Clin Proteomics. 2024 Feb 29;21(1):16. doi: 10.1186/s12014-024-09464-x.
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.
目前，接受治疗患者的抗体浓度测量主要通过酶联免疫吸附测定（ELISA）来确定，而该方法仍存在已知的缺点。因此，我们的目标是建立一种靶向质谱测定法，能够对英夫利昔单抗高变区和相互作用区的肽段进行可重复的绝对定量。
对英夫利昔单抗的肽段进行胰蛋白酶消化，随后在与TSQ Altis三重四极杆质谱仪联用的Vanquish Horizon超高效液相色谱仪上进行分离。使用稳定同位素合成肽进行归一化和绝对定量。采用覆盖0.25 - 50μg/ml范围的校准曲线进行定量。
我们证明了肽段选择、消化所用水解酶的选择以及消化时间对肽段绝对产量有重大影响（肽段1为28 - 44%，肽段2为64 - 97%）。此外，我们表明，用于绝对定量的生成校准曲线在几个月内具有高度的可重复性和稳健性（最低定量限1为0.72μg/ml，最低定量限2为1.00μg/ml）。与ELISA值相比，通过质谱获得的两个靶向肽段的绝对值结果通常较低。
在本研究中，采用了半自动化工作流程，并对8名患者及相应复制品（n = 3 - 4）进行了测试。我们证明了校准曲线在患者样本中英夫利昔单抗绝对定量方面的稳健实施，变异系数范围为0.5%至9%。综上所述，我们开发了一个平台，能够快速（样本制备2天，每个样本测量时间30分钟）且稳健地定量患者体内英夫利昔单抗抗体浓度。质谱的使用也便于将该方法直接扩展以纳入其他抗体肽段。
