PYX-201 is an investigational antibody drug conjugate (ADC) with an engineered, fully human IgG1 antibody, a cleavable chemical linker, and a toxin (Aur0101) with an average drug-antibody ratio (DAR) of ∼4. A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated to determine the presence in human plasma, of free payload Aur0101 from PYX-201 to assess drug safety and efficacy. Aur0101 and its deuterated internal standard (IS), Aur0101_d₈, were extracted from 25 µL of human plasma using a solid liquid extraction (SLE) method. Chromatographic analysis was carried out on a Waters Acquity UPLC BEH C18 (2.1 mm x 50 mm, 1.7 µm, 130 A) column. Quantitation of free Aur0101 was conducted on a Sciex triple quadrupole mass spectrometer API 6500+ using multiple reaction monitoring (MRM) mode via positive electrospray ionization. The calibration curve was linear over the concentration range of 25.0 to 12,500 pg/mL with correlation coefficient, r² ≥ 0.9988. The intra-assay %RE was between -4.3% to 14.3% with % CV was ≤ 6.2%. The inter–assay %RE was between -0.2% to 9.5% with % CV was ≤ 6.1%. The average analyte recovery was 89.7% and the average IS recovery was 88.7%. Aur-0101 was found to be stable in human plasma and human whole blood under various tested conditions with and without the presence of PYX-201. To our knowledge, this is the first published fully validated assay for free, unconjugated Aur0101 in any matrix, from any species. This assay has been successfully applied to clinical sample analysis to support clinical studies.

PYX-201 是一种在研抗体药物偶联物 (ADC)，具有工程化的全人 IgG1 抗体、可裂解的化学接头和平均药物抗体比 (DAR) 约为 4 的毒素 (Aur0101)。开发了一种灵敏、快速的液相色谱-串联质谱 (LC-MS/MS) 方法并经过充分验证，以确定人血浆中是否存在 PYX-201 的游离有效负载 Aur0101，以评估药物安全性和有效性。Aur0101 及其氘代内标 (IS)，Aur0101_d ₈使用固液萃取 (SLE) 方法从 25 µL 人血浆中提取。色谱分析在 Waters Acquity UPLC BEH C18（2.1 mm x 50 mm，1.7 µm，130 A）柱上进行。在 Sciex 三重四极杆质谱仪 API 6500+ 上使用多反应监测 (MRM) 模式通过正电喷雾电离对游离 Aur0101 进行定量。校准曲线在 25.0 至 12,500 pg/mL 的浓度范围内呈线性，相关系数 r ²  ≥ 0.9988。测定内 %RE 介于 -4.3% 至 14.3% 之间，% CV ≤ 6.2%。检测间 %RE 介于 -0.2% 至 9.5% 之间，% CV ≤ 6.1%。平均分析物回收率为 89.7%，平均内标回收率为 88.7%。在各种测试条件下，无论是否存在 PYX-201，Aur-0101 在人血浆和人全血中均保持稳定。据我们所知，这是首次发表的针对任何基质、任何物种的游离、未结合的 Aur0101 进行完全验证的检测方法。该测定已成功应用于临床样品分析以支持临床研究。