Iwamoto Noriko, Takanashi Megumi, Hamada Akinobu, Shimada Takashi
Life Science Research Center, Shimadzu Corporation.
Biol Pharm Bull. 2016 Jul 1;39(7):1187-94. doi: 10.1248/bpb.b16-00230. Epub 2016 Apr 28.
Presently, monoclonal antibodies (mAbs) therapeutics have big global sales and are starting to receive competition from biosimilars. We previously reported that the nano-surface and molecular-orientation limited (nSMOL) proteolysis which is optimal method for bioanalysis of antibody drugs in plasma. The nSMOL is a Fab-selective limited proteolysis, which utilize the difference of protease nanoparticle diameter (200 nm) and antibody resin pore diameter (100 nm). In this report, we have demonstrated that the full validation for chimeric antibody Rituximab bioanalysis in human plasma using nSMOL proteolysis. The immunoglobulin fraction was collected using Protein A resin from plasma, which was then followed by the nSMOL proteolysis using the FG nanoparticle-immobilized trypsin under a nondenaturing condition at 50°C for 6 h. After removal of resin and nanoparticles, Rituximab signature peptides (GLEWIGAIYPGNGDTSYNQK, ASGYTFTSYNMHWVK, and FSGSGSGTSYSLTISR) including complementarity-determining region (CDR) and internal standard P14R were simultaneously quantified by multiple reaction monitoring (MRM). This quantification of Rituximab using nSMOL proteolysis showed lower limit of quantification (LLOQ) of 0.586 µg/mL and linearity of 0.586 to 300 µg/mL. The intra- and inter-assay precision of LLOQ, low quality control (LQC), middle quality control (MQC), and high quality control (HQC) was 5.45-12.9% and 11.8, 5.77-8.84% and 9.22, 2.58-6.39 and 6.48%, and 2.69-7.29 and 4.77%, respectively. These results indicate that nSMOL can be applied to clinical pharmacokinetics study of Rituximab, based on the precise analysis.
目前，单克隆抗体（mAbs）疗法在全球销售额巨大，并且开始面临生物类似药的竞争。我们之前报道过，纳米表面和分子取向限制（nSMOL）蛋白水解是血浆中抗体药物生物分析的最佳方法。nSMOL是一种针对Fab片段的选择性有限蛋白水解方法，它利用了蛋白酶纳米颗粒直径（200 nm）与抗体树脂孔径（100 nm）的差异。在本报告中，我们展示了使用nSMOL蛋白水解对人血浆中嵌合抗体利妥昔单抗进行生物分析的全面验证。使用蛋白A树脂从血浆中收集免疫球蛋白部分，然后在50°C的非变性条件下，使用固定有FG纳米颗粒的胰蛋白酶进行nSMOL蛋白水解6小时。去除树脂和纳米颗粒后，通过多反应监测（MRM）同时定量利妥昔单抗特征肽（GLEWIGAIYPGNGDTSYNQK、ASGYTFTSYNMHWVK和FSGSGSGTSYSLTISR），包括互补决定区（CDR）和内标P14R。使用nSMOL蛋白水解对利妥昔单抗进行的这种定量显示，定量下限（LLOQ）为0.586 μg/mL，线性范围为0.586至300 μg/mL。LLOQ、低质量控制（LQC）、中质量控制（MQC）和高质量控制（HQC）的批内和批间精密度分别为5.45 - 12.9%和11.8%、5.77 - 8.84%和9.22%、2.58 - 6.39%和6.48%、2.69 - 7.29%和4.77%。这些结果表明，基于精确分析，nSMOL可应用于利妥昔单抗的临床药代动力学研究。
