Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor
Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor
复制标题
DOI：
10.1208/s12248-010-9218-9
复制
发表时间：
2010-12-01
期刊：
影响因子：
4.5
通讯作者：
Lee, Jean W.
中科院分区：
文献类型：
--
作者：
Hall, Michael P.;Gegg, Colin;Lee, Jean W.
蛋白质治疗候选物的体内生物转化（例如，蛋白质水解）的知识揭示了影响稳定性的结构缺陷。这些信息有助于开发和确认配体结合测定法，该方法对生物活性部分（包括完整分子和代谢物）具有所需的特异性，从而进行适当的PK分析。此外，这些信息可以用于重新设计结构，以消除体内的缺陷，从而设计出最稳定的候选结构。我们开发了一种配体结合质谱（LBMS）的战略方法，利用抗人Fc免疫亲和力捕获和分层质谱分析，研究融合蛋白与人Fc（“肽体”）融合的肽的生物转化。LBMS结合了配体捕获的选择性和质谱的特异性和详细的分子水平信息。在本文中，我们展示了LBMS在三种靶向血小板生成素受体的肽体AMG531 （romiplostim）、AMG195（线性）和AMG195（环）上的临床前应用。数据表明，配体捕获提供了良好的样品清理和完整肽体和代谢物的浓度，以便随后通过基质辅助激光解吸电离飞行时间质谱法进行查询，以确定体内蛋白水解点。需要通过纳米级液相色谱与电喷雾电离质谱相结合的更高分辨率分析来鉴定异质代谢物。AMG531有5个蛋白水解点，AMG195有2个蛋白水解点（线性），而AMG195在大鼠中是最稳定的结构。我们推荐使用LBMS来评估所有新型融合蛋白在早期临床前阶段的生物转化和体内稳定性。
The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.