a Process Development, Catalent Pharma Solutions, Inc , Bloomington , IN , USA.
MAbs. 2019 Aug/Sep;11(6):1089-1100. doi: 10.1080/19420862.2019.1625676. Epub 2019 Jul 4.
Although peroxide and leachable metal-induced chemical modifications are among the most important quality attributes in bioprocess development, there is no mainstream characterization method covering all common modifications theoretically possible on therapeutic proteins that also gives consistent results quickly. Here, we describe a method for rapid and consistent global characterization of leachable metals- or peroxide-stressed immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). Using two independent protease digestions, data-independent acquisition and data-dependent acquisition liquid chromatography high-resolution mass spectrometry, we monitored 55 potential chemical modifications on trastuzumab, a humanized IgG1 mAb. Processing templates including all observed peptides were developed on Skyline to consistently monitor all modifications throughout the stress conditions for both enzymatic digestions. The Global Characterization Data Processing Site, a universal automated data processing application, was created to batch process data, plot modification trends for peptides, generate sortable and downloadable modification tables, and produce Jmol code for three-dimensional structural models of the analyzed protein. In total, 53 sites on the mAb were found to be modified. Oxidation rates generally increased with the peroxide concentration, while leachable metals alone resulted in lower rates of modifications but more oxidative degradants. Multiple chemical modifications were found on IgG1 surfaces known to interact with FcɣRIII, complement protein C1q, and FcRn, potentially affecting activity. The combination of Skyline templates and the Global Characterization Data Processing Site results in a universally applicable assay allowing users to batch process numerous modifications. Applying this new method to stability studies will promote a broader and deeper understanding of stress modifications on therapeutic proteins.
尽管过氧化物和可浸出金属诱导的化学修饰是生物工艺开发中最重要的质量属性之一，但目前还没有一种主流的表征方法能够涵盖理论上所有可能对治疗性蛋白产生的常见修饰，并且无法快速得到一致的结果。在这里，我们描述了一种快速且一致的方法，用于对可浸出金属或过氧化物应激的免疫球蛋白（IgG）1 单克隆抗体（mAb）进行全面表征。我们使用两种独立的蛋白酶消化、数据非依赖性采集和数据依赖性采集液相色谱高分辨率质谱法，监测了曲妥珠单抗（一种人源化 IgG1 mAb）上的 55 种潜在化学修饰。包括所有观察到的肽的处理模板是在 Skyline 上开发的，用于在两种酶消化过程中一致地监测所有修饰条件下的所有修饰。创建了“全局特征数据处理站点”（Global Characterization Data Processing Site），这是一个通用的自动化数据处理应用程序，用于批量处理数据、绘制肽的修饰趋势、生成可排序和可下载的修饰表，并为分析蛋白的三维结构模型生成 Jmol 代码。总的来说，mAb 上有 53 个位点被修饰。氧化速率通常随过氧化物浓度的增加而增加，而过氧化物单独作用则导致修饰率较低，但氧化降解产物较多。在已知与 FcγRIII、补体蛋白 C1q 和 FcRn 相互作用的 IgG1 表面上发现了多种化学修饰，这可能会影响其活性。Skyline 模板和 Global Characterization Data Processing Site 的组合产生了一种普遍适用的检测方法，允许用户批量处理大量修饰。将这种新方法应用于稳定性研究将促进对治疗性蛋白应激修饰的更广泛和更深入的理解。
