Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC, UMR7178, F-67087 Strasbourg, France.
IRPF, Centre d'Immunologie Pierre-Fabre (CIPF), F-74160 Saint-Julien-en-Genevois, France.
J Proteome Res. 2021 Jan 1;20(1):923-931. doi: 10.1021/acs.jproteome.0c00664. Epub 2020 Oct 4.
Host cell proteins (HCPs) are a major class of bioprocess-related impurities generated by the host organism and are generally present at low levels in purified biopharmaceutical products. The monitoring of these impurities is identified as an important critical quality attribute of monoclonal antibody (mAb) formulations not only due to the potential risk for the product stability and efficacy but also concerns linked to the immunogenicity of some of them. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), mass spectrometry (MS)-based approaches have been emerging as powerful and promising alternatives providing qualitative and quantitative information. However, a major challenge for liquid chromatography (LC)-MS-based methods is to deal with the wide dynamic range of drug products and the extreme sensitivity required to detect trace-level HCPs. In this study, we developed powerful and reproducible MS-based analytical workflows coupling optimized and efficient sample preparations, the library-free data-independent acquisition (DIA) method, and stringent validation criteria. The performances of several preparation protocols and DIA versus classical data-dependent acquisition (DDA) were evaluated using a series of four commercially available drug products. Depending on the selected protocols, the user has access to different information: on the one hand, a deep profiling of tens of identified HCPs and on the other hand, accurate and reproducible (coefficients of variation (CVs) < 12%) quantification of major HCPs. Overall, a final global HCP amount of a few tens of ng/mg mAb in these mAb samples was measured, while reaching a sensitivity down to the sub-ng/mg mAb level. Thus, this straightforward and robust approach can be intended as a routine quality control for any drug product analysis.
宿主细胞蛋白（HCP）是由宿主生物产生的主要一类与生物工艺相关的杂质，通常在纯化的生物制药产品中以低水平存在。这些杂质的监测被确定为单克隆抗体（mAb）制剂的一个重要关键质量属性，不仅因为它们对产品稳定性和功效的潜在风险，还因为其中一些与免疫原性有关。虽然总体 HCP 水平通常通过酶联免疫吸附测定（ELISA）进行监测，但基于质谱（MS）的方法已经作为强大且有前途的替代方法出现，提供定性和定量信息。然而，基于液相色谱（LC）-MS 的方法的一个主要挑战是处理药物产品的宽动态范围和检测痕量 HCP 所需的极端灵敏度。在这项研究中，我们开发了强大且可重复的基于 MS 的分析工作流程，结合了优化和高效的样品制备、无库的数据独立采集（DIA）方法和严格的验证标准。使用一系列四种市售药物产品评估了几种制备方案和 DIA 与经典数据依赖采集（DDA）的性能。根据所选方案，用户可以访问不同的信息：一方面，数十种鉴定的 HCP 的深度分析，另一方面，主要 HCP 的准确和可重复（变异系数（CVs）<12%）定量。总体而言，在这些 mAb 样品中测量到的每种 mAb 几十纳克/毫克的总 HCP 量，而达到了亚纳克/毫克 mAb 级别的灵敏度。因此，这种简单而稳健的方法可以作为任何药物产品分析的常规质量控制。
