Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme‐linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D‐Western blot or immunoaffinity‐purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA‐based immunocapture with protein identification by liquid chromatography–tandem mass spectrometry (LC–MS/MS): ELISA‐MS. ELISA‐MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti‐Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species‐specific isotype control antibody. We propose that ELISA‐MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA‐MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.

中文翻译：

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一种评估宿主细胞蛋白 ELISA 抗体覆盖率的新方法——结合基于 ELISA 的免疫捕获和质谱法。

监测宿主细胞蛋白 (HCP) 是重组生物药物生产中最重要的分析要求之一，以确保产品纯度和患者安全。酶联免疫吸附试验 (ELISA) 是监测 HCP 清除率的标准方法。重要的是要验证 ELISA 的关键试剂 HCP 抗体是否涵盖了可能存在于纯化原料药中的广泛 HCP。当前用于评估 HCP 抗体覆盖率的覆盖率方法基于 2D-Western 印迹或免疫亲和纯化结合 2D 凝胶电泳，并且存在一些局限性。在本研究中，我们提出了一种新的覆盖方法，将基于 ELISA 的免疫捕获与通过液相色谱-串联质谱 (LC-MS/MS) 进行的蛋白质鉴定相结合：ELISA-MS。大肠杆菌HCP 抗体，避免当前覆盖分析方法的局限性，并利用 MS 分析的优势。获得的结果包括每个 HCP 抗体所涵盖的单个 HCP 的列表。这种新方法显示出高灵敏度、高重现性，并通过包含物种特异性同种型对照抗体来严格控制非特异性结合。我们建议 ELISA-MS 将成为现有覆盖方法的有价值的补充甚至替代。ELISA-MS 将增加选择最佳 HCP ELISA 的可能性，从而改善 HCP 监测并产生具有最低可实现风险的最终 HCP 概况。总体而言，这将有利于制药行业和患者安全。