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Host cell protein detection gap risk mitigation: quantitative IAC-MS for ELISA antibody reagent coverage determination

机译：宿主细胞蛋白质检测间隙风险减缓：用于ELISA抗体试剂覆盖率测定的定量IAC-MS

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Host cell proteins (HCPs) must be sufficiently cleared from recombinant biopharmaceuticals during the downstream process (DSP) to ensure product quality, purity, and patient safety. For monitoring of HCP clearance, the typical method chosen is an enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-HCP antibodies obtained from an immunization campaign. This polyclonal reagent is a critical factor for functionality and confidence of the ELISA. Therefore, it is important to ensure that the pool of ELISA antibodies covers a broad spectrum of the HCPs that potentially could persist in the final drug substance. Typically, coverage is determined by gel-based approaches. Here, we present a quantitative proteomics approach combined with purification of HCPs by immunoaffinity chromatography (qIAC-MS) for assessment of ELISA coverage. The cell culture fluid (CCF) of a mock fermentation and a recombinant monoclonal antibody product were characterized in detail to investigate whether the HCPs used for immunization of animals accurately represent HCPs that are relevant to the process. Using the qIAC-MS approach, the ELISA antibody coverage was determined for mock fermentation and product CCF, as well as several different DSP intermediates. Here, the use of different controls facilitated the identification and quantification of HCPs present in the polyclonal reagent and those that nonspecifically bound to IAC material. This study successfully demonstrates that the described qIAC-MS approach is not only a suitable orthogonal method to commonly used 2D SDS-PAGE-based analysis for evaluating ELISA antibody coverage, but that it further identifies HCPs covered as well as missed by the ELISA, enabling an improved risk assessment of HCP ELISA.
机译：在下游工艺（DSP）期间必须从重组生物制药中充分清除宿主细胞蛋白（HCP），以确保产品质量，纯度和患者安全性。为了监测HCP间隙，所选择的典型方法是使用从免疫活动获得的多克隆抗HCP抗体的酶联免疫吸附测定（ELISA）。这种多克隆试剂是ELISA功能性和置信度的关键因素。因此，重要的是要确保ELISA抗体的池涵盖潜在的HCP的广谱，这些HCP可能会持续到最终药物。通常，覆盖率由基于凝胶的方法决定。这里，我们提出了定量蛋白质组学方法，其通过免疫亲和性色谱（QIAC-MS）与HCP的纯化进行评估，以评估ELISA覆盖率。详细表征了模拟发酵和重组单克隆抗体产物的细胞培养液（CCF），以研究用于免疫动物的HCP准确地代表与该过程相关的HCP。使用QIAC-MS方法，测定ELISA抗体覆盖物用于模拟发酵和产物CCF，以及几种不同的DSP中间体。这里，使用不同对照的使用促进了多克隆试剂中存在的HCP的鉴定和定量，以及非特异性地结合IAC材料的HCP。该研究成功地证明了所描述的QIAC-MS方法不仅是常用的2D SDS-PAGE的分析的合适的正交方法，用于评估ELISA抗体覆盖率，但它进一步识别涵盖的HCP以及由ELISA错过的HCP，实现HCP ELISA的提高风险评估。

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