Host cell proteins (HCPs) constitute a major class of process‐related impurities, whose substantial clearance must be demonstrated by suitable analytical methods to warrant product quality and reduce potential safety risks for patients. In this regard, enzyme linked immunosorbent assays (ELISAs), which primarily rely on the quality of the HCP reference standard (immunogen) and HCP‐specific polyclonal antibodies, are considered the gold standard for HCP monitoring. For the qualification of the employed antibodies, two‐dimensional (2D) western blots (2D‐WBs) are the preferred technique to determine the coverage, though a number of practical constraints are well recognized. By using several orthogonal approaches, such as affinity‐based mass spectrometry and indirect ELISA, the present study revealed potential detection gaps (i.e., noncovered HCPs) of conventional 2D‐WBs, which can be primarily attributed to two different root causes: (i) low amounts of proteins or antibodies being unable to overcome the detection limit and (ii) western blot artifacts due to the loss of conformational epitopes through protein denaturation hindering HCP‐antibody recognition. In contrast, the lack of specific antibodies against certain (particularly, low molecular weight) HCPs, as proposed in previous studies, seems to play only a minor role. Together, these findings imply that CHO‐HCP ELISA antibodies are better than qualification studies by 2D‐WBs indicate.

中文翻译：

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质疑二维蛋白质印迹确定的覆盖率值：关于抗 HCP ELISA 试剂表征的关键研究

宿主细胞蛋白 (HCP) 是与工艺相关的一大类杂质，必须通过适当的分析方法证明其实质性清除，以保证产品质量并降低患者的潜在安全风险。在这方面，主要依赖于 HCP 参考标准（免疫原）和 HCP 特异性多克隆抗体的质量的酶联免疫吸附试验（ELISA）被认为是 HCP 监测的金标准。对于所用抗体的鉴定，二维 (2D) 蛋白质印迹 (2D-WBs) 是确定覆盖率的首选技术，尽管许多实际限制已得到广泛认可。通过使用几种正交方法，例如基于亲和力的质谱法和间接 ELISA，本研究揭示了潜在的检测差距（即，传统 2D-WBs 的未覆盖 HCP)，这主要归因于两个不同的根本原因：(i) 少量蛋白质或抗体无法克服检测限和 (ii) 由于构象表位的丢失而导致的蛋白质印迹伪影通过蛋白质变性阻碍 HCP 抗体识别。相比之下，缺乏针对某些（特别是低分子量）HCP 的特异性抗体，正如先前研究中提出的那样，似乎只起次要作用。总之，这些发现表明 CHO-HCP ELISA 抗体优于 2D-WB 的鉴定研究表明。(i) 少量蛋白质或抗体无法克服检测限；(ii) 由于蛋白质变性导致构象表位丢失，阻碍 HCP 抗体识别，导致蛋白质印迹伪影。相比之下，缺乏针对某些（特别是低分子量）HCP 的特异性抗体，正如先前研究中提出的那样，似乎只起次要作用。总之，这些发现表明 CHO-HCP ELISA 抗体优于 2D-WB 的鉴定研究表明。(i) 少量蛋白质或抗体无法克服检测限；(ii) 由于蛋白质变性导致构象表位丢失，阻碍 HCP 抗体识别，导致蛋白质印迹伪影。相比之下，缺乏针对某些（特别是低分子量）HCP 的特异性抗体，正如先前研究中提出的那样，似乎只起次要作用。总之，这些发现表明 CHO-HCP ELISA 抗体优于 2D-WB 的鉴定研究表明。