AbstractHost 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.