Immunoreactivity profiling of Anti-Chinese hamster ovarian host cell protein antibodies by isobaric labeled affinity purification-mass spectrometry reveals low-recovery proteins

•A novel isobaric labeling affinity purification-mass spectrometry (iLAP-MS) method enabling sensitive and reliable immunoreactivity profiling of anti-host cell protein (HCP) antibodies was developed.•The iLAP-MS method enabled the identification of low-recovery HCPs with low affinity to anti-HCP antibodies, which are difficult to accurately quantify by immunoassay.•Lower molecular weight, lower isoelectric point, and lower hydrophobicity were revealed to be the common characteristics of the low-recovery HCPs. We evaluated the immunoreactivity profiles of eight commercial anti-host cell protein (anti-HCP) antibodies from different host animals and their antigens used for immunization by an isobaric labeled affinity purification-mass spectrometry (AP-MS) method. As a result, 34 proteins with high abundance but low recovery from harvest cell culture fluid were identified. Since they are likely to be underestimated in biopharmaceutical quality assessment, the features common to these proteins were investigated. Compared to other immunoprecipitated HCP proteins, proteins exhibiting lower molecular weight (ΔMW = -14600), lower isoelectric point (ΔpI = -0.86), and lower hydrophobicity (ΔGRAVY = -0.13) were enriched. This AP-MS method provides important information for HCP control strategies using immunological methods and is expected to contribute to the development of safe biopharmaceutics.