Comprehensive Characterization of Relationship Between Higher-Order Structure and FcRn Binding Affinity of Stress-Exposed Monoclonal Antibodies

In biopharmaceutical development, information regarding higher-order structure (HOS) is important to verify quality and characterize protein derivatives. In this study, we aimed to characterize the association between HOS and pharmacokinetic property of a stress-exposed monoclonal antibody (mAb). Purity, primary structure, thermal stability, and HOS were evaluated for mAbs exposed to heat, photo-irradiation, and chemical oxidation. To investigate conformation of stress-exposed mAbs, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was utilized. No distinct difference in secondary or tertiary structure between stress-exposed and non-stressed samples was found by conventional spectroscopic techniques. In binding activity with the neonatal Fc receptor (FcRn), however, a marked decline was observed for force-oxidized mAb and a slight decline was observed for heat- and photodegraded mAbs. Using differential scanning calorimetry, a change in thermal stability was observed in the C(H)2 domain for all the stress-exposed samples. Using HDX-MS analyses, individual regions with altered conformation could be identified for heat-degraded and force-oxidized samples. These findings indicate that comprehensive study is important for detecting conformational changes and helpful for predicting biophysical property, and that the evaluation of HOS using several analytical techniques is indispensable for confirming biopharmaceutical quality.