Change of charge variant composition of trastuzumab upon stressing at physiological conditions

•High-resolution separation of charge variants of the monoclonal antibody trastuzumab by cation-exchange chromatography and elution with pH gradient buffers after forced degradation studies.•Modifications in charge variants were assigned by LC-MS peptide mapping.•Stressed trastuzumab showed similar HER2-binding compared to the original antibody.•No significant changes in binding properties were observed for different Fcγ receptors between stressed and non-stressed trastuzumab. Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.