Monoclonal antibodies (MAbs) are therapeutic proteins with important applications in many disease areas. The systematic and phase dependent characterization are critical to develop safe and efficacious biopharmaceutical drugs according to regulatory guidelines. Different modes of chromatography are the key approaches to support these characterization efforts. In this presentation, I will describe the finding of a unique stop codon mutation by characterizing unknown PR-HPLC peptide peaks for pre-IND clone screening. In addition, I will also present the detailed characterization of reversed phase chromatography unknowns for a late state development project prepared for BLA filing. Furthermore, the application of UHPLC for antibody characterization will be also discussed.  These examples indicate chromatography separations are indispensable tools to understand the characteristic of antibodies and reveal novel antibody modifications.