An overview of Pfizer’s development strategy and technologies for heightened characterization of recombinant proteins will be presented. Because many biotherapeutics are complex glycoproteins, they require modern analytical technologies and orthogonal methods to generate product knowledge. The use of high-resolution mass spectrometry has been the cornerstone of our analytical strategy. A case study is presented that delves into the characterization of unique, low-level O-glycosylation observed for a recombinant protein. Using a combination of cation exchange chromatography (CEX-HPLC) to enrich the charge-based isoforms and mass analysis by ESI-QTOF MS, several unique charge variants can be detected based on the intact protein molecular mass. To determine the identity of these charge variants and the location of the modifications, peptide mapping, b-elimination and fragmentation (MS/MS) were utilized.