Integrating ultra‐high‐performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in‐depth characterization of complex fusion proteins.

Rationale: Fc‐fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product‐specific methods over conventional generic/platform methods. Methods: Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra‐high performance liquid chromatography (UHPLC–MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in‐depth fusion protein characterization. Results: The amino acid sequence coverage, peptide mapping, and post‐translational modifications of etanercept analogues were analyzed by UHPLC–MS. The post‐translational modification results were complemented by imaged capillary isoelectric focusing to produce quality research on etanercept analogues. Conclusions: The developed workflow integrating UHPLC–MS and icIEF provided an innovative strategy for characterizing complex fusion proteins in the process of quality control and manufacturing. [ABSTRACT FROM AUTHOR]