Discussions

The structural complexity and heterogeneity of Fc-fusion protein analysis require a set of analytical tools to be properly characterized. The analytical methods were used to confirm the primary structure of the Fc-fusion proteins, to evaluate the main PTMs, such as charge/size variants and glycan profiles, and to determine the main physicochemical properties of the Fc-fusion proteins, such as identity, purity, and integrity. The identity of an Fc-fusion protein can be determined by a variety of analytical methods, including ion-exchange chromatography (IEX), imaged capillary isoelectric focusing (icIEF), hydrophobic interaction chromatography (HIC), reversed-phase liquid chromatography (RPLC), and peptide mapping using liquid chromatography-mass spectrometry (LC-MS). Among them, IEX and icIEF are mostly used because the distribution of charge variants of an Fc-fusion protein provides a distinctive fingerprint of the protein.