The fusion of an Fc moiety to a therapeutic protein is widely applied as a half-life extension strategy. However, unlike monoclonal antibodies, Fc-fusion proteins have been shown to be more susceptible to protease-mediated catabolism. The resultant catabolites may still be pharmacologically active and therefore require characterization. We combined intact protein LC-MS and digestion LC-MS/MS methods to both characterize the biotransformation of the fusion protein, Fc-FGF21, and quantify the intact molecule and its major catabolites in rat serum. The digestion LC-MS/MS assay and intact protein LC-MS assay determined that there were four major catabolites formed in vivo: one amino acid (dC1), two amino acids (dC2), or three amino acids (dC3) clipped off from the C-terminus, and a truncated fragment. By 72 h post dosing, only 66% of the intact protein remained. The digestion method was developed with a sensitivity of 20 ng/mL-10 times more sensitive than the intact protein method at 200 ng/mL. While the digestion approach proved more sensitive, the intact LC-MS method was primarily employed for understanding the biotransformation of the Fc-FGF21 fusion protein in the rat in vivo study. Graphical abstract.
Keywords: Fc-FGF21; LC-MS; digestion; immunoaffinity; intact protein.