Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method

Abstract

An Fc fusion protein expressed in Escherichia coli contains Met1 and Asp2 residues at the N terminus and an active peptide attached to the C terminus of the Fc region. Due to the unique amino acid sequence of Fc, many commonly used proteolysis methods have severe drawbacks for characterizing degradations of Met1 and Asp2 residues. A novel method has been developed to effectively characterize the degradations by employing a limited endoproteinase Glu-C digestion. The limited digestion generates a dimeric peptide of (Met1-Glu14)2 due to specific cleavage at the residue Glu14 of the N terminus. This peptide together with its degraded products, including Met1 oxidation and Asp2 isomerization, can be identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The optimization of digestion procedure and linearity of quantification are also described. This approach was successfully used in a photostability study to assess the product stability of an Fc fusion peptibody.