Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method |
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Authors: | Yu Lei Xiao Gang Zhang Jifeng Remmele Richard L Eu Mingda Liu Dingjiang |
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Affiliation: | Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. |
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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. |
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