Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method
Section snippets
Materials
Five Fc fusion peptibodies were produced at Amgen (Thousand Oaks, CA, USA). The Fc fusion peptibody samples were statically thawed at room temperature and stored at 4 °C prior to digestion. Endoproteinase Glu-C was purchased from Roche (Indianapolis, IN, USA). HPLC-grade solvents were obtained from Burdick & Jackson (Muskegon, MI, USA). Trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL, USA). Urea was purchased from J.T. Baker (Phillipsburg, NJ, USA).
The bulk peptibody materials
Limited proteolysis with endoproteinase Glu-C
A limited proteolysis procedure was applied to protein characterization for posttranslational and chemical modifications of therapeutic proteins such as antibodies and Fc fusion peptibodies. The approach often provides easy sample preparation and quick detection of specific residue degradations, and it simplifies the analysis and characterization of therapeutic Fc fusion peptibodies and antibodies [11], [12], [13], [14]. Therefore, it is advantageous over many conventional peptide mapping
Conclusions
Limited Glu-C proteolysis followed by LC–MS/MS is a simple and effective approach to identify and quantify the covalent degradations at the N-terminal Met1 and Asp2 residues of Fc fusion peptibody. It can be complementary to the other peptide mapping methods for evaluating Fc fusion peptibody stability during production, development, storage, and transportation. The single Met and double Met oxidation detection and quantification with this method are very useful in understanding Met1 oxidation
Acknowledgment
The authors thank Holly Z. Huang for the help during the photo stability study, Priti Baker for using the method and sharing her isomerization data during the method development.
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