Elsevier

Analytical Biochemistry

Volume 428, Issue 2, 15 September 2012, Pages 137-142
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2012.06.002Get rights and content

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.

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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