Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method.
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.
Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. leiy@amgen.com
Lei
L
Yu
Gang
G
Xiao
Jifeng
J
Zhang
Richard L
RL
Remmele
Mingda
M
Eu
Dingjiang
D
Liu
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli; D007536:Isomerism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D013312:Stress, Physiological; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
D010455:Peptides; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D008715:Methionine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase