Premium
Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by ‘bottom‐up’ mass spectrometry with electron transfer dissociation
Author(s) -
Mukherjee Raju,
Adhikary Laxmi,
Khedkar Anand,
Iyer Harish
Publication year - 2010
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4464
Subject(s) - deamidation , chemistry , mass spectrometry , tandem mass spectrometry , chromatography , electron transfer dissociation , succinimide , asparagine , aspartic acid , isomerization , amide , biochemistry , amino acid , enzyme , catalysis
Aspartic acid formed by nonenzymatic deamidation of asparagine often isomerizes to isoaspartic acid through a succinimide intermediate. Accumulation of isoaspartic acid initiates aggregation and degradation in proteins. Deamidation at the antigen‐binding region reduces the efficacy and also upregulates immunogenicity of monoclonal antibodies. We report an improved ‘bottom‐up’ tandem mass spectrometric method to detect and decipher the position of isoaspartate formation in therapeutic immunoglobulin gamma in a single chromatographic run. Differentiation between aspartate and isoaspartate residues through collision‐induced tandem mass spectrometry is formidable due to their identical mass. Signature backbone cleavage ions, c n + 57 and z l–n − 57, produced upon radical‐mediated fragmentation, were used to delineate the site of isomerization. It is more conclusive than monitoring the relative peak intensity and the decrease in hydrophobicity of the isoaspartate‐containing peptide in a chromatographic elution. Collectively, this methodology provides a useful tool to monitor deamidation and isomerization in biopharmaceuticals during their production, downstream processing and storage. Copyright © 2010 John Wiley & Sons, Ltd.