Probing deamidation in therapeutic immunoglobulin gamma (IgG1) by 'bottom-up' mass spectrometry with electron transfer dissociation.
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.
Mukherjee
Raju
R
Research and Development, Biocon Limited, 20th KM, Hosur Road, Bangalore-560100, India. raju.mukherjee@biocon.com
Adhikary
Laxmi
L
Khedkar
Anand
A
Iyer
Harish
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001702:Biopharmaceutics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D026581:Isoaspartic Acid / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*
D007074:Immunoglobulin G; D026581:Isoaspartic Acid; D001224:Aspartic Acid