Identification and Characterization of Deamidation Sites in the Conserved Regions of Human Immunoglobulin Gamma Antibodies

• Published in: Analytical chemistry (Washington) Vol. 77; no. 18; pp. 6004 - 6011
• Main Authors: Chelius, Dirk, Rehder, Douglas S, Bondarenko, Pavel V
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.09.2005
• Subjects: Amino Acid Sequence; Amino acids; Analytical chemistry; Animals; Arginine - chemistry; Aspartic Acid - chemistry; Cell Line; Chemical degradation; Chemical reactions; Chemistry; Chromatographic methods and physical methods associated with chromatography; Cricetinae; Cricetulus; Exact sciences and technology; Human subjects; Humans; Immunoglobulin gamma-Chains - analysis; Immunoglobulin gamma-Chains - chemistry; Immunoglobulin gamma-Chains - immunology; Immunoglobulins; Liquid chromatography; Mass spectrometry; Molecular Sequence Data; Other chromatographic methods; Peptides; Spectrometric and optical methods; Tandem Mass Spectrometry; Human; Peptides; HPLC chromatography; IgG; Deamidation; Monoclonal antibody; Digestion; Chemical degradation; Alkylation; Protein; Residue; Aminoacid; DNA; Mass spectrometry MS/MS; Aspartic acid
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac050672d

Deamidation of asparagine residues of biological pharmaceuticals is a major cause of chemical degradation if the compounds are not formulated and stored appropriately. The mechanism of this nonenzymatic chemical reaction has been studied in great detail; however, the identification of deamidation sites in a given protein remains a challenge. In this study, we identified and characterized all deamidation sites in the conserved region of a recombinant monoclonal antibody. The conserved region of this antibody is shared by all human IgGs with the exception of minor differences in the hinge region. Our high-performance liquid chromatography method could separate the succinimide, isoaspartic, and aspartic acid isoforms of peptide fragments generated using trypsin. Each of the isoforms was unambiguously identified using tandem mass spectrometry. Deamidation at the identified four sites was slow for the intact, folded antibody at accelerated degradation conditions (pH 7.5 and 37 °C). Deamidation was enhanced after reduction, alkylation, and tryptic digestion, indicating that the three-dimensional structure of the antibody reduced deamidation. Furthermore, after the reduction, alkylation, and tryptic digestion, only 4 of a possible 25 asparagine residues showed deamidation, demonstrating the effect of the primary amino acid sequence, especially the −1 and +1 amino acids flanking the deamidation site. For instance, the amino acid motifs SNG, ENN, LNG, and LNN were found to be more prone to deamidation, whereas the motifs GNT, TNY, YNP, WNS, SNF, CNV, SNT, WNS, FNW, HNA, FNS, SNK, GNV, HNH, SNY, LNW, SNL, NNF, DNA, GNS, and FNR showed no deamidation. Our findings should help predict deamidation sites in proteins and peptides and help develop deamidation-resistant biological therapeutics.