Identification and Characterization of Deamidation Sites in the Conserved Regions of Human Immunoglobulin Gamma Antibodies
- 18 August 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (18), 6004-6011
- https://doi.org/10.1021/ac050672d
Abstract
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.Keywords
This publication has 10 references indexed in Scilit:
- Quantification of the isomerization of Asp residue in recombinant human αA-crystallin by reversed-phase HPLCJournal of Pharmaceutical and Biomedical Analysis, 2003
- Crystal Structure of a Neutralizing Human IgG Against HIV-1: A Template for Vaccine DesignScience, 2001
- The effects of alpha‐helix on the stability of Asn residues: Deamidation rates in peptides of varying helicityProtein Science, 1999
- Deamidation and isoaspartate formation during in vitro aging of recombinant tissue plasminogen activator.Journal of Biological Chemistry, 1994
- Spontaneous degradation of polypeptides at aspartyl and asparaginyl residues: Effects of the solvent dielectricProtein Science, 1993
- Chemical Pathways of Peptide Degradation. IV. Pathways, Kinetics, and Mechanism of Degradation of an Aspartyl Residue in a Model HexapeptidePharmaceutical Research, 1993
- Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides.Journal of Biological Chemistry, 1991
- Stability of Protein PharmaceuticalsPharmaceutical Research, 1989
- Effect of protein conformation on rate of deamidation: Ribonuclease AProteins-Structure Function and Bioinformatics, 1989
- THE COVALENT STRUCTURE OF AN ENTIRE γG IMMUNOGLOBULIN MOLECULEProceedings of the National Academy of Sciences, 1969