Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies.
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 degrees 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.
Chelius
Dirk
D
Department of Pharmaceutics, Amgen, Inc., Thousand Oaks, California 91320, USA.
Rehder
Douglas S
DS
Bondarenko
Pavel V
PV
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry
D007142:Immunoglobulin gamma-Chains; D001224:Aspartic Acid; D001120:Arginine