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

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 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.

Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies

“…Ramachandran plot analysis, which is used to calculate peptide φ and ψ angles, revealed that the backbone geometry of HC Asn57 was outside of the theoretically allowed region, which indicated that the structure model around HC Asn57 may have been built incorrectly. 19 As shown in Figure 7A, the backbone carbonyl of HC Asn57 in the original structure model did not match the calculated electron density map. The CDR-H2 loop structure model was refined by flipping the original HC Asn57 backbone carbonyl and then fitting the rest of the loop.…”

“…These results were consistent with previous reports. 19,20 It should be noted that HC Asn315 is not considered a deamidation hot spot even though up to 8% deamidation was detected in unstressed mAbs because HC Asn315 contains the sensitive NG sequence motif and much of the deamidation detected at this site occurred during peptide map analysis (unpublished observations). Similar method related artifacts were also reported previously.…”

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

“…Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on-line RP-HPLC and mass spectrometry (MS) [9,24].…”

In vivo characterization of therapeutic monoclonal antibodies

“…After administration, the accumulated succinimide hydrolyzes to Asp and/or isoAsp under the physiologic pH. In mAbs, deamidation has been reported in the Fc regions [4,5] and the CDRs [6][7][8][9][10][11][12]. Deamidation in the CDR regions could affect drug efficacy [10,12].…”