Formation of Pyroglutamic Acid from N-Terminal Glutamic Acid in Immunoglobulin Gamma Antibodies

Abstract

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

N-terminal Glutamate to Pyroglutamate Conversion in Vivo for Human IgG2 Antibodies

“…Glutamate conversion to pE does not result in a charge state difference, so this change might be missed using simple chromatography-based release assays. To monitor glutamate cyclization, more complex assays, such as peptide mapping, would be required (5,6). Whether pE is monitored and controlled for a given therapeutic antibody should be based on its impact to safety and efficacy.…”

“…To generate N-terminal pE for identification and quantification purposes, the purified mAbs were incubated in PBS, pH 7.4, at 37°C for up to 1 month. These conditions previously have been used to form pyroglutamate from N-terminal glutamate on selected antibodies (6). Pyroglutamate was identified by peptide mapping with protease Lys-C.…”

“…Pyroglutamate was identified by peptide mapping with protease Lys-C. The identification of the modification of the N-terminal peptide was based on the loss of 18 mass units that was specific for the N-terminal residue (MS/MS analysis; not shown) (6,7). Fig.…”

Increased aggregation propensity of IgG2 subclass over IgG1: Role of conformational changes and covalent character in isolated aggregates

“…Further, there are disulfide bonds that hold the structure together: intradomain disulfide bonds, light chain to heavy chain disulfide bonds, and inter-heavy chain disulfide bonds. In addition to the complexity described above, antibodies undergo several post-translational modifications, such as pyroglutamic acid formation at the N-terminus, 14 Cterminal lysine removal, 15 and addition of polysaccharide chains. 16 The complexity of these molecules complicates our ability to study their mechanisms of aggregation.…”

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

“…Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. 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.…”