Determination of Fab−Hinge Disulfide Connectivity in Structural Isoforms of a Recombinant Human Immunoglobulin G2 Antibody

Abstract

The detection and characterization of unexpected disulfide-mediated structural variants of human immunoglobulin G2 (IgG2) antibodies was recently the subject of two copublications. In this paper, we present data to confirm the previously reported structures and elucidate the complete disulfide connectivity of each variant through the application of a novel analytical methodology. In this manner, the data illustrate the presence of at least five structural variants, including the classical structure with independent Fab domains and a hinge region. Multiple subvariants of the IgG2-A/B and IgG2-B structures are identified; these subvariants of each structure differ through the order of attachment of Fab peptides to the sequential hinge cysteines. Furthermore, the connectivity of a novel subvariant of IgG2-B containing an intrachain disulfide linkage in the lower hinge region is elucidated. The results presented in this paper reveal that the population of IgG2 disulfide structural variants is yet more complex than recently reported.

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

“…IgG2 isotypes have four disulfide pairs in the hinge (compared with two in IgG1 and IgG4). These disulfides have been shown to generate multiple structural variants during protein expression, [45][46][47] rearrange in vivo 48 and can affect the activity of the antibody. IgG1 and IgG2 but not IgG4 isotypes can bind to the 131 His allotype of FcγRII and, in the context of an anti-CD3 antibody, induce T cell proliferation.…”

Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations

“…The hinge region of IgG4 molecules can be conformationally stabilized by a single amino acid substitution of Ser with Pro, like in IgG1 and IgG2 hinges (CPPC) (6). Several disulfide isoforms have been detected for the human IgG2 antibodies (8,9,11,12). Human IgG2 antibodies can also form dimers involving hinge region Cys residues and show increased avidity (13).…”

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

“…For example, disulfide bond variants within the lower hinge region of the native B isoform reported elsewhere would not be differentiated using this method. 32 …”

“…Electrospray ionization MS/MS characterization of IgG2 hinge disulfides can also be difficult due to the large size of hinge peptides (5 – 10 kDa, depending on disulfide configuration and number of missed cleavages) that generate low-intensity, multiply charged peak clusters that complicate the deconvolution of the MS/MS spectra. 11,12,32 Quantification of IgG2 disulfide isoforms requires a separate reverse-phase chromatography or charge distribution analysis of the intact protein. 11,12 The advantage of using matrix-assisted laser desorption/ionization (MALDI)-MS/MS for IgG2 disulfide-bonded (DSB) peptide analysis is the predominance of singly-charged ions in the MALDI spectra, which considerably simplifies data analysis and renders it compatible with both semi-quantitative output and automation from a single LC-MS/MS analysis.…”