Human IgG2 Antibodies Display Disulfide-mediated Structural Isoforms

Abstract

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C L ) and the heavy chain C H 1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C H 1-C Lhinge) 2 complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either or light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

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

“…For example, the LC212-HC129 bond was detected only in the A and A/B forms using this method, which is consistent with previous reports about the LC-HC connectivity in these isoforms. 11,12 Most native intra-chain and inter-chain disulfide bonds (12 total for an IgG2) were detected in all isoforms. However, the intra-chain HC22-HC96 could not be detected in neither the A nor the B isoforms, suggesting possible trypsin missed cleavage at this site or low ionization of this particular DSB peptide.…”

“…This LC method was used to collect the disulfide isoform fractions for tryptic digestion, and the lack of baseline resolution of the peaks results in some expected cross-contamination from neighboring species. Peaks were assigned based on previous publications 11 , 12 …”

“…12,24,64,65 The LC-MALDI-TOF/TOF approach described here uses the Bruker DisulfideDetect software to automatically output semi-quantitative maps of the singly disulfide-bonded peptides within a single analysis. This method takes advantage of the partial reductive fragmentation of peptides at the disulfide bond by in-source decay in the MALDI source.…”

“…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.…”

“…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. 36–38 …”