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(L)-hinge)(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 kappa or lambda 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.

FIGURE 1.

Schematic drawings of the human IgG subtypes indicating the originally proposed disulfide connections. IgG1 is the only isotype where the LC was proposed to connect via first Cys residue in the genetic hinge region.

FIGURE 2.

Separation of recombinant derived IgG2 by nonreducing CE-SDS (top), RP-HPLC (middle), and CEX-HPLC (bottom).

FIGURE 3.

RP-HPLC analysis of a human recombinant IgG2 mAb before and after protein A purification. a, direct analysis of the IgG2 mAb in cell culture medium after removal of cellular components by centrifugation. The labeled peaks (1-3) containing the IgG2 isoforms were confirmed by direct mass spectral analysis. Additional proteinaceous species were observed during the RP analysis due to host cell contaminants. b, analysis of the same IgG2 mAb after protein A purification.

FIGURE 4.

Schematic representation of the expected disulfide-containing peptides in the IgG2 mAb obtained by Lys-C digestion under nonreducing conditions. The positions of half-cystines are shown with numbering for this particular antibody molecule. Only half of the molecule is presented. The nomenclature for disulfide bond-containing peptides is as follows: H1, heavy chain Lys-C peptide 1; H7-8, heavy chain Lys-C peptides 7 and 8, uncleaved due to a Pro residue in the sequence; H1/H4, dipeptide connected by one disulfide bond; (H11-12)₂, dipeptide connected by four disulfide bonds; and H6/H7-8/L12, tripeptide connected by two disulfide bonds.

FIGURE 5.

LC/ESI-MS/MS analysis of recombinant IgG2 mAb after Lys-C digest. Top trace in blue: nonreduced UV trace. The peaks corresponding to expected disulfide-linked peptides are labeled 1-7 with 4a and 4b as well as 6a and 6b representing incomplete digests at Lys-Pro positions. The peaks corresponding to additional observed disulfide-linked peptides are labeled a-e. Bottom trace in red: reduced UV trace. The peaks corresponding to Cys containing peptides are labeled A-O.

FIGURE 6.

Expected disulfide-linked peptides involving inter-chain disulfide bonds. These peptides constitute the P1 group in the nonreduced Lys-C peptide map in Fig. 5.

FIGURE 7.

Schematic of the major variant inter-chain connected peptides of the P2 and P3 groups in the nonreduced Lys-C map in Fig. 5. Peptides within each box are all linked to one another through Cys residues shown in the peptide sequences. Disulfide patterns are not illustrated.

FIGURE 8.

Proposed disulfide structural isoforms of human IgG2 antibodies with varying disulfide connections involving the hinge.

FIGURE 9.

Top panel: Lys-C peptide maps of collected individual fractions of recombinant mAb separated by CEX-HPLC. Fractions CEX-1, CEX-2, CEX-3, and CEX-4 were collected as indicated in the chromatogram shown in the bottom panel of Fig. 2. Bottom panel: expanded view of P1, P2, and P3, which shows the positions of the inter-chain disulfide-connected peptides. Note: the peak labeled with (H11-12)₂ contains a co-eluted heavy chain peptide: H5.

FIGURE 10.

Overlay of RP-HPLC chromatogram (214 nm) peaks (gray) and disulfide form distribution determined by peptide mapping. The inset table details the presence (+) and absence (-) of the three types of peptides in each of the IgG2 disulfide isoforms. (P1 peptides are represented in blue, P2 peptides in green, and P3 peptides in red).

FIGURE 11.

Top panel: Lys-C peptide maps of recombinant IgG2 mAb, human myeloma IgG2 with κ LC, human myeloma IgG2 with λ LC, and human IgG derived from normal serum. Bottom panel: expanded view of the areas P2 and P3, which shows the region of the maps containing the novel inter-chain disulfide connected peptides.