we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1␤ ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 M guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 Ͼ Ͼ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2 composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "bloodlike" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

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.

Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules. A QbD strategy requires deep understanding at the molecular level of the attributes that are crucial for safety and efficacy and for insuring that the desired quality of the purified protein drug product is met at the end of the manufacturing process. A mass spectrometry (MS)-based approach to simultaneously monitor the extensive array of product quality attributes (PQAs) present on therapeutic molecules has been developed. This multi-attribute method (MAM) uses a combination of high mass accuracy / high resolution MS data generated by Orbitrap technology and automated identification and relative quantification of PQAs with dedicated software (Pinpoint). The MAM has the potential to replace several conventional electrophoretic and chromatographic methods currently used in Quality Control to release therapeutic molecules. The MAM represents an optimized analytical solution to focus on the attributes of the therapeutic molecule essential for function and implement QbD principles across process development, manufacturing and drug disposition.