Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody.

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.

Development of a chip-based capillary gel electrophoresis method for quantification of a half-antibody in immunoglobulin G4 samples.

A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).