Disulfide bond characterization of endogenous IgG3 monoclonal antibodies using LC-MS: an investigation of IgG3 disulfide-mediated isoforms

摘要

The use of monoclonal antibodies (mAbs) for the manufacture of innovator and biosimilar biotherapeutics has increased tremendously in recent years. From a structural perspective, mAbs have high disulfide bond content, and the correct disulfide connectivity is required for proper folding and to maintain their biological activity. Therefore, disulfide linkage mapping is an important component of mAb characterization for ensuring drug safety and efficacy. The native disulfide linkage patterns of all four subclasses of IgG antibodies have been well established since the late 1960s. Among these IgG subtypes, disulfide mediated isoforms have been identified for IgG2 and IgG4, and to a lesser extent in IgG1, which is the most studied IgG subclass. However, no studies have been carried out so far to investigate whether different IgG3 isoforms exist due to alternative disulfide connectivity. In an effort to investigate the presence of disulfide-mediated isoforms in IgG3, we employed a bottom-up mass spectrometry approach to accurately determine the disulfide bond linkages in endogenous human IgG3 monoclonal antibody, and our results show that no such alternative disulfide bonds exist. While many antibody-based drugs are developed around IgG1, IgG3 represents a new, and in some cases, more desirable drug candidate. Our data represent the first demonstration that alternative disulfide bond arrangements are not present in endogenous IgG3; and therefore, they should not be present in recombinant forms used as antibody-based therapeutics.