A Simultaneous Semiquantification of Eight Immunoglobulin Isotypes in Mouse Serum by LC-MS/MS for Application of Immunogenicity Assessment of Therapeutic Antibodies.

Immunogenicity assessment is critical in the development of biotherapeutics. Conventionally, this evaluation has been facilitated by employing ligand binding assays (LBAs). However, the development of immunogenicity assays by LBA can present certain challenges in some cases, and alternative assay platforms can be advantageous. In this study, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was employed to assess the immunogenicity using E6011 as a model drug in mice. The study identified signature peptides of eight mouse immunoglobulin (Ig) isotypes, including IgA, IgE, IgG1, IgG2a, IgG2b, IgG2c, IgG3, and IgM. The anti-E6011 antibody (ADA) present in mouse serum was bound to biotinylated E6011, and the immunocomplex was captured by avidin beads. The captured ADA was released and digested, thereby producing signature peptides of each Ig isotype for detection. The assay's reproducibility was demonstrated by the analysis of replicate samples. The assay was subsequently applied to real samples from mice administered repeated doses of E6011, and it was found that only IgG1 was detected in the postdose samples. The results obtained by the LC-MS/MS assay were consistent with those by the LBA, suggesting that the developed LC-MS/MS-based immunogenicity assay is useful and can be used as an alternative assay for immunogenicity assessments.