Abstract
There is an urgent demand to develop new technologies to characterize immunogenicity to biotherapeutics. Here, we developed an immunocapture LC-MS assay to isotype and semi-quantify monkey anti-drug antibodies (ADAs) to fully human monoclonal antibody (mAb) drugs. ADAs were isolated from serum samples using an immunocapture step with the Fab of the full-length mAb cross-linked to magnetic beads to minimize matrix interference. A positive monoclonal antibody control against the human immunoglobulin kappa light chain was used as a calibration standard for ADA quantitation. The final LC-MS method contains 17 multiple reaction monitoring (MRM) transitions and an optimized 15-min LC method. The results suggested that IgG1 was the most abundant isotype in ADA-positive samples. IgG2 and IgG4 were identified at lower levels, whereas IgG3 and IgA levels were only observed at very minor levels. In addition, levels of total ADA measured by the LC-MS assay were comparable to results obtained using a traditional ligand binding assay (LBA). The LC-MS ADA assay enabled rapid immunogenicity assessment with additional isotype information that LBAs cannot provide.
Highlights
Biotherapeutics, such as monoclonal antibodies, have been rapidly growing in the pharmaceutical market
A study of 121 US Food and Drug Administration (FDA)–approved biological products revealed that 89% of products showed anti-drug antibodies (ADAs) incidence, with almost half reporting some impact on efficacy, only 26% reported impacts on PK [4]
The isolated ADAs were digested with trypsin to generate proteolytic peptides and analyzed in the triple quadrupole mass spectrometer using an multiple reaction monitoring (MRM) method (Fig. 1)
Summary
Biotherapeutics, such as monoclonal antibodies (mAbs), have been rapidly growing in the pharmaceutical market. These therapeutics have the potential to elicit an unwanted immune response that can result in the production of anti-drug antibodies (ADAs) [1,2,3]. The binding of ADAs to a biological product can decrease the half-life of the product and/or have neutralizing activity. A study of 121 US Food and Drug Administration (FDA)–approved biological products revealed that 89% of products showed ADA incidence, with almost half reporting some impact on efficacy, only 26% reported impacts on PK [4]. ADAs can be induced by patientrelated factors including genetic profile, immune status, or disease status [7]
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