Simultaneous isotyping and semi-quantitation of anti-drug antibodies to an IgG1 biotherapeutic using hybrid LBA-LC-MS/MS.

BACKGROUND: Commonly, ligand-binding platforms are being used for immunogenicity assessment, but with the recent advent of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for protein quantification, this technology has become an alternative for the measurement of anti-drug antibodies (ADAs), when combined with an immunocapture step to extract them out of the biological sample. METHOD: The monoclonal antibody adalimumab was immobilized on magnetic beads to isolate ADAs against this drug from serum samples. Multiple repetitions of immunopurification were used to minimize nonspecific binding and improve drug tolerance while maintaining sufficient recovery. A subsequent tryptic digestion released peptides, from which unique peptide sequences, originating from the constant region of seven ADA subclasses, were selected. These were then analyzed by LC-MS/MS against (unextracted) subclass-specific reference standards for semi-quantification. RESULTS: With two immunocapture and two immunopurification steps, the method simultaneously measures the ADA subclasses IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA within their relevant ranges, with good repeatability and drug tolerance, and limited interference of endogenous immunoglobulins. The method was successfully applied for the analysis of serum samples of subjects dosed with adalimumab. CONCLUSION: Hybrid LBA-LC-MS/MS is a viable platform for measuring ADAs and adds value, especially when ADA isotyping is needed.