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* = Presenting author

P552 Development and validation of a tandem liquid chromatographic mass spectrometric assay for the measurement of vedolizumab levels in patient serum

C. Christ*1, A. Dignass2, 3, F. Hartmann2, 3, J. Stein3, 4

1Immundiagnostik AG, Bensheim, Germany, 2Agaplesion Markus Krankenhaus, Frankfurt/Main, Germany, 3Interdisciplinary Crohn Colitis Centre Rhein-Main, Frankfurt/Main, Germany, 4DGD Clinics Sachsenhausen, Frankfurt/Main, Germany

Background

Vedolizumab (VLZ), an α4β7 integrin antagonist, is a therapeutic monoclonal antibody approved for use in moderate-to-severe ulcerative colitis and Crohn’s disease. As for TNFa agonists, measurement of serum levels is indispensable for the optimisation of VLZ therapy. Typically, ligand binding assays (LBA), such as enzyme-linked immunosorbent assays (ELISAs), are used to measure serum antibody levels. However, LBAs may have significant limitations, including procurement or generation of the binding reagents, interference from matrix components, and drug antibodies. To overcome these limitations, we have developed a reliable tandem liquid chromatographic mass spectrometric (LC-MS/MS) method to quantify VLZ serum levels.

Methods

Serum analyses were performed on an ultraperformance liquid chromatography (UPLC)-MS/MS system (Ultimate 3000 Binary RSLC system, Thermo Scientific, Waltham, Massachusetts, United States) coupled to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, Waltham, Massachusetts, United States) with an H-ESI II ionisation source. The analytical column was an Acquity UPLC BEH Shield RP C18 2.1mm × 50mm column with 1.7μm particle size. The mobile phases were KM9600LMA and KM96LMB. VLZ was extracted from serum by a modified ‘pellet digestion’ technique. Tryptic peptides for quantification were identified for VLZ using a selective reaction monitoring mass spectrometry method.

Results

Sensitivity for VLZ was determined to be 1.6μg/mL. The standard curves showed high reproducibility and sensitivity. Intra- and inter-assay precision were 10.46% and 7.67%, respectively, and accuracy was within 20%. There was no significant interference from lipemic, haemolysed, or rheumatoid factor (Rf) serum.

Conclusion

A general LC-MS/MS method approach using immunocapture was developed, qualified, and applied to VLZ quantification in serum, with an affinity purification time of one hour. The method has been demonstrated to be accurate, precise and robust. The LC-MS/MS method may also be applied to other protein-based drugs to accurately detect serum drug levels.