MSACL 2026 Abstract

Feasibility of LC–MS/MS Based Therapeutic Drug Monitoring for Vedolizumab and Ustekinumab Using Commercial PK and ADA Kits

Amol O. Bajaj (1), Tracie Profaizer (1), Stephen Merrigan (1), Dorothee Lebert (3), Julio Delgado (1,2)
(1) ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA,(2) Department of Pathology, University of Utah, Salt Lake City, Utah, USA,(3) Promise Proteomics, 7 Parvis Louis Neel, CS20050, 38040 Grenoble Cedex, France.

Amol Bajaj, MS (Presenter) ARUP

Presenter Bio: I am a Scientist III in R&D Mass Spectrometry at the ARUP Institute for Clinical and Experimental Pathology. I hold an MS in Biotechnology from the University of Houston Clear Lake. My primary focus is on developing innovative analytical strategies that enhance the capability of clinical laboratories to perform challenging assays. Currently, I am working on methodologies for detecting ceramides in serum/plasma, which are biomarkers for cardiovascular diseases.
I have also developed high-resolution mass spectrometric methods for the characterization and identification of microorganisms, including mycobacteria. Mycobacterium tuberculosis, responsible for one of the most devastating infectious diseases, claimed nearly 1.3 million lives in 2020 and infected approximately 10 million people. This pathogen can remain dormant within the human host, posing significant diagnostic challenges.
In addition to my work with mycobacteria, I have recently developed and validated a Thiopurine metabolites assay. Thiopurine drugs, used to treat conditions such as acute leukemia, inflammatory bowel disease (IBD), and other autoimmune diseases, are critical in managing these conditions. With IBD now a global health concern affecting over 5 million people worldwide, and autoimmune diseases like rheumatoid arthritis affecting millions in the USA, the need for precise and reliable assays has never been greater.
I am also deeply interested in the development and validation of methods for proteins and peptide biomarkers. I have successfully developed techniques for detecting intact insulin growth factors 1 (IGF1) and 2 (IGF2) in human serum, which play a crucial role in childhood growth and have anabolic effects in adults.

Relevant Financial Disclosures (within past 24 months, reported on May 20, 2026) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Vedolizumab and ustekinumab are monoclonal antibody therapies widely used for inflammatory bowel disease, and therapeutic drug monitoring (TDM) has been associated with improved clinical management. Quantitation from immunoassay-based drug and anti drug antibody testing can be limited by analytical interference and inter assay variability. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) directly measures drug specific peptides and offers improved analytical specificity. This study evaluated the feasibility of implementing commercial LC–MS/MS pharmacokinetic (PK) and anti drug antibody (ADA) kits in a clinical laboratory.

METHODS:
Commercial LC–MS/MS PK and ADA kits were evaluated on a Sciex 6500+ triple quadrupole platform. For PK evaluation, an internal standard is added at the beginning of sample preparation, enabling normalization of biases arising from different steps of the protocol (i.e., extraction, digestion). This internal standard is a Stable-Isotopically-Labelled mAb (SIL-mAb), with a sequence highly similar to that of the targeted mAb. However, the SIL-mAb is specifically labelled with [13C6,15N2] Lys and [13C6,15N4] Arg. Aliquots of human plasma or serum samples (20 µL) were mixed with SIL-mAb. Then, the mAb of interest and its SIL form are immunocaptured, extracted, and digested with an enzyme to generate peptides.

For ADA evaluation in human plasma or serum samples (100 µL), the strategy relies on the fact that ADAs are antibodies, so they have two antigen-binding sites that can be used to form a “bridge” between the t-mAbs and their corresponding SIL-mAbs. After elution and trypsin digestion, SIL-peptides are detected only if free ADAs is present in the plasma/serum.

The digested sample plate is then transferred to the LC autosampler, and the samples were analyzed using a 6500+ LC-MS/MS, equipped with a 1290 series HPLC (Agilent). Chromatographic separation was performed using an Accucore Vanquish C18+ column (1.5 µm, 5 cm, 2.1 mm ID, Thermo Fisher Scientific). Quantification was performed using a six-point calibration curve (0.5–100 µg/mL for both vedolizumab PK and ustekinumab PK and 10–400 ng/mL for both vedolizumab ADA and ustekinumab ADA), monitoring four mass transitions for vedolizumab PK and three for ustekinumab PK and their respective IS and monitoring two peptides, three mass transitions for vedolizumab ADA and four for ustekinumab ADA and their respective IS.

Analytical performance was assessed through studies of linearity, accuracy, precision, and sensitivity. Method comparison was performed using deidentified residual clinical specimens against a national reference laboratory using LC-MS/MS methodology for vedolizumab PK and ELISA for ustekinumab PK. The ADA evaluation included deidentified residual clinical specimens and contrived specimens.

RESULTS:
Vedolizumab PK demonstrated a strong correlation to a reference laboratory LC–MS/MS method across 84 de-identified patient samples with a Deming regression slope of 1.40 (95% CI 1.34–1.48) and R of 0.976. Bland–Altman analysis showed a mean positive bias of 10 µg/mL (46%). Linearity was demonstrated across 2–100 µg/mL using spike in and dilution studies. Analytical precision from pooled patient samples showed total CVs of 3% at low concentrations (4-6 µg/mL) and <4% at mid to high concentrations (30–50 µg/mL). Sensitivity assessments near the LLOQ demonstrated CVs ≤10%.
Ustekinumab PK demonstrated linearity from 2.5–80 µg/mL. Comparison with a reference laboratory immunoassay method across 44 de-identified patient samples showed a Deming regression slope of 1.13 (95% CI 0.96–1.30) and an R of 0.899. Bland–Altman analysis showed a mean positive bias of 0.28 µg/mL (5%). Precision studies showed total CVs of 10% at low concentrations (3–4 µg/mL), 4% at mid concentrations (7-9 µg/mL), and 13% at higher concentrations (15-18 µg/mL).

Vedolizumab LC–MS/MS ADA assays detected high titer antibodies and were linear from 11–500 ng/mL for both short and long signature peptides, with average calibrator recoveries near 100%. Ustekinumab ADA demonstrated linearity from 20–470 ng/mL. Precision across ADA concentration pools ranged from 2–10% CV at low to mid-levels and 8–16% CV at higher concentrations. Reduced sensitivity at lower ADA levels was observed.

CONCLUSION:
Commercial LC–MS/MS kits for vedolizumab and ustekinumab PK demonstrated adequate analytical performance and an acceptable workflow, supporting progression toward formal validation for longitudinal TDM. ADA assays were analytically feasible and capable of detecting high titer antibodies but require additional optimization and broader clinical evaluation before routine implementation. LC–MS/MS represents a robust alternative to immunoassays for biologic TDM, offering improved specificity and resistance to analytical interference.