Automated sample-preparation and mass spectrometry-based assays for free and total Infliximab: Enabling therapeutic drug monitoring of monoclonal antibodies

Melissa J Sam; David G Harman; Catherine M Toong; William A Donald

doi:10.1016/j.aca.2026.345222

Automated sample-preparation and mass spectrometry-based assays for free and total Infliximab: Enabling therapeutic drug monitoring of monoclonal antibodies

Anal Chim Acta. 2026 Apr 22:1396:345222. doi: 10.1016/j.aca.2026.345222. Epub 2026 Feb 11.

Authors

Melissa J Sam¹, David G Harman², Catherine M Toong³, William A Donald⁴

Affiliations

¹ School of Chemistry, University of New South Wales, Sydney, NSW, Australia; Immunology Laboratory, NSW Health Pathology, Liverpool, NSW, Australia; Ingham Institute of Applied Medical Research, Liverpool, NSW, Australia; South Western Sydney Clinical School, University of New South Wales, Liverpool, NSW, Australia. Electronic address: melissa.sam@health.nsw.gov.au.
² School of Science, Western Sydney University, Campbelltown, NSW, Australia. Electronic address: d.harman@westernsydney.edu.au.
³ Immunology Laboratory, NSW Health Pathology, Liverpool, NSW, Australia; Ingham Institute of Applied Medical Research, Liverpool, NSW, Australia; South Western Sydney Clinical School, University of New South Wales, Liverpool, NSW, Australia; Immunology Department, Liverpool Hospital, Liverpool, NSW, Australia. Electronic address: catherine.toong@health.nsw.gov.au.
⁴ School of Chemistry, University of New South Wales, Sydney, NSW, Australia. Electronic address: w.donald@unsw.edu.au.

PMID: 41791804
DOI: 10.1016/j.aca.2026.345222

Abstract

Background: Therapeutic drug monitoring (TDM) of biologics is commonly used in the management of inflammatory disease. However, current clinical tests are confounded by the interaction of drug and anti-drug antibodies (ADAs), which prevent detection on standardized tests. The direct measurement of ADAs with ELISA leaves up to 25% of ADAs undetected, and no standard tests exist for measurement of ADAs in complex with drug in serum. Although there have been reports exploring biologic ADA interactions, high-throughput workflows for translation into the clinic are lacking.

Results: We report, for the first time, a high-throughput workflow capable of quantifying therapeutic antibodies bound to ADAs in patient serum, applied to infliximab (IFX) for TDM. Using a 96 well plate-based, rapid, targeted dual liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, free and bound IFX were measured as a marker of previously undetected anti-infliximab (ATI) development. The method is optimized for on-plate digestion and automated with robotic sample handling to enable routine adoption of trough IFX in clinical practice. The free IFX assay results agreed well with ELISA and expanded the quantitation range (0.5-50 μg/mL). The total IFX assay quantified IFX regardless of ATI presence. In patient samples, ∼49% showed >20% higher total vs. free IFX, consistent with undetected ATIs.

Significance: The dual-assay enables routine quantification of biologic-ADA interactions in a cost effective, high throughput LC-MS/MS workflow. It addresses key limitations of immunoassays and may improve detection of immunogenicity in patients receiving biologic therapy. The method is generalizable to other therapeutic antibodies affected by ADA interference.

Keywords: Anti-Drug antibody; Anti-Infliximab; Drug-tolerant; ELISA; Free IFX; Inflammatory bowel disease; Infliximab; LC-MS/MS; TDM; Total IFX.

MeSH terms

Antibodies, Monoclonal* / blood
Antibodies, Monoclonal* / immunology
Automation
Chromatography, Liquid
Drug Monitoring* / methods
Humans
Infliximab* / analysis
Infliximab* / blood
Infliximab* / immunology
Tandem Mass Spectrometry

Substances

Infliximab
Antibodies, Monoclonal