Development of Immunocapture-LC/MS Assay for Simultaneous ADA Isotyping and Semiquantitation

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Therapeutic proteins can trigger immune responses, leading to anti-drug antibodies (ADAs) that compromise patient safety and drug efficacy.
• Current methods like ELISA and ECLIA are widely used for ADA detection but may lack specificity.
• Liquid chromatography-mass spectrometry (LC/MS) offers high specificity, selectivity, and a wide dynamic range for biotherapeutic analysis.

Purpose of the Study:

• To develop and validate a novel immunocapture-LC/MS methodology for simultaneous isotyping and semiquantitation of ADAs in human plasma.
• To leverage the advantages of LC/MS for more precise ADA characterization during drug development.

Main Methods:

• An immunocapture strategy using biotinylated drug or anti-drug antibody immobilized on streptavidin magnetic beads to isolate ADAs and drug-ADA complexes from human plasma.
• Post-isolation, ADAs are released, digested with trypsin, and specific universal peptides are detected using LC/MS.
• Data analysis involves cut-point determination and calibration curves for semiquantitation.

Main Results:

• The methodology successfully demonstrated proof-of-concept by detecting pre-existing ADAs in human plasma.
• The method allows for simultaneous isotyping and semiquantitation of ADAs.
• LC/MS provides enhanced specificity and selectivity compared to traditional immunoassays.

Conclusions:

• The developed immunocapture-LC/MS method is a sensitive and specific approach for ADA detection, isotyping, and semiquantitation.
• This technique offers a valuable tool for assessing immunogenicity during therapeutic protein and peptide drug development.
• The methodology enhances the understanding of anti-drug antibody responses, contributing to improved drug safety and efficacy assessments.