Hybridization LC-MS/MS: An alternative platform to enable specific and sensitive quantification of Antibody-ASO...

Area of Science:

• Biopharmaceutical Analysis
• Mass Spectrometry
• Oligonucleotide Therapeutics

Background:

• Antibody-antisense oligonucleotide (Ab-ASO) conjugates combine antibody targeting with gene modulation.
• Accurate quantification of Ab-ASO conjugates is crucial for understanding their therapeutic behavior.
• Existing ligand-binding assays (LBAs) have limitations in dynamic range and reagent complexity.

Purpose of the Study:

• To develop and qualify a robust hybridization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying Ab-ASO conjugates.
• To enable sensitive and selective measurement of Ab-ASO conjugates in biological matrices.
• To provide an alternative quantification platform with improved characteristics over LBAs.

Main Methods:

• Employed sequence-specific hybridization extraction using a biotinylated DNA probe for selective enrichment of Ab-ASO conjugates.
• Utilized discovery proteomics to identify and select high-specificity surrogate peptides from the antibody moiety.
• Performed tryptic digestion followed by LC-MS/MS analysis for quantitation of the selected surrogate peptides.

Main Results:

• Successfully qualified the LC-MS/MS assay in mouse serum across a concentration range of 10.0 - 10,000 ng/mL.
• Demonstrated high analytical agreement with a benchmark ligand-binding assay (LBA) in a pharmacokinetic study.
• The developed method offers a significantly superior dynamic range and simplified reagent needs compared to LBAs.

Conclusions:

• The hybridization LC-MS/MS strategy provides a robust, sensitive, and selective platform for Ab-ASO conjugate quantitation.
• This method overcomes limitations of traditional LBAs, offering a wider dynamic range and reduced reagent complexity.
• The developed LC-MS/MS approach is valuable for pharmacokinetic and biodistribution studies of Ab-ASO therapeutics.