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

Antibody-antisense oligonucleotide (Ab-ASO) conjugates are an innovative class of therapeutics that merge the gene-modulation capabilities of ASOs with the tissue-targeting properties of monoclonal antibodies. Sensitive, selective, and reliable methods to quantify Ab-ASO conjugates in biological samples are critical to understand their pharmacokinetics, pharmacodynamics, toxicity, and biodistribution properties. While ligand-binding assay (LBA) approach is common, it is often limited by the need for multiple custom reagents and narrower dynamic range. In this work, we present a robust hybridization LC-MS/MS strategy for the quantitation of Ab-ASO conjugates using sequence-specific hybridization extraction. Discovery proteomics was utilized to identify and select high-specificity surrogate peptides for the antibody moiety. The method leverages a biotinylated complementary DNA probe to selectively enrich the conjugate through Watson-Crick base pairing, followed by tryptic digestion and LC-MS/MS quantitation of surrogate peptides. The assay was successfully qualified in mouse serum over the range of 10.0 - 10,000 ng/mL and applied to a single-dose pharmacokinetic study, demonstrating high analytical agreement with a benchmark LBA strategy. The LC-MS/MS approach provides a superior dynamic range, simplifies reagent requirements and offers an LC-MS based quantitation platform that was previously unavailable for Ab-ASO conjugate quantitation.