Hybridization LC-MS/MS platform quantifies Antibody-ASO conjugates with superior dynamic range and simplified reagents

Accurate and sensitive quantification of Antibody-Antisense Oligonucleotide (Ab-ASO) conjugates is crucial for understanding their pharmacokinetics, pharmacodynamics, toxicity, and biodistribution. Ab-ASOs represent an innovative therapeutic class, combining the precise gene-modulation of ASOs with the targeted delivery of monoclonal antibodies. Current standard ligand-binding assay (LBA) approaches for these complex bioconjugates often suffer from limitations, including the need for multiple custom reagents, a narrower dynamic range, and potential issues with specificity, hindering comprehensive drug development.

Researchers developed a robust hybridization LC-MS/MS strategy for quantifying Ab-ASO conjugates. The method employs a biotinylated complementary DNA probe for sequence-specific hybridization extraction, selectively enriching the conjugate via Watson-Crick base pairing. Following enrichment, tryptic digestion was performed, and specific surrogate peptides for the antibody moiety were quantified using LC-MS/MS. The assay was qualified in mouse serum over a broad concentration range and applied to a single-dose pharmacokinetic study, comparing its performance against a benchmark LBA strategy to validate its analytical agreement.

The novel hybridization LC-MS/MS method successfully quantified Ab-ASO conjugates in mouse serum, demonstrating high analytical agreement with a benchmark LBA strategy. The assay exhibited a significantly improved dynamic range, spanning from 10.0 ng/mL to 10,000 ng/mL. This represents a substantial improvement over traditional LBA methods, which often have more restricted quantification limits. The platform also simplified reagent requirements, reducing the need for multiple custom reagents typically associated with LBAs. Surrogate peptides, identified through discovery proteomics, ensured high specificity for the antibody component of the conjugate. The method's successful application in a single-dose pharmacokinetic study further validated its utility for in vivo analysis. This LC-MS based quantitation platform addresses a critical gap, previously unavailable for comprehensive Ab-ASO conjugate analysis. > The assay was successfully qualified in mouse serum over the range of 10.0 - 10,000 ng/mL, demonstrating a superior dynamic range and high analytical agreement with a benchmark LBA strategy.