Bioanalysis of free antisense oligonucleotide payload from antibody-oligonucleotide conjugate by hybridization LC-MS/MS

Abstract

Background: Antisense oligonucleotides (ASOs) have been conjugated to various moieties, such as peptides, antibodies or Fab regions of antibodies, to enhance their delivery to target tissues. The quantitation of free ASO (ASO payload) is critical to characterize its pharmacokinetics/pharmacodynamics (PK/PD) properties and biodistribution after delivery of the peptide/antibody/Fab ASO conjugates.Results: We developed a hybridization-based LC-MS/MS methodology for quantification of free ASO in tissues in the presence of Fab-ASO and ASO with linker (ASO-linker).Conclusion: The developed method was applied to measure accurately the free ASO concentrations in liver and gastrocnemius in mice that were dosed with Fab-ASO. This methodology has also been applied to free ASO bioanalysis for other antibody-ASO and Fab-ASO conjugates in various tissues and plasma/serum samples.

Keywords: Fab–ASO conjugates; LC-MS/MS; antibody–ASO conjugates; antibody–oligonucleotide conjugates (AOCs); antisense oligonucleotides (ASOs); bioanalysis; hybridization.

Figure 1.

Scheme of ASO1, ASO1-linker and Fab–ASO1.

Figure 2.

Effect of proteinase K digestion and hybridization extraction on Fab–ASO1 and ASO1-linker. (A) Peak area of ASO1 in Fab–ASO1 and ASO1-linker with no treatment, hybridization only and proteinase K digestion followed by hybridization; (B) Peak area of ASO1-linker in Fab–ASO1 and ASO1-linker with no treatment, hybridization only and proteinase K digestion followed by hybridization.

Figure 3.

Recovery of ASO1 in mouse plasma and brain homogenate at different MRDs.

Figure 4.

LC-MS/MS chromatograms of ASO1 and the IS, ASO-IS from (A) double blank, (B) blank spiked with ASO-IS only, (C) LLOQ and (D) ULOQ in mouse liver and gastrocnemius combine homogenate. IS: Internal standard; LLOQ: Lower limit of quantitation; ULOQ: Upper limit of quantitation.