(T1030-01-05) A Validated Hybridization LC-MS/MS Method for Quantitative Bioanalysis of HT-KIT, a Vivo-Morpholino Antisense Oligonucleotide

Purpose: Vivo-morpholinos are phosphorodiamidate morpholino oligonucleotides (PMOs) conjugated to cell-penetrating moieties offering enhanced cellular uptake and metabolic stability for efficient in vivo gene modulation. These molecules overcome key limitations of traditional gene knockdown approaches and have broad applications in disease modeling and therapeutic development. Despite their potential, quantitative bioanalysis of vivo-morpholinos remains limited. Key challenges include inefficient recovery using conventional extraction methods and poor chromatographic performance due to electrostatic interactions between the positively charged conjugated PMOs and negatively charged LC column surfaces. These issues hinder the development of sensitive and selective LC-MS/MS methods for pharmacokinetic applications. To address these challenges, a hybridization LC-MS/MS was developed and validated to quantify HT-KIT, an octa-guanidine-conjugated vivo-morpholino in development for cancers driven by KIT dysregulation. The method combines sequence-specific hybridization extraction with optimized chromatographic and mass spectrometric conditions, enabling accurate and reproducible quantification from small-volume serum samples. The assay met all regulatory validation standards and was applied to preclinical toxicokinetic studies.

Methods: Mouse serum samples (25 µL) were fortified with HT-KIT (1.00 - 250.00 ng/mL) and digested with Proteinase K under mild buffer conditions to release bound analyte. A full-length biotinylated probe complementary to HT-KIT was used to form an analyte–probe–bead complex via streptavidin magnetic capture. After multiple washes, HT-KIT was thermally released, and a linear PMO internal standard was added post-extraction. Chromatographic separation was achieved on a Waters XSelect Premier CSH C18 column (50 × 2.1 mm, 2.5 µm) with a mobile phase containing heptafluorobutyric acid (HFBA). Detection was performed using a SCIEX 6500+ triple quadrupole mass spectrometer in positive ESI/MRM mode.

Results: Reversed-phase ion-pairing LC methods traditionally used for oligonucleotide analysis are incompatible with the highly cationic nature of conjugated PMOs like HT-KIT. To address this, the stationary and mobile phases were optimized. A Waters XSelect Premier CSH C18 column, with a positively charged surface, was selected to minimize electrostatic interactions that otherwise impair peak shape and analyte retention. Heptafluorobutyric acid (HFBA) was incorporated into the mobile phase to improve chromatographic resolution between the analyte and the internal standard. To overcome HFBA-induced ion suppression, a post-column infusion of ammonium bicarbonate was introduced, resulting in a stable and enhanced MS response. Under these optimized conditions, HT-KIT and its internal standard were baseline-resolved, achieving an LLOQ of 1.00 ng/mL using just 25 µL of mouse serum (Figure 1). Hybridization extraction was optimized by evaluating probe lengths (15-, 20-, and 25-mer) and bead types. The full-length 25-mer probe yielded the most consistent recovery. Cytiva Streptavidin Mag Sepharose beads provided quantitative recovery with minimal non-specific binding, in contrast to Dynabeads MyOne C1. The method was validated over 1.00 – 250.00 ng/mL using a 1/x² weighted linear regression. Accuracy and precision met all acceptance criteria at LLOQ (1.00 ng/mL), low (3.00 ng/mL), mid (125.00 ng/mL), and high (187.50 ng/mL) QC levels (Table 1). No significant matrix effect was observed in seven lots of mouse serum, including one hemolyzed. HT-KIT was stable for 28 days at −80°C/−20°C, 24 hours at room temperature, and through four freeze–thaw cycles.

Conclusion: A hybridization LC-MS/MS method was successfully developed and validated for the quantification of HT-KIT, an octa-guanidine-conjugated PMO, in mouse serum over a 1.00 – 250.00 ng/mL range using only 25 µL of sample. The method demonstrated excellent linearity, accuracy, precision, specificity, and analyte stability in alignment with regulatory guidelines. This workflow provides a robust analytical platform for the preclinical bioanalysis of conjugated PMOs and can support the PK/TK assessment of vivo-morpholino therapeutics in drug development.

Figure 1. The representative chromatograms of extracted (A) blank mouse serum, (B) LLOQ at 1.00 ng/mL, (C) ULOQ at 250.00 ng/mL, and (D) Internal Standard.

Table 1. Inter-Run Precision and Accuracy for HT-KIT in Mouse Serum