(M0930-10-64) Quantification of Oligonucleotide Therapeutics in Plasma Using a Generic Kit-Based Approach

Purpose: As drug development efforts focus on large molecules, such as oligonucleotides therapeutics, such as oligonucleotide, the desire to employ LC/MS in bioanalysis is also increasing. LC-MS oligonucleotide sample preparation and quantification presents many challenges. There is no single workflow and the various workflow options can be complex and laborious, making it difficult for a scientist to achieve success. This work describes the quantification of several modified oligonucleotide therapeutics including: an antisense oligodeoxynucleotide phosphorothioate, O-methylated antisense oligonucleotide, a GalNAc -SiRNA conjugate, and lipid modified oligonucleotide, from biological fluids using a kit-based approach and generic protocol.

Methods: Using a prototype sample preparation and solid phase extraction (SPE) kit with supplied protocol, oligonucleotides (e.g. antisense oligodeoxynucleotide phosphorothioate, O-methylated antisense oligonucleotide, a GalNAc -SiRNA conjugate, and lipid modified) were extracted from plasma and urine samples (12.5-300 µL) using a proteinase K digestion sample pretreatment and anion-exchange SPE. LC-MS/MS quantification of signature peptides was performed using a triple quadrupole MS system. Chromatographic separation was achieved using a UHPLC system with a sub-2 µm fully porous 130Å, 2.1 mm x 50 mm column (55°C), at a flow rate of 0.6 mL/min using a linear gradient with 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Final injection volume was 5-15 µL with a subsequent analysis time of 5 minutes.

Results: The prototype sample preparation kit and protocol were used to successfully recover ( >75%) and reproducibly quantify oligonucleotide from plasma and urine quantification. Sensitivity, linearity, accuracy and precision data met typical method validation requirements with mean accuracies ranging from 85-115% and mean % CVs ±15% Standard curves were linear over 3.5 orders of magnitude.

Conclusion: This generic kit-based approach was successfully use to quantify multiple oligonucleotide therapeutics from biomatrices with minimal to no sample prep method development whilst achieving high oligonucleotide recovery >75% with sample volumes as low as 12.5 µL The universal, kit-based approach allows scientists to achieve high sensitivity with a simple step-wise protocol and standardized, pre-measure reagents, ensuring both the sensitivity and reproducibility required in discovery studies to make time sensitive and critical project decisions.