(T1230-09-52) A Sensitive Solution for Quantification of Anti-sense Oligonucleotides (ASOs) in Rat Plasma Using High-Resolution Mass Spectrometry

Purpose: Oligonucleotide therapeutics and gene therapies are rapidly gaining attention as their potency improves and delivery mechanism are refined. Modalities such as ASOs are becoming more important due to their high specificity and ability to reach formerly untreatable targets. Therefore, sensitive and robust methods for quantitative analysis of oligonucleotides are needed to support the development of ASO therapeutics. Ligand binding assays using fluorescence detection can achieve very low detection limits. However, the linear dynamic range (LDR) is limited, and differentiation of oligonucleotides from their impurities and metabolites is often a challenge, resulting in a lack of specificity in complex systems. While high-resolution, time-of-flight mass spectrometry (TOF-MS) has been used for quantification, it can be hard to reach the high sensitivity of a triple quadrupole system because of duty cycle limitations inherent to the mass analyzer design. In this study, a novel TOF mass spectrometer with duty-cycle enhancements was employed for quantification of ASOs. A 10-fold improvement in sensitivity was observed compared to traditional TOF instruments. In addition, microflow LC was used to further improve sensitivity by an order of magnitude compared to standard flow LC methods.

Methods: In this experiment, fomivirsen, nusinersen, eluforsen, a model fully phosphorothioated 2’O-methylated oligonucleotide and an internal standard oligonucleotide were analyzed using an MRM-HR method. Calibration curves were measured in triplicate for all analytes by spiking the analytes into plasma extract in the concentration range between 0.01 and 300 ng/mL.

Results: Exceptional linearity, accuracy and precision were achieved for all analytes. LLOQs and linear calibration curve ranges were determined based on the requirements that the %CV of the calculated mean of the concentration is below 20% at the LLOQ and below 15% for all higher concentrations, while the accuracy of the calculated mean is between 80% and 120% at the LLOQ and between 85% and 115% for the higher concentrations. The LLOQ for nusinersen was 10 pg/mL, while the LLOQs for the other ASOs was 30 pg/mL. LDR was 3.5 to 4 orders of magnitude. In addition, impurities could be detected, and their concentration relative to the main products estimated, based on the TOF-MS data.

Conclusion: Overall, low-pg/mL LLOQs for ASOs in rat plasma were achieved using the microflow LC method on a novel TOF mass spectrometer with duty-cycle enhancements.