(T1230-01-02) A Validated Assay for LC-MS/MS Analysis of an siRNA Therapeutic Drug Using Microsampling Devices

Purpose: The recent increase in siRNA therapeutic technology has created a new wave of pharmaceutical innovation, one that is patient-centric and that requires a robust method of analysis. These drugs present unique challenges to overcome, but by using micro sampling devices we can create clinical trials that are non-invasive and accessible to all patient populations. This work presents a validated method to extract siRNA from dried blood samples collected with a Tasso device and its analysis via LC-MS/MS. This process allows for both decentralized and clinic supported work to be done with in-home collection devices which generate reliable analytical data.

Methods: Fresh human whole blood was spiked with siRNA to create liquid blood standards and calibrators. The blood from these liquid calibrators was then slowly pipetted into a Tasso M20 Microsampling device and left to dry with desiccant in a mylar bag.  Each pad of the device forms one aliquot equal to 17.5uL of liquid blood. To extract the siRNA from the Tasso pad, we added TPER (Tissue Protein Extraction Reagent) at a 10:1 ratio to the aliquot volume of the device, then shaken for 3.5 hours. Following digestion, QC samples above the calibration range concentration were diluted using TPER reagent. All samples then received 300µL of Clarity OTX Lysis Loading Buffer and the solution was mixed at ambient for 0.5 hours. The resolubilized siRNA from the digestion process was then extracted with a Clarity OTX 25mg solid phase extraction plate, which then followed an analyte optimized Clarity OTX SPE procedure (1). Eluent from the SPE procedure was evaporated and reconstituted in water containing HFIP and TEA to match mobile phase conditions used in the LCMS analysis. The liquid handling method used aqueous and organic mobile phases of 1000:10:1 Water:HFIP:TEA and 1000:10:1 Methanol:HFIP:TEA. These underwent a gradient elution method which started at 10% organic concentration and ramped to 95% organic concentration over a 3.5-minute period, then held at 95% organic for 1.5 minutes, while flowing at 200µL a minute. Chromatography was performed using a Shimazu Nexera LC handler coupled to a Prominence oven and a Thermo DNAPac 4µm 2.1x50mM column. Quantitative analysis was performed on a Sciex API 5000 mass spectrometer in negative ionization mode following the m/z -9 charge state on the anti-sense strand of the siRNA target analyte. High Q3 mass transitions were followed instead of the commonly used phosphorothioate (pSO₂), 5’ phosphate group, or other low Q3 mass transitions to avoid issues with endogenous or exogenous interferences present in small mass product ion transitions.

Results: An analytical range for the method was established at 100 – 20,000 ng/mL. The acceptance criteria for calibration standards and quality control samples were set to 20% (25% at LLOQ) for %RE and %CV. Three accuracy and precision runs were then performed using dried VAMS QC samples and calibrators. The inter-run %RE from all three AP runs ranged from -11.86% to 2.05% and the inter-run %CV was < 10.5 % at all QC levels. Room temperature long term stability was performed at 10 weeks against fresh samples dried a bench top for 24hrs. Stability results were acceptable with %RE from 4.24% to 6.5% and %RE from -5.26% to 9.33%. this stability also inherently proves room temperature stability for the extraction process. Extracts showed acceptable reinjection reproducibility after 119 hours at 4℃. Hematocrit levels at 35% and 55% were assessed at low, high, and Dilution QC levels representing the blood cell content that may exist in disease state patient samples. No significant impact of the hematocrit at 35%, but a slight high bias at Low and high QC levels for 55% hematocrit was noticed but mitigated by further dilution in the 10x dilution QC.

Conclusion: This method was found to be suitable for validation of extraction and recovery of siRNA from the Tasso VAMS device. Acceptance was within standard BMV guidance criteria. Some of the known concerns of microsampling devices is storage stability and reliable device recovery, both of which are shown to not be impactful when using this robust validated method of analysis. This evaluation of these devices lays a foundation for protocols to assess other therapeutics in the siRNA class of pharmaceuticals, its potential applicability to other drug classes, and as an alternative to other VAMS collection methods.