(M1330-01-05) Application of Mitra Microsampling for the Quantitative Bioanalysis of Antisense Oligonucleotides

Monday, April 24, 2023

1:30 PM – 2:30 PM ET

Presenting Author(s)

Kevork Mekhssian, MSc

Scientific Director
Altasciences
Laval, Quebec, Canada

Main Author(s)

MC

Ming-Luan Chen, Ph.D.

Principal Scientist
Altasciences
Laval, Quebec, Canada

Co-Author(s)

MD

Muskaan Dutt

Analyst
Altasciences
Laval, Quebec, Canada
Kevork Mekhssian, MSc

Scientific Director
Altasciences
Laval, Quebec, Canada
Anahita Keyhani, PhD

Senior Director Scientific Operations Mass Spectrometry
Altasciences
Laval, Quebec, Canada

Purpose: Volumetric Absorptive Microsampling (VAMS^®) has recently emerged as an alternative method to venous sampling, offering the convenience of home sampling of small volumes of whole blood ( < 20mL). Coupled with liquid chromatography and mass spectrometry (LC-MS) analysis, this technique has been mainly used to support small molecule pharmacokinetic (PK) clinical studies, with limited data available on the application and utility of this approach to other therapeutic classes such as oligonucleotides. In this work, we describe the first application of VAMS® with Mitra microsampling device (Neoteryx) for the quantification of an Antisense Oligonucleotide (ASO) using an automated, high throughput sample preparation workflow coupled with LC-MS/MS detection. Due to the limited sample volume and to discriminate closely related metabolites (i.e. n-1, n-2), the hybridization LC-MS platform was used to achieve the required high sensitivity and specificity levels. The analytical performance of this novel workflow was evaluated for Fomivirsen (FME), a synthetic 21-mer ASO, used in the treatment of cytomegalovirus retinitis (CMV) in immunocompromised patients.

Methods: Human whole blood samples fortified with FME were absorbed onto 10µL Mitra VAMS^® and dried at room temperature in the presence of desiccant for at least 24 hours. Effective desorption of the ASO from the Mitra tips by Impact-Assisted Extraction (IAE) was established by screening various buffer solutions to optimize recovery, while overcoming the bias from the sample hematocrit (HCT) level. For selective extraction, hybridization of the single-stranded ASO with a complimentary biotinylated capture probe, coupled to streptavidin-coated magnetic beads was automated using the KingFisher^TMFlex 96-well sample processor. A heat step was used to elute the ASO and the resultant extract was separated on an Acquity Oligonucleotide BEH C18 column (50 x 2.1mm, 1.7 µm) using acetonitrile (ACN) gradient elution with ion-pairing mobile phases composed of dibutylamine (DBA), dimethylcyclohexylamine (DMCHA), and hexafluoro-2-methyl-2-propanol (HFMIP). The analyte FME and IS (n+2 analog) were monitored by MRM on a SCIEX Triple Quad 6500+.

Results: Various buffers and surfactants were screened for the effective desorption of FME and IS from Mitra VAMS^® using IAE. Addition of detergents in the desorption buffer significantly improved extraction of ASOs from the Mitra sorbent, probably due to enhanced protein solubility and decreased surface tension which helps buffer permeation into the porous Mitra tips. Extraction solution containing 20 mM Tris, 20 mM EDTA, 100 mM NaCl, 0.5% NP-40, pH 8.0 was selected to characterize the assay performance. The calibration curve was constructed with a range of 5.00 – 1000.00 ng/mL using peak area ratios of FME to the IS and applying a weighted (1/x²) linear regression. Representative chromatograms (BL, LLOQ, ULOQ, IS) are shown in Figure 1. Precision and accuracy of the method were evaluated after replicate analysis of QC samples at LLOQ (5.00 ng/mL), Low (15.00 ng/mL), Medium (500.00 ng/mL), and High (750.00 ng/mL) concentrations. Precision was determined by the coefficient of variation (% C.V.) calculated from four replicate QC measurements. Accuracy was reported as the percent nominal (% Nominal), calculated as the percent deviation of the observed sample concentration from its theoretical value. QC samples in all extracted batches met the acceptance criteria for precision and accuracy. The assay was demonstrated to be independent of blood HCT in a range from 0% to 66% HCT levels (Table 1). The long-term stability of the ASO on Mitra tips was demonstrated for 23 days at ambient temperature, and no impact from putative metabolites (n-1, n-2) was observed in the assay (Table 2).

Conclusion: A bioanalytical method for the determination of FME ASO in human blood was successfully developed with an analytical range from 5.00 to 1000 ng/ml by using Mitra VAMS^®coupled with hybridization LC-MS/MS. The method is linear, precise, and accurate with all evaluable bioanalytical assessments meeting the acceptance criteria. The application of Mitra VAMS^® to the quantification of therapeutic ASOs from micro samples would facilitate a more convenient and accurate remote sampling and can be applied to decentralized clinical trials and patient-centric drug development.

Figure 1. Representative chromatograms of extracted blank (A), extracted LLOQ at 5.00 ng/mL FME (B), extracted ULQ at 1000.00 ng/mL FME (C) and (D) extracted IS.

Table 1. Impact of HCT level on the quantitation of Fomivirsen by Mitra VAMS

Table 2. Impact of Metabolite interference on the quantitation of Fomivirsen by Mitra VAMS