(T0930-06-36) Development of a Novel LC-MS/MS Method for Quantitation of a Therapeutic Aptamer in Rat Plasma

Purpose: Aptamer-based drugs, with their high selectivity and low immunogenicity, are playing promising roles in drug development. Due to their larger molecular weight (usually >35 nt) compared to other oligonucleotide drugs (20-25 nt), the pharmacokinetic profiles of aptamers are routinely analyzed using Hybridization-based ELISA, RT-PCR, or HPLC-UV [1,2]. However, these methods struggle to distinguish between inactive metabolites from the full-length active parent drug, and can be easily interfered by the co-existing large molecules in biological matrices. The current study aims to develop a sensitive and robust LCMS/MS method for quantifying a 41 nt therapeutic aptamer in plasma, enabling its precise detection in biological fluids for future studies.

Methods: The analyte is a therapeutic aptamer (C₄₅₀H₅₉₆N₁₅₁O₂₇₈P_41, MW:13831.6) synthesized by WuXi AppTec. The internal standard was a 38 nt DNA with a similar structure modification as the analyte. Sample preparation involved extraction using a Clarity OTX SPE 96-well plate (Phenomenex). We optimized the extraction method by comparing the recovery from the standard extraction method, as the manufacturer’s protocol recommended, with various modifications. These modifications included changes to the loading mixture composition, elution buffer composition and volume, washing procedure, and dryness procedure. Extraction was conducted using a positive pressure manifold (Waters), and the treated samples were subsequently analyzed via LC-MS/MS. Mobile phase A consisted of water with 10 mM DIEA and 30 mM HFIP, while organic phase B consisted of 5 mM DIEA and 15 mM HFIP in 90% acetonitrile. Gradient elution was employed at a flow rate of 300 μL/min, utilizing a Shimadzu LC-40 system equipped with a Waters Oligonucleotide BEH Column (2.1×100 mm, 1.7 µm). A SCIEX QTRAP 6500+ at the negative ESI mode was used to monitor the intensity of the transition m/z 691/303 for the analyte and m/z 852/398 for the internal standard. The stability of the analyte in the plasma and whole blood has also been investigated in various experimental conditions.

Results: Following optimization, we found that a loading composition with plasma sample and lysis buffer ratio at 1:2 (v:v), a wash buffer of 50 mM ammonia acetate (pH 5.5) with 50% methanol, and an 0.5 mL elution buffer with 10% tetrahydrofuran (THF) yielded the optimal recoveries for both the analyte (80.2±2.5%) and the internal standard (107±11%). Increasing the elution buffer volume (up to 1.6 mL), the plasma to lysis buffer ratio (up to 1:8) and the THF percentage (up to 40%) did not improve the analyte recovery. Wash buffer with no methanol, 10% methanol, and 50% methanol in 50 mM Ammonia Acetate (pH 5.5) were compared for their extraction efficacy. Despite the slight loss ( < 10%) of the analyte during the wash procedure using 50% methanol in 50 mM ammonia acetate (pH 5.5), the solution is crucial for significantly reducing the matrix effect and improving the reproducibility of the assay. The dryness procedure yields a higher signal intensity but introducing greater variability, which might be attributed to the uncontrollable composition of the dried eluent. Therefore, eluent was directly injected without dryness. The finalized method met regulatory criteria for linearity, accuracy (interbatch bias: -4 to 8%) and precision (interbatch CV: 4.7 to 6.2%) within the calibration range of 50 – 2000 ng/mL. There is no interference between the analyte and the matrix, nor between the analyte and the internal standard. The analyte demonstrated stability in plasma for at least 24 days at -65 °C, 20 hours at room temperature, and for 2 freeze-thaw cycles. In fresh whole blood, its room temperature stability is 1 hour. The processed sample stability was at least 20 hours in a 4 °C autosampler. Maximum injection size is 106.

Conclusion: To the best of our knowledge, this study is the first report of a bioanalytical method for therapeutic aptamer utilizing an LC-MS/MS system. Our method provides an LLOQ at 3.6 nM of the aptamer, covering its in vitro efficacy concentration range. This method has the potential for the analysis of preclinical samples in future toxicokinetic and pharmacokinetic studies. Furthermore, our findings on the SPE-based extraction showed that the volume of the elution buffer and the solution employed for the wash step are most influential to the recovery and extraction efficiency. Such technical information from the successful optimization may provide valuable insights for the development of bioanalytical methods for other aptamers in the future.

References: 1.Wang L, Ji C. Advances in quantitative bioanalysis of oligonucleotide biomarkers and therapeutics. Bioanalysis. 2016 Jan 1;8(2):143-55.
2.Xiao X, Li H, Zhao L, Zhang Y, Liu Z. Oligonucleotide aptamers: Recent advances in their screening, molecular conformation and therapeutic applications. Biomedicine & Pharmacotherapy. 2021 Nov 1;143:112232.