2023 FDA Science Forum
Validation of HILIC-HRMS Method for Oligonucleotide Analysis
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Contributing OfficeCenter for Drug Evaluation and Research
Abstract
Background
Synthetic oligonucleotide therapeutics (ONTs) are an evolving class of drug products with the potential to treat rare and life-threatening medical events as well as common chronic diseases. However, ONTs have unique regulatory challenges largely attributed to their molecular complexity, not only in the intended sequence but in product-related impurities. Historically, ion-pair reversed phase (IPRP) liquid chromatography (LC) coupled with mass spectrometry (MS) have been widely used in oligonucleotide analysis. Recently, hydrophilic interaction liquid chromatography (HILIC) that is IP reagent-free and more MS compatible has received recognition and may be a promising approach for ONT characterization. Nevertheless, challenges remain including method robustness, reproducibility and analytical sensitivity.
Purpose
This study aims to validate the recently developed HILIC-high resolution (HR) MS method following regulatory guidances.
Methods
A custom-synthesized full-length product (FLP, a modified 18-mer RNA) with or without spiked impurities was used. HRMS data were acquired using a Tribrid orbitrap mass spectrometer. LC-MS data were processed using software including BioPharma Finder (BPF) and Skyline.
Results
The calibration curve range covering 5-orders of magnitude from 0.2 fmol to 20 pmol was evaluated with selected column loads. LLOQ was determined at a column load of 0.04 pmol with a % CV < 12%. The calibration curve covering the low concentration end (from LLOQ to 2 pmol) displayed an excellent linearity with R2 > 0.999. The calibration curve covering the high concentration end (from 2 to 20 pmol) also displayed a great linearity with R2 > 0.998 and precision with % CV < 2% over the entire range. The high column loads (up to 25 pmol) were precise with % CV < 3% and were free of carry over and peak shape distortion, indicating a ULOQ could be set at 20 pmol or above that enables the detection of low-level impurities from high column loads of FLP. Future studies will investigate matrix effects or potential interference by spiking separable or coeluting impurities.
Conclusions
A HILIC-HRMS method developed for quantifying oligonucleotides was validated for precision, sensitivity, calibration curve range, linearity and accuracy. The developed method is robust, sensitive and accurate for analysis of complex oligonucleotides and impurities.
