(T1230-08-52) LC and LCMS Solutions for Sample Preparation, and Data Analytics to Advance Oligonucleotide Therapy Development

Purpose: Nucleic acid drugs, such as antisense oligonucleotides, are capable of targeting genes both inside and outside of cells to treat diseases at the genetic level. Unlike conventional small molecule drugs, they can target the underlying genetic causes of diseases, making them a promising next-generation therapeutic option. However, impurities, including shorter length components and protecting groups, can arise during the chemical synthesis of nucleic acid drugs, necessitating the proper separation and purification of the target oligonucleotide. This study introduces an analytical method for separating and analyzing oligonucleotides of different lengths using ion-exchange chromatography, under the assumption that shorter length components are impurities resulting from the synthesis process. To achieve optimal analytical performance, an inert ultra-high-performance liquid chromatography (UHPLC) system was utilized. Furthermore, we incorporated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an innovative automated data analysis software to facilitate oligonucleotide development and ensure quality control.

Methods: In ion-exchange chromatography, a mobile phase with varying salt concentration or pH is used to separate and elute samples. For this study, an aqueous solution of sodium hydroxide with sodium perchlorate was chosen as the mobile phase due to its high elution strength. The separation mechanism in ion-exchange chromatography is based on the number of phosphate groups in the oligonucleotide, allowing for accurate identification and quantification of various oligonucleotide sequences. To compare the quantitative results obtained, a triple quadrupole mass spectrometer (TQMS) is used. Since oligonucleotide therapeutics with 20 or more bases have a molecular mass of 6000 or higher, it is crucial to use a high-performance mass spectrometer like Q-TOF for accurate molecular mass determination. Furthermore, the TQMS is employed for quantitative analysis as it typically has a broad dynamic range. To streamline the analysis process and facilitate the identification and quantification of oligonucleotides with different base pairs, automated data analytics software is used.

Results: HPLC is a widely used analytical technique in which stainless steel is preferred due to its corrosion-resistant and pressure-proof properties. However, it should be noted that stainless steel can interact with compounds containing phosphate groups through metallic affinity, which may affect the analysis. Separation of shorter oligonucleotides (less than 20 bases) can be easily achieved by HPLC, but it becomes increasingly challenging for longer sequences. During the analysis, it was found that the metal-based method failed to detect a peak at 1 g/mL and although the peak intensity was detectable at 2.5 g/mL, it was not enough to accurately quantify the sample. Due to metal adsorption, there was a large deviation in the quantitation values of QC controls, as expected due to the low linearity of the calibration curve. On the other hand, the calibration curve obtained with the "metal-free" method showed excellent linearity (r² = 0.9999). To overcome accurately identify and overcome other challenges, a high-resolution ion-pair reversed-phase (IP-RP) LC-MS system was developed for high-resolution oligonucleotide separation and mass analysis. The system was able to achieve a high level of accuracy, with the most abundant mass accuracy being less than 2.5 ppm for all targets. Oligonucleotides were quantified using both high-resolution and TQ mass spectrometers using MRM acquisition mode. By focusing on the m/z range for production scans, higher sensitivity was achieved, and the mass spectra extracted from the scan mode data the "ReSpect" algorithm, available in the LabSolutions Insight Explore™ software, was used to deconvolute the spectra. The software can also identify and quantify the oligonucleotides.

Conclusion: In summary, this study demonstrates that the adoption of a metal-free flow path, inert UHPLC system, and ion-pairing buffer system has resulted in highly sensitive, accurate, and robust analytical results for the separation and identification of oligonucleotides. The developed method allows for high-resolution separation and high-accuracy mass analysis of oligonucleotides, guaranteeing an error of less than 2.5 ppm. The analytical results were found to be reproducible and accurate, with linearity observed across a range of 1 - 1000 ng/mL. By utilizing the LCMS (Q-TOF) mass spectrometer, the molecular mass of oligonucleotides can be accurately determined with an error of 0.05 ppm. Overall, this analytical method has the potential to be a valuable tool in various fields, including molecular biology, genetic engineering, and pharmaceutical development.