(M1230-09-57) A Workflow for Purity Determination, Intact Mass Measurement, and MS/MS Sequencing of Oligonucleotide Impurities Detected in Synthetic Oligonucleotides

Purpose: Oligonucleotides are a rapidly expanding category of therapeutics that are easy to manufacture, fully synthetic, and capable of targeting various disease pathways, far beyond what is capable with mAbs and small molecules. The preferred mass spectrometry-based method for oligonucleotide analysis has been reversed-phase chromatography employing various ion-pairing reagents and modifiers in negative ESI-MS mode (IP-RP LC-MS). Purity analysis relies on UV detection of all chromatographically separated oligonucleotide impurities. In contrast, the intact mass measurement and the sequencing of oligonucleotide impurities rely on a mass spectrometer's ability to match the UV detector's sensitivity for the analysis of very low-level impurities ( < 0.1%). This poster will show how IP-RP-chromatography successfully separated a synthetic 21-mer oligonucleotide and its impurities using a Q-Tof LC/MS system fitted with a novel chromatographic LC using a modified surface across its entire fluidic path. High-resolution ( > 20,000) ESI-MS, MS/MS, and MS^E (with targeted and untargeted fragmentation) oligonucleotide MS and MS/MS spectra were acquired in negative ion mode.

Methods: IA 21-mer synthetic-oligonucleotide, bearing 13 modified nucleotides, having the sequence: GCC UCA GTC TGC TTC GCA CCT was obtained from ATDBio (Southampton, UK). A 2’-O-(2-methoxyethyl) - (2’-MOE) modification was attached to two guanosines (G) and one adenosine (A). Also, in addition to the attachment of the 2’-MOE functional group to one uridine (U) and six cytidines (C), the nucleobases of these two nucleotides were further modified by the attachment of a 5-Methyl (5-Me) group to produce one 2’-MOE 5-Me uridine (U) and six 2’-MOE 5-Me cytidines (C). Out of the nine cytidine residues present in the 21-mer sequence, three cytidines were modified only at the nucleobase by the addition of a 5-Me functional group (C). Finally, there were eight nucleotides left unmodified: one adenosine (A), three guanosines (G), and five deoxythymidines (T). Stock solutions were prepared in DI water at a concentration of 1 µM (or 2.4 µg/mL), from which a 10 µL volume was injected, which corresponds to loading 10 picomoles of the 21-mer oligonucleotide on-column.

Results: An accurate, automated assignment of putative impurities detected in synthesized oligonucleotides with UV and MS detectors was achieved. Mass accuracy obtained for intact mass measurements of oligos and their impurities on a QTof mass spectrometer is better than 10 ppm. The MS/MS spectra acquired for six oligonucleotide impurities, along with the MS/MS spectra acquired for a 21-mer extensively modified FLP were processed using a Sequence App to confirm their expected sequences.

Conclusion: The workflow described here demonstrates the capability to achieve excellent sequence coverage (83.3%) even for low abundance oligonucleotide impurities, down to ~ 0.05% relative abundance levels.

References: 1. An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides, 2020, Waters application note, P/N 720006820EN. 2. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier, 2021, Waters application note, P/N 720007301EN. 3. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using the BioAccord System with ACQUITY Premier System and New Automated INTACT Mass Aplication, 2022, Waters application note, P/N 720007546EN. 4. CONFIRM Sequence: A waters_connect Application for Sequencing of Synthetic Oligonucleotides and Their Impurities, 2022, Waters application note, P/N 720007677EN.