Simple Analysis of Impurities in Oligonucleotide Therapeutics Using a Single Quadrupole Mass Spectrometer

Význam tématu

Oligonucleotide therapeutics represent a growing class of drug modalities requiring precise quality control. Impurities such as truncated sequences or degradation products can affect efficacy and safety. Combining high-resolution chromatography with mass spectrometry offers accurate identification and quantification of these variants, which is critical for regulatory compliance and drug development.

Cíle a přehled studie / článku

The study demonstrates a streamlined workflow for analyzing a model antisense oligonucleotide and its related impurities using a Nexera™ XS inert UHPLC system coupled to a LCMS-2050 single quadrupole mass spectrometer. Key objectives include chromatographic separation of full-length and truncated sequences, mass spectral deconvolution to estimate molecular weights, and evaluation of method accuracy and ease of use.

Použitá metodika a instrumentace

Samples comprised a 20-mer oligonucleotide (full-length product, FLP) and three deletion impurities (n-1, n-3, n-10). Chromatographic separation employed:

• Nexera XS inert UHPLC system
• Shim-pack Scepter Claris C18-300 column (100×2.1 mm, 1.9 µm)
• Mobile phases: A) 100 mmol/L HFIP and 10 mmol/L TEA in water, B) methanol
• Gradient: 10% B (0–15 min) → 35% B (15–20 min) → 40% B (20–22 min) → 90% B (20.1–22 min) → 10% B (22.1–26 min)
• Flow rate: 0.3 mL/min, column temperature: 60°C, injection volume: 6 µL, UV detection at 190–400 nm

Mass spectrometry conditions:

• LCMS-2050 single quadrupole with heated DUIS ion source (ESI/APCI hybrid)
• Negative ion mode, interface voltage -3.0 kV
• Scan range: m/z 550–2000
• Nebulizing gas: 3.0 L/min, drying gas: 5.0 L/min, heating gas: 7.0 L/min
• Desolvation temperature: 450°C, DL temperature: 200°C

Použitá instrumentace

• Nexera XS inert UHPLC system
• Shim-pack Scepter Claris C18-300 column
• LCMS-2050 single quadrupole mass spectrometer with DUIS source

Hlavní výsledky a diskuse

Chromatograms showed four distinct peaks in UV (260 nm) and TIC traces in the order n-10, n-3, n-1 and FLP. Mass spectra revealed multiply charged ions for each species, which were deconvoluted to yield molecular weights of 3552 (vs. theoretical 3553), 5986 (5987), 6776 (6776) and 7169 (7169) for n-10, n-3, n-1 and FLP, respectively. Mass errors were minimal, demonstrating high accuracy and reliability of the LCMS-2050 system.

Přínosy a praktické využití metody

The workflow enables rapid, sensitive profiling of oligonucleotide impurities with straightforward operation, making it suitable for routine quality control in pharmaceutical R&D and manufacturing environments. Users with limited mass spectrometry experience can obtain precise molecular weight information to confirm known impurities or flag unexpected variants.

Budoucí trendy a možnosti využití

Advanced impurity identification and sequence confirmation may be achieved by integrating quadrupole time-of-flight (Q-TOF) LC-MS systems and dedicated bioinformatics software. Future developments may focus on high-throughput automation, expanded modification mapping and real-time monitoring of synthesis and degradation pathways.

Závěr

The combination of inert UHPLC separation and the LCMS-2050 single quadrupole mass spectrometer offers a robust, user-friendly platform for accurate impurity analysis of oligonucleotide therapeutics. The method achieves baseline separation, precise mass determination and minimal mass errors, supporting effective quality control workflows.

Reference

1. Hattori T, Kato N, Nakazono J. Simple Analysis of Impurities in Oligonucleotide Therapeutics Using a Single Quadrupole Mass Spectrometer. Application News No. 01-00656-EN, Shimadzu Corporation, First Edition Nov. 2023.
2. Efficient Method Development of Oligonucleotides by Reversed-Phase Ion-Pair Chromatography. Application News No. 01-00558-EN, Shimadzu Corporation.
3. An Oligonucleotide Impurity Analysis Workflow Using LabSolutions Insight Biologics Software. Application News No. 01-00595A-EN, Shimadzu Corporation.