Rapid monitoring of the purification process of oligonucleotide impurities using a benchtop MALDI-TOF MS system

Significance of the Topic

Oligonucleotide therapeutics represent a rapidly expanding class of drug candidates with substantial clinical and commercial promise. Their manufacture by solid-phase phosphoramidite chemistry inherently generates a series of sequence-related impurities (shortmers, phosphorothioate adducts), which must be removed to meet stringent purity criteria. Traditional LC-MS techniques provide detailed impurity profiles but are time-consuming and less amenable to in-process monitoring. A benchtop MALDI-TOF MS approach offers a rapid, reproducible alternative suitable for real-time control of oligonucleotide purification processes.

Objectives and Study Overview

This study aimed to develop and validate a fast, robust MALDI-TOF MS method for profiling oligonucleotide impurities during both batch ion-exchange purification and continuous multi-column solvent gradient purification (MCSGP). Key objectives included identifying optimal sample preparation protocols, evaluating reproducibility and quantitation performance, and demonstrating applicability to multiple purification cycles of a synthetic 18-mer antisense oligonucleotide model.

Methodology

A chemically synthesized 18-mer model MOE-ASO containing fully phosphorothioated internucleotide linkages was used. Crude and purified samples were processed by:

• Dried-droplet two-layer matrix application using 2,4,6-trihydroxyacetophenone (THAP) in ethanol.
• Deposition of alternating matrix and sample layers (0.5 µL each) on the MALDI plate to enhance crystallization homogeneity.
• Acquisition of multiple replicates (eight spectra per fraction) to support principal component analysis (PCA) and assess reproducibility.

Instrumentation

• Benchtop MALDI-8030 linear MALDI-TOF mass spectrometer operated in dual-polarity mode at 25 °C.
• LCMS-9050 Q-TOF MS for orthogonal confirmation of impurity assignments.
• Ion-exchange chromatography systems:
    • Batch purification: BioPro IEX SmartSep Q (20 µm, 100 × 5 mm, YMC) with 20 mM Tris-HCl/3 M NaCl gradient.
    • Continuous chromatography (MCSGP): Contichrom CUBE system equipped with two identical columns, internal recycling of side fractions, inline dilution.

Results and Discussion

• The THAP two-layer matrix method significantly improved spot homogeneity, resulting in enhanced signal reproducibility and relative quantitation of shortmer impurities (N-1 to N-15).
• PCA of MALDI spectra distinguished crude from purified samples and confirmed consistent impurity removal across ten continuous chromatography cycles.
• Batch purification reached ≥ 93 % purity (confirmed by RP-IP UV analysis), with efficient elimination of small-chain impurities.
• MCSGP provided stable impurity profiles over multiple cycles, demonstrating high yield and purity through counter-current solvent gradient purification and internal recycling.

Benefits and Practical Applications

The MALDI-TOF MS method delivers:

• Rapid analysis (< minutes per sample) enabling in-process monitoring and timely decision-making.
• High reproducibility through optimized sample preparation, reducing dependence on extensive LC-MS runs.
• Compatibility with both batch and continuous chromatographic workflows for quality control in oligonucleotide manufacturing.

Future Trends and Applications

Emerging developments may include:

• Integration of MALDI-TOF MS with automated sample handling for high-throughput in-line monitoring.
• Advanced software algorithms for improved quantitative accuracy and impurity identification.
• Extension to other oligonucleotide chemistries (siRNA, miRNA, aptamers) and complex bioconjugates.
• Real-time coupling with MCSGP systems for closed-loop process optimization.

Conclusion

The optimized benchtop MALDI-TOF MS protocol offers a fast, reliable tool for monitoring oligonucleotide purification, complementing traditional LC-MS and enabling enhanced process control. Its application to both batch and continuous chromatography underscores its versatility for quality assurance in oligonucleotide therapeutic production.

References

No formal literature list provided in the source document.