Reliable Purification of Full-Length Product and Enhanced Isolation and Detection of Impurities With Ready to Use XBridge Premier Oligonucleotide BEH™ C18 OBD™ Preparative Columns

Significance of the Topic

Synthetic oligonucleotide therapeutics are emerging precision medicines that require rigorous purification strategies to ensure safety and efficacy. Impurities such as truncated sequences or chemically modified variants must be isolated for toxicological evaluation, and any sample loss or inconsistent recovery can bias downstream studies. High-performance preparative chromatography with minimized non-specific adsorption is therefore essential for accurate impurity profiling.

Study Objectives and Overview

This application note compares traditional stainless-steel preparative columns to Waters XBridge Premier Oligonucleotide BEH C18 OBD columns equipped with MaxPeak High Performance Surfaces (HPS). The goals were to assess:

• Recovery and reproducibility of early-eluting impurities in preparative IP-RPLC
• Consistency of fraction collection across injections without prior surface passivation
• Scalability from analytical to preparative workflows using matched column chemistries

Methodology and Used Instrumentation

An ion-pair reversed-phase preparative HPLC method was employed to fractionate a crude 20-mer oligonucleotide into 2-minute windows (8.7–26.7 min). Two 10 mm × 150 mm C18 columns (traditional XBridge vs. MaxPeak HPS-coated XBridge Premier OBD) were tested under identical conditions (4.5 mL/min flow rate, 60 °C, 100 mM TEAA mobile phase). Collected fractions were dried, reconstituted, and analyzed on an ACQUITY Premier Oligonucleotide BEH C18 UPLC column (2.1 × 50 mm, 1.7 µm) with UV/MS detection.

Used Instrumentation:

• Preparative HPLC: Waters Arc Quaternary Solvent Manager-R pumps, 3767 Sample Manager, 2489 UV/Vis detector, FractionLynx software
• Analytical UPLC-UV/MS: ACQUITY UPLC I-Class PLUS, RDa detector, waters_connect software
• Columns: XBridge Oligonucleotide BEH C18 OBD Prep vs. XBridge Premier Oligonucleotide BEH C18 OBD Prep (MaxPeak HPS-coated)

Key Results and Discussion

The MaxPeak HPS-coated preparative column delivered more consistent retention profiles from the first injection, eliminating the need for sacrificial conditioning injections. Early-eluting impurities (8.7–14.7 min window) were detected only with the HPS column and exhibited over 100 % higher recovered peak area versus the stainless-steel column. Analytical UV/MS of collected fractions revealed unique mass values recovered exclusively from the HPS column, confirming mitigation of metal-surface adsorption. Overall, the HPS hardware improved reproducibility and impurity detection.

Benefits and Practical Applications

• Enhanced recovery and consistency for impurity profiling without extended conditioning
• Predictable scale-up from analytical ACQUITY Premier columns to preparative XBridge Premier OBD columns
• Improved column longevity and corrosion resistance via organosilica-protected surfaces

Future Trends and Applications

Oligonucleotide purification workflows will benefit from further integration of inert surface technologies and automated fraction collection. Emerging preparative methods may expand to larger oligonucleotides, modified backbones, and conjugated constructs. Combining HPS hardware with novel stationary phases and advanced mobile-phase chemistries can further reduce non-specific interactions and enhance throughput for next-generation therapeutics.

Conclusion

MaxPeak High Performance Surface-coated preparative columns offer a reliable solution for full-length oligonucleotide purification and impurity isolation. By eliminating metal-surface adsorption, these columns deliver consistent recovery of critical early-eluting components, streamline scale-up, and improve confidence in toxicity studies and QA/QC workflows.

References

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