Maximize Your Resources and Simplify Scale-up with AdvanceBio Oligonucleotide Columns

Significance of the topic

Ion pair reversed-phase chromatography is a cornerstone technique for the analysis and purification of synthetic oligonucleotides in biopharmaceutical research and manufacturing. High-resolution separation of full-length products from closely related impurities is critical for accurate characterization, quality control, and scale-up in therapeutic oligonucleotide production.

Objectives and overview of the study

The study compares the performance of Agilent AdvanceBio Oligonucleotide superficially porous columns (4 µm particle size) against Waters XBridge BEH C18 OBD fully porous columns (5 µm) at both analytical and semipreparative scales. The goal is to assess resolution, yield, purity, and sample capacity in the purification of a 22-mer 2’-O-methylated RNA oligonucleotide.

Methodology and instrumentation

Two gradient methods were evaluated:

• Method 1 – constant ion-pair reagent concentration with eluent B containing 90 % acetonitrile for efficient column cleanup.
• Method 2 – varying ion-pair reagent concentration with eluent B composed of 80 % aqueous TEAA and 20 % acetonitrile.

The same crude oligonucleotide sample (20 mg/mL) was injected at analytical (4.6 × 150 mm) and semipreparative scales (10 × 150 mm), loading 3.2 mg per run. Gradient profiles were adapted from Tables 1–3 to maintain comparable conditions across columns.

Used instrumentation

• Agilent 1290 Infinity II high-speed pump
• Agilent 1290 Infinity II multisampler with thermostat
• Agilent 1290 Infinity II multicolumn thermostat set at 60 °C
• Agilent 1290 Infinity II diode array detector (260 nm)
• Agilent 1260 Infinity bio-inert analytical-scale fraction collector

Main results and discussion

• Analytical scale separations on the AdvanceBio column exhibited sharper peaks and higher resolution of closely eluting impurities compared to the Waters BEH C18 column.
• Method 1 provided more rigorous column washing and minimized carryover, yielding enhanced reproducibility over Method 2.
• At the semipreparative scale, the AdvanceBio column delivered greater sensitivity, higher purity fractions, and superior product recovery, with no significant compromise in loading capacity.
• Fraction analysis under Method 2 further confirmed a larger number of high-purity fractions obtained with the superficially porous column.

Benefits and practical applications of the method

Adopting superficially porous AdvanceBio columns enables biopharma laboratories to:

• Achieve higher-resolution purification of synthetic oligonucleotides.
• Increase yield and purity without reducing sample throughput.
• Use a fully scalable platform from analytical to preparative dimensions, reducing method transfer risks.

Future trends and potential applications

Ongoing developments in superficially porous particle chemistries and ion-pair reagents promise further improvements in separation efficiency. Integration with mass spectrometry and automated fraction collection workflows will accelerate process development for novel oligonucleotide therapeutics. Scaling calculators and software tools will streamline gradient optimization and transfer to production-scale columns.

Conclusion

The Agilent AdvanceBio Oligonucleotide superficially porous column chemistry on the 1290 Infinity II platform outperforms conventional fully porous C18 media for oligonucleotide purification. It offers enhanced resolution, higher sensitivity, scalable dimensions, and reliable cleanup for both analytical and semipreparative workflows, making it an ideal choice for demanding biopharma applications.

Reference

• Agilent publication 5994-7478EN
• Agilent publication 5994-4877EN