The Secret to Pure Oligos: Why Anion Exchange Chromatography Rules at Scale
Oligonucleotide therapeutics are on the rise. More and more short DNA and RNA drugs are entering clinical development and even gaining FDA approval. But this progress comes with a challenge: how do you purify these molecules reliably and at scale?
How do you get from crude synthesis to a highly pure therapeutic candidate?
👉 The answer is anion exchange chromatography (AEX).
Up to now, we’ve highlighted the amazing chemistry behind oligonucleotide analysis, and in a recent post we already broke down the basics of AEX (Oligos Made Easy Part 2: Secrets of AEX Analysis) In this blog post, we're going to focus on large-scale purification by AEX and the challenges involved. We'll exemplary show you how to purify an oligo (poly dT) efficiently on a litre scale, – with excellent yield and purity.
Why purification matters
After chemical synthesis, crude oligonucleotides contain a variety of by-products and impurities. For clinical programs or pharmaceutical use, though, only highly pure material is acceptable. There are several HPLC techniques available, but not all of them are suitable for scale-up and fail when it's time to move to production. What’s needed is a purification strategy that scales up effortlessly.
Why Anion Exchange Chromatography stands out
AEX is a proven solution for oligonucleotide purification. Here’s why:
💡 High loading capacity – process large sample volumes with ease
💡 Outstanding separation efficiency – achieve clinical-grade purity
💡 Scalable and robust – from R&D to full GMP manufacturing
💡 Eco-friendlier – aqueous buffers instead of harsh solvents
Real-world success: Oligo(dT) at scale
The goal: purify an oligo (poly dT ), a versatile molecule that is used for mRNA isolation or as a primer in cDNA synthesis, on a liter scale. So, to get the samples ready, we had to cleave them from the solid support, remove the protecting groups with ammonia and neutralize the sample before the chromatography separation. The process ran smoothly on the AZURA® Prep HPLC system, which is designed for high-throughput purification with precise gradient control, UV monitoring and automated fraction collection. We used a strong anion exchange resin with gradient elution with a phosphate buffer and sodium bromide as the counterion. Sodium bromide is used to make stainless steel devices last longer, as chloride is more aggressive. Typically for best resolution 10 to 15 µm AEX particles are used.
AZURA preparative HPLC system
The numbers tell the story.
🚀Before purification: 89% purity
✅ After purification with AEX: 95% purity, 90% yield
Chromatogram of oligonucleotide purification from 1 l sample
Analytical chromatogram of purified and pooled oligo (dT) after AEX
For more information and details about the method, please check out our AppNote:
Oligonucleotide Purification via Ion Exchange
Obviously, there are always pros and cons, and AEX is no exception. It does have its challenges:
❓ Phosphodiester versus Phosphorothioate Separation: Not sufficiently achievable; long or modified sequences reduce separation quality.
❓Aggregation: Secondary structures can form aggregates, lowering purification efficiency.
❓ Endotoxins: Aqueous conditions increase risk of process-related endotoxins.
The bottom line
Manufacturing oligonucleotide-based drugs requires scalable and reliable purification strategies and a robust process for research, clinical, and GMP production. Anion exchange chromatography proves to be a powerful tool.Oligonucleotide therapeutics are too valuable to risk with inefficient purification. Anion exchange chromatography delivers the purity, yield, and scalability that today’s biopharma industry demands.
Whether you’re scaling up for clinical trials or optimizing GMP production, AEX with AZURA® Prep systems offers a clear path to success.
👉 Ready to unlock next-level purification for your oligonucleotides?
Get in touch with us today — let’s turn your oligos into tomorrow’s therapies.
For further information on this topic, please contact our author: krop@knauer.net
If you're planning to set up or optimize your oligonucleotide workflow, feel free to contact us at sales@knauer.net. Stay tuned for more exciting insights into the Oligonucleotide world in our “Oligos Made Easy” series.
