Oligonucleotides
In AIEX or IP-RP modes. A continuous twin-column process that recovers full-length ASOs, siRNA & GalNAc conjugates batch chromatography discards—fewer side-fraction losses, fewer in-process controls.
Published case results: +50% yield (60→90%+) on 20-mer | +38.8 pp yield (52.7→91.5%) on GalNAc conjugate | +13 pp yield (80→93%) on conjugated siRNA AIEX
Sources: YMC ChromaCon Oligo Application Note; Weldon et al., J. Chromatogr. A 2022; Weldon et al., Org. Process Res. Dev. 2025
HPLC purification of oligonucleotides can use anion exchange (AIEX), ion-pair reversed-phase (IP-RP), or reversed-phase (RP) chromatography to separate full-length product from closely related synthesis impurities. In conventional batch purification, narrow product collection windows can protect purity but leave valuable full-length material in side-fractions that may require re-chromatography.
MCSGP addresses this limitation by continuously recycling impure side-fractions between two columns. The process uses the same stationary phases, buffers, and solvents as the existing batch method, allowing established oligonucleotide chromatography conditions to serve as the starting point for continuous process development.
Oligonucleotide therapeutics are surging — with 24 FDA-approved drugs, 200+ candidates in clinical trials, and blockbuster siRNAs driving demand to multi-kilogram scales. Purification by anion exchange (AIEX) or ion-pair reversed-phase (IP-RP) chromatography is the critical downstream bottleneck.
Batch chromatography forces a painful trade-off: high purity or high yield — not both. Oligonucleotide crude feeds contain hundreds of closely related impurities — N-1 shortmers, N+1 longmers, phosphodiester (PO) failures, depurination products, and phosphorothioate diastereomers — many differing by as little as 1 Da in a 7,000+ Da molecule. Achieving target purity requires a narrow center-cut, discarding valuable full-length product in side-fractions. Recovering that product means costly re-chromatography — with all the associated solvent, buffer, and QC burden.
For high-volume APIs demanded by cholesterol-lowering siRNAs, hepatitis B ASOs, and GalNAc-conjugated therapies, this inefficiency is unsustainable.
MCSGP (Multi-column Countercurrent Solvent Gradient Purification) eliminates the purity-yield trade-off in oligonucleotide chromatography. Two identical columns continuously recycle impure side-fractions — recovering full-length oligonucleotide product that batch processes discard. It uses the same AIEX, IP-RP, and RP resins, buffers, and solvents as your existing batch oligonucleotide method.
AutoPeak® dynamic process control monitors oligonucleotide elution profiles in real-time via UV and conductivity, automatically adjusting collection windows to maintain consistent output quality — enabling robust, unattended 24/7 oligonucleotide purification. Without AutoPeak® control, studies have shown yields can drop to as low as 20% under process variability — AutoPeak® eliminates this risk.
Same chemistry. Better results. MCSGP works with the AIEX, IP-RP, and RP stationary phases and mobile phases already used in your oligonucleotide batch process — your existing resin and eluent conditions are the starting point for MCSGP development. No change to your chromatographic chemistry is needed.
+13–74%
Yield Increase
90%+
Typical MCSGP Yield
87% ↑
Throughput Increase
Zero
Re-chromatography Runs
42.5% ↓
Synthesis Scale Reduction
2×
Productivity Gain
| Parameter | Batch Oligonucleotide Chromatography | MCSGP with AutoPeak® |
|---|---|---|
| Purity-Yield Trade-off | Inherent — narrow cuts sacrifice yield | Eliminated — high purity AND yield simultaneously |
| Product Yield | 50–80% | 90–95% (+13–74% absolute) |
| Re-chromatography | Required for side-fractions | Eliminated — internal recycling |
| Buffer/Solvent Consumption | High | Comparable or reduced per gram product |
| IPC/QC Burden | Testing every side-fraction, every batch | Massively reduced — one sample per MCSGP cycle |
| Throughput | Limited by re-chromatography cycles | Up to 87% higher (continuous 24/7) |
| Process Control | Manual monitoring | Automated (AutoPeak® UV-based PAT) |
| Robustness | Sensitive to drift and crude feed variability | AutoPeak® dynamically compensates |
| Operator Requirement | Requires supervision | Designed for unsupervised operation |
| Upstream Synthesis Impact | Fixed synthesis scale | Higher yield enables up to 42.5% synthesis downscaling |
| Stationary Phase / Eluents | Standard AIEX / IP-RP / RP | Same — no change required |
ASOs with phosphorothioate (PS) backbones generate complex diastereomer mixtures and broad chromatographic peaks that make batch purification especially challenging. MCSGP continuously recovers full-length product from overlapping PS impurity shoulders — delivering yields of 90%+ at target purity with AIEX or IP-RP chromatography.
siRNA sense and antisense strands require high-purity individual strand purification before duplex annealing. Published MCSGP data on a conjugated siRNA sense strand showed yield improvement from 80% to 93% with AIEX, with 87% higher throughput — critical for the multi-kilogram scales demanded by cholesterol-lowering and hepatitis therapies.
GalNAc conjugation enables liver-targeted delivery and is used in the latest FDA-approved oligonucleotide therapies. The bulky GalNAc cluster adds purification complexity. MCSGP with RP-HPLC demonstrated +73.6% relative yield improvement for a GalNAc-DNA-LNA gapmer, enabling 42.5% synthesis downscaling.
Combine orthogonal oligonucleotide purification steps — AIEX for charge-based separation and IP-RP for hydrophobicity-based separation — in a single automated 2D chromatography workflow. RP-AEX sequence eliminates the need for in-line dilution and produces the sodium counterion form required for formulation.
ICH-required oligonucleotide impurity characterization (N-1, N+1, PO failures, depurination products) typically takes weeks of repetitive analytical runs. N-Rich automated on-column enrichment isolates milligram quantities of target oligonucleotide impurities up to 80× faster.
Ready to Transform Your Oligonucleotide Purification?
This application note presents a systematic approach for optimization of MCSGP with regards to the performance parameters Yield, Productivity and Eluent Consumption, and gives recommendations on the priority of the parameters.
This application note provides a simple step-bystep flowchart template to guide the development of batch gradient conditions that result in optimal input data for the MCSGP wizard.
This application note demonstrates a significant improvement in yield and productivity by converting an optimized batch chromatography method to Multi-column Counter-current Solvent Gradient Purification (MCSGP) for the manufacturing of a therapeutic oligonucleotide.
First publication presenting a Process Characterization and Process Performance Qualification approach for regulatory-compliant Process Validation of MCSGP, demonstrated for Bivalirudin purification.
An MCSGP process is developed for purification of a sugar-conjugated siRNA therapeutic using anion exchange chromatography, demonstrating yield improvements from 80% to 93% compared to single-column chromatography.
