The rapid advancement of oligonucleotide therapeutics is pushing the boundaries of drug design, precision, and delivery. As researchers innovate new modalities and chemistries—from antisense oligonucleotides (ASOs) to guide RNAs (gRNAs) and small interfering RNAs (siRNAs)—the demand for efficient and scalable purification technologies has never been greater. DuPont is meeting this challenge head-on with its robust portfolio of AmberChrom™ XT chromatography resins, designed to optimize purification processes across oligonucleotide classes.

 

The Role of RP-HPLC in Oligonucleotide Purification

Reverse-phase high-performance liquid chromatography (RP-HPLC) is a cornerstone of oligonucleotide purification. DuPont's AmberChrom™ XT20 and XT30 polymeric resins, known for their mechanical strength and chemical resistance, deliver high-resolution separations even in challenging conditions.

In recent tests, purification was performed on pre-packed AmberChrom™ Profile™ columns using either ion-exchange (IEX) or ion-pairing reverse phase (IP-RP) chromatography. This flexible setup enabled the efficient processing of oligonucleotide loads ranging from 10 to 20 mg per mL of resin.

 

High Purity in DMT-On ASO Purification

Phosphoramidite-based synthesis commonly yields DMT-On products—oligonucleotides capped with dimethoxytrityl (DMT) protecting groups. These groups engage strongly with the hydrophobic AmberChrom™ resins, producing a clean separation between the full-length product (FLP) and shorter hydrophilic impurities.

In a study using AmberChrom™ XT30 resin to purify a 20-mer DMT-On ASO, a sharp FLP peak was observed. The pooled product achieved high purity and concentration, starting from a feed purity of approximately 83%. This clear separation supports downstream applications requiring high-quality therapeutics.

 

Meeting the Challenge of DMT-Off and Complex Modalities

AmberChrom™ resins also excel in purifying DMT-Off products and more structurally complex oligonucleotides. In the DMT-Off ASO example, using ion-pairing agents helped mediate the interaction between the negatively charged oligonucleotide and the hydrophobic resin, maintaining high yield and purity (93% purity and 86% yield).

Further trials on longer and more difficult feeds—including a 91-mer sgRNA and a monomethoxytrityl (MMT)-On siRNA—demonstrated the flexibility of the platform. The XT20 resin delivered >96% purity in a one-step purification of the sgRNA with just 30% crude purity, while the MMT-On siRNA purification achieved 91% purity with a 90% yield.

 

Summary of Results

 

 

Flexible Solutions for Research and Manufacturing

DuPont offers both pre-packed HPLC columns and bulk resin formats, enabling seamless scale-up from research labs to commercial production. With its chemically inert and physically durable resin platform, DuPont provides an ideal solution for oligonucleotide purification challenges across modalities.

To explore DuPont's AmberChrom™ XT chromatography resins or to request samples, visit: www.dupontwatersolutions.com/life-sciences

 

READ MORE HERE