Poster

Solvent-Free Purification Of D84-mer Oligonucleotide Using Capto™ PlasmidSelect Resin

Source: Cytiva
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The growing demand for long oligonucleotides in gene therapies, especially with tools like CRISPR-Cas9, necessitates advanced purification techniques to achieve high-purity products. Traditional methods, such as anion exchange (AIEX) and reversed-phase chromatography (RPC), face limitations in resolving longer oligos due to size-related coupling inefficiencies and high failure sequence content.

This study introduces a scalable, solvent-free downstream process for purifying longer oligonucleotides, employing aqueous buffer systems and two key resins: Capto™ PlasmidSelect and Capto Q ImpRes. The process begins with separating full-length, tritylated oligos from failure sequences using Capto PlasmidSelect resin, achieving 81% purity. Next, detritylation is conducted in batch mode with acetic acid, reaching 90% detritylated full-length oligo purity within 45 minutes.

The final polishing step employs Capto Q ImpRes resin for anion exchange purification, effectively removing residual impurities and shortmer sequences. This step results in a final purity of 98% for the detritylated full-length oligo. The entire process yields an overall recovery of 69% from initial synthesis to final purification, with consistent results scalable using systems like ÄKTA pilot™ 600 and ÄKTA ready™.

This solvent-free, high-purity process overcomes traditional challenges, ensuring efficiency, simplicity, and scalability for the production of long oligonucleotides. The methods described here enable researchers to meet the increasing demand for high-quality oligos, essential for advancing gene therapy applications.

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