# Mechanistic Insights Into the Retention and Separation Mechanism of Poly(Ethylene Glycol)‐Modified Short Oligonucleotides in Anion‐Exchange Chromatography

## TL;DR

This study explores how adding PEG to short DNA molecules affects their purification through anion-exchange chromatography, revealing how PEG changes their behavior without altering their charge.

## Contribution

The study provides mechanistic insights into how PEGylation affects the chromatographic behavior of oligonucleotides through steric and hydration effects.

## Key findings

- PEGylation shifts elution to lower salt concentrations, especially for shorter oligomers and mid-position modifications.
- PEGylation reduces the ion-exchange equilibrium constant, indicating altered local interaction environments.
- PEGylation weakens the retention-dependent decrease in plate height, suggesting modified mass-transfer characteristics.

## Abstract

Purification is a critical step in the development of synthetic short DNA for pharmaceutical applications. PEGylation enhances stability and pharmacokinetics but introduces steric and hydration effects that affect chromatographic behavior without altering the nominal DNA charge. This study investigated the retention and mass‐transfer behavior of thymine‐based poly(dT) oligomers (9–95 bases) modified with poly(ethylene glycol) (PEG) using anion‐exchange chromatography with a mechanistic ion‐exchange framework. By systematically varying PEG molecular weight and modification site, we examined how PEG‐induced changes in molecular size and local interaction environment are reflected in mechanistic retention parameters. PEGylation shifted elution to lower salt concentrations, with more pronounced effects observed for shorter oligomers and mid‐position modifications. Model‐based analysis revealed that the effective number of binding sites remained unchanged after PEG modification, indicating preserved charge‐based binding stoichiometry. In contrast, PEGylation reduced the ion‐exchange equilibrium constant, reflecting changes in the local interaction environment. HETP analysis showed that unmodified poly(dT) exhibited a strong retention‐dependent decrease in plate height, whereas this dependence was weaker for PEGylated DNA, suggesting that PEGylation modifies intraparticle mass‐transfer characteristics through combined steric hindrance and charge‐shielding effects. These results provide mechanistic insights into the chromatographic behavior of PEGylated oligonucleotides and a rational basis for optimizing their purification.

Mechanistic analysis revealed unchanged binding‐site stoichiometry (B) and altered interaction strength (K
e) for PEGylated DNA.