Ion-Pair Reversed-Phase LC Method Development for sgRNA Impurity Analysis Using Biozen Oligo Column

By
  • Lara Nercessian
  • Claire Butré
  • Damien Mouvet
  • Jürgen Niesser (Phenomenex)
  • Arnaud Delobel
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Ion-Pair RP‑LC Method for sgRNA Impurity Analysis

Single guide RNAs (sgRNAs) require precise impurity profiling, particularly for truncated species such as N‑1 generated during synthesis. Long RNA analysis is challenging due to size and secondary structure, making high‑resolution separation essential. This work applies an Analytical Quality by Design (AQbD) strategy, including risk assessment and DoE, to optimise ion‑pair reversed‑phase LC conditions. The final method, developed using the Biozen Oligo column, enables reliable routine sgRNA purity determination.

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Key Highlights

  • Focus on quantifying closely related impurities, including N‑1 truncated species.
  • IP‑RP LC chosen for high‑resolution separation of long RNA oligonucleotides.
  • AQbD and DoE used to identify and optimise critical chromatographic parameters.
  • Final method provides reproducible performance for routine sgRNA purity assessment.

How does this support your analytical workflow?

  • Improves separation and quantification of sgRNA impurities.
  • Enhances method understanding and robustness through AQbD.
  • Supports consistent routine analysis across purification stages.
  • Provides high‑resolution LC conditions tailored to long RNA structures.

Applications

  • sgRNA purity testing
  • Truncated impurity analysis (e.g., N‑1)
  • High‑resolution RNA chromatography in development and QC

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