HILIC Analysis of CRISPR-Cas9 Gene Editing Tools on a Low Adsorption LC Flow Path

Importance of the Topic

Oligonucleotide-based therapeutics such as mRNA and CRISPR-Cas9 components require precise analytical methods to characterize sequence, modifications, and quality attributes. Hydrophilic interaction liquid chromatography (HILIC) offers a sustainable alternative to ion-pairing reversed-phase LC for separating highly polar oligonucleotides. However, adsorption to stainless-steel surfaces in the flow path can impair sensitivity and reproducibility. This study addresses these challenges by comparing conventional and deactivated stainless-steel HILIC columns for gene editing RNA analysis.

Study Objectives and Overview

• Compare adsorption and performance of a standard stainless-steel HILIC-Z column with a chemically deactivated HILIC-Z column in the analysis of sgRNA and Cas9 mRNA digests
• Evaluate the impact of terminal phosphate chemistry on adsorption using RNase T1 and RNase 4 digestions, and subsequent CNP and CIP treatments

Methodology

• RNA Samples: Synthetic 50 nt RNA, 104 nt sgRNA, and 4522 nt Cas9 mRNA
• Enzymatic Digestion: RNase T1 for guanosine-specific cleavage; RNase 4 for uridine-specific cleavage
• Terminal Phosphate Treatments: 2',3'-cyclic phosphodiesterase (CNP) to convert cyclic to linear 3' phosphate; calf intestinal alkaline phosphatase (CIP) to remove linear phosphate groups
• HILIC Conditions: Zwitterionic stationary phase; acetonitrile–water mobile phases with 20 mM ammonium acetate; gradients optimized for small and large RNA fragments

Instrumentation

• Agilent 1290 Infinity III Bio LC System with low-adsorption flow path
• Zwitterionic HILIC-Z columns: standard stainless-steel and Ultra-Inert deactivated (2.1 × 150 mm, 2.7 µm)
• DAD detection at 260 nm with 4-nm bandwidth

Main Results and Discussion

• Conventional stainless-steel HILIC-Z required extensive conditioning (25 injections) to stabilize performance; deactivated column was stable from the first injection
• Standard column exhibited lower peak areas, increased peak tailing, and variable recoveries; deactivated column delivered sharper peaks and higher intensities across all RNA samples
• Fragments with linear 3' phosphate termini showed the greatest adsorption; cyclic or 3'-hydroxyl termini reduced nonspecific interactions
• Additional CIP treatment mitigated adsorption on the standard column but did not fully match deactivated column performance

Benefits and Practical Applications

• Improved sensitivity and reproducibility in RNA mapping workflows for mRNA therapeutics and CRISPR components
• Reduced need for mobile phase modifiers or repeated passivation steps, simplifying method development and maintenance
• Enhanced peak capacity and MS compatibility due to minimized metal-induced adsorption

Future Trends and Applications

• Implementation of inert or alternative metal alloys (e.g., MP35N) in LC hardware to further reduce adsorption
• Integration with high-resolution MS for comprehensive oligonucleotide sequencing and variant analysis
• Development of standardized low-adsorption methods for large-scale QC in biopharma and gene therapy development

Conclusion

Deactivated stainless-steel HILIC columns substantially improve the analysis of gene editing and mRNA therapeutics by reducing nonspecific adsorption and enhancing chromatographic performance. Terminal phosphate chemistry critically influences adsorption behavior, and enzymatic treatment can partly mitigate interactions on standard hardware. Adoption of inert column technologies streamlines RNA mapping workflows, providing reliable tools for quality control and advanced characterization in biopharmaceutical research.

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