Advancing Quality Control for CRISPR-Cas9 Systems: Separation of CRISPR Components using Ion Exchange Chromatography

Recently, the first CRISPR/Cas9 edited cell therapy was approved for treatment of sickle cell anemia, with other CRISPR-based therapies in the pipeline. However, despite the growing market for CRISPR-based therapeutics, technologies for all-in-one quality control analysis of CRISPR components and ribonucleoprotein (RNP) complexes are not well-established. In order to ensure safe and effective CRISPR drug substances, thorough quality control assessment of CRISPR reagents and characterization of RNP complex formation is needed. Current methods for characterization of CRISPR/Cas components are limited and do not typically allow for simultaneous characterization of apo-Cas, apo-sgRNA, and RNP species. Here, we exploit the differing charges of apo-Cas, apo-sgRNA, and RNP in order to achieve separation of CRISPR components by ion exchange (IEX) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. In this work, we have applied a corrosion resistant instrument equipped with UV detection and low adsorption flow path components. Anion exchange and cation exchange columns were coupled, allowing for simultaneous retention of Cas9 on the cation exchange column and sgRNA on the anion exchange column. A number of practical considerations for the analysis of CRISPR components by IEX chromatography were determined during method development. Considerations include UV detector flow cell/analyte compatibility, sample diluent, system suitability, choice of mobile phase buffer and ionic strength, and the impact of sgRNA sample heterogeneity. Adequately addressing these considerations will remain of critical importance as more CRISPR drug substances enter the drug development pipeline.