Technique Versatile in Evaluating Quality Attributes of CRISPR-Based Therapeutics
CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, is a revolutionary gene-editing tool that has sparked excitement since its discovery in 2011. Since then, its contributions to agriculture, the discovery of emerging pathogens, and disease diagnostics and therapeutics have been immense.
Recently, the first CRISPR edited cell therapy-based therapeutic was approved for treatment of sickle cell anemia, with other CRISPR-based therapies in the pipeline. Despite the growing market for CRISPR-based therapeutics, technologies for comprehensive quality control analysis of CRISPR components and ribonucleoprotein (RNP) complexes are not well-established. To ensure safe and effective CRISPR drug substances, thorough quality control assessment of CRISPR reagents and characterization of RNP formation is needed. Current methods for characterization of CRISPR/Cas components are limited and do not typically allow for simultaneous characterization of proteins that include the apo-Cas, apo-sgRNA, and RNP species. In collaboration with researchers from Waters Corporation, researchers from MRIGlobal have developed a solution.
Innovation to Ensure Safety and Efficacy
While Cas9 carries a positive charge, sgRNA is highly negatively charged, and the RNP complex exhibits an intermediate charge state. By leveraging the different charges of these proteins, we have achieved separations of CRISPR components by ion exchange (IEX) high-performance liquid chromatography (HPLC) paired with ultraviolet (UV) detection. In this work, we have used a chromatography system with high performance surfaces that mitigate loss of metal-sensitive analytes 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. By coupling, the Cas9, sgRNA, and RNP complexes were separated in a single run, allowing for the stoichiometry of the overall mixture to be monitored. In addition, charge variants for Cas9 and sgRNA single components were characterized.
Benefits of this Method
The use of coupled cation/anion exchange chromatography can probe charge states of the components in a single method, revealing information about the components not seen in use of other methods. This new method also allows for stoichiometric evaluation of the RNP formation from the individual components. Separately, cation exchange and anion exchange can be used to evaluate CRISPR components from a quality perspective. When coupled, the two column chemistries can serve as an all-in-one method for an analysis of CRISPR components and RNPs that gives valuable insights into RNP complexation efficiency and optimization of CRISPR reagent ratios.
Further, our work here demonstrates the capability of coupled cation/anion exchange chromatography to serve as an all-in-one method to evaluate charge heterogeneity in individual CRISPR components and perform stoichiometric analysis of RNP formation. This will be a versatile technique for various applications, including evaluation of critical quality attributes of CRISPR-based therapeutics and optimization of CRISPR reagents.
Read the full MRIGlobal paper at “Coupled IEX Chromatography for Stoichiometric Analysis of CRISPR Ribonucleoprotein Complexes” and more about “CRISPR Gene Editing” on the MRIGlobal blog. You can also read the Waters Corporation paper at “Coupled IEX Chromatography for Stoichiometric Analysis of CRISPR Ribonucleoprotein Complexes.”
