SEC Column Coatings for CRISPR Molecule Peak Resolution
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Solution Overview
Problem
Size exclusion chromatography (SEC) methods face challenges in separating CRISPR-related molecules due to non-specific secondary interactions with stationary phase materials and metallic hardware, leading to peak broadening, tailing, and loss of resolution, particularly for biomolecules like Cas proteins, which impede accurate quantitation and recovery.
Innovation Solution
The use of a chromatography system with metallic flow path components coated with an organosilane layer and a stationary phase material modified with hydroxy-terminated polyethylene glycol (PEG) to reduce secondary interactions, allowing for improved separation and recovery of CRISPR-related molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional stainless-steel chromatography hardware and conventional stationary phase materials are used, then the system is simple and cost-effective, but non-specific secondary interactions occur between CRISPR molecules and the hardware/surfaces, causing peak broadening, tailing, and loss of resolution
Solution Approach 1:
An organosilane coating layer is introduced as an intermediary between the stainless-steel hardware and the CRISPR molecules. This coating layer acts as a mediator that prevents direct contact between the molecules and the metallic surfaces, thereby eliminating non-specific secondary interactions while maintaining the structural integrity of the hardware. The coating serves as a protective barrier that allows the hardware to retain its mechanical strength without adversely interacting with the analytes.
Solution Approach 2:
The surface chemistry of the hardware is changed by applying an organosilane coating. This parameter change transforms the surface properties from metallic and potentially interactive to chemically modified and inert. The coating alters the surface energy, charge distribution, and chemical composition of the hardware surfaces, preventing the formation of non-specific interactions with CRISPR molecules while maintaining the physical structure of the chromatography system.
2Manufacturing precision
If extensive mobile phase optimization is performed to reduce secondary interactions, then separation quality improves, but the process becomes tedious and time-consuming
Solution Approach 1:
The organosilane coating serves as a permanent intermediary that continuously prevents non-specific interactions during the separation process. By establishing this protective barrier beforehand, the need for extensive mobile phase optimization to compensate for secondary interactions is eliminated. The coating provides a stable, reproducible surface that maintains consistent separation quality without requiring tedious method development.
Solution Approach 2:
The organosilane coating is applied to the hardware surfaces before the chromatography experiment begins. This preliminary action prepares the surfaces in advance to prevent non-specific interactions during the actual separation process. By pre-establishing the protective coating, the method development time is reduced because the separation quality is already optimized by the coating rather than requiring extensive mobile phase tuning.
3Adaptability or versatility
If CRISPR molecules with basic residues and acidic domains are analyzed, then the full range of CRISPR protein functionality is captured, but strong electronegativity causes adsorptive losses from metallic column hardware
Solution Approach 1:
The organosilane coating acts as an intermediary layer between the CRISPR molecules and the metallic hardware. This coating prevents direct electrostatic and adsorptive interactions between the electronegative acidic domains of the proteins and the metallic surfaces. The coating maintains the full range of protein functionality for analysis while preventing analyte loss through adsorption to the hardware.
Solution Approach 2:
The surface properties of the hardware are changed by the organosilane coating, which alters the electrical and chemical parameters of the hardware surfaces. This parameter change reduces the electronegativity and adsorptive properties of the metallic surfaces, preventing binding of CRISPR molecules with acidic domains. The coating enables comprehensive analysis of CRISPR protein functionality while maintaining high analyte recovery.
4Ease of manufacture
If fusion protein domains or affinity tags are present on CRISPR proteins, then the proteins can be enriched through nickel affinity procedures, but recovery is compounded by interactions with column hardware
Solution Approach 1:
The organosilane coating serves as an intermediary that prevents non-specific interactions between CRISPR proteins with affinity tags and the metallic column hardware. This coating allows the proteins to maintain their enrichment capability through nickel affinity procedures while preventing additional losses from hardware interactions. The coating ensures that only specific affinity-based binding occurs, not non-specific adsorption.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables rapid and efficient chromatographic monitoring of Cas proteins, ensuring stability and purity, with enhanced peak shape, resolution, and reproducibility, facilitating the assessment of CRISPR-associated protein monomers and associated RNA molecules.
Implementation Method 1
Secondary interactions can be especially problematic with biomolecules, particularly larger structures, because they have a capacity (via their size and structural order) to form microenvironments that can adversely interact with separation components and flow path surfaces
Implementation Method 2
Without wishing to be bound by any particular theory, it is believed that the electronegativity of this motif may be responsible for the challenging chromatographic separations of the protein, and the strong electronegativity would be predicted to show affinity for and adsorptive losses from metallic column hardware
Implementation Method 3
Secondary interactions, such as ionic and hydrophobic interactions, can cause undesired effects including peak broadening, tailing, and loss of resolution and separation efficiency
Implementation Method 4
Size exclusion chromatography (SEC) is a common separation technique that employs differences in hydrodynamic radii to separate solubilized analytes on an immobilized stationary phase
Data Source
AI summary
The present disclosure is directed to methods for performing size exclusion chromatographic (SEC) separations. Embodiments of the present disclosure feature methods for improved separations of biomolecule analytes, such as CRISPR-related proteins, nucleotides, and ribonucleoprotein complexes, in SEC, for example, by using hydroxy-terminated polyethylene glycol surface modified stationary phase materials and/or C2/PEG surface modified column hardware.