Solid-Surface Affinity Ligands for Gentle Nucleic Acid Removal
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Solution Overview
Problem
Current methods for purifying biological preparations, such as monoclonal antibodies, face challenges in efficiently separating and removing DNA and RNA from complex mixtures without damaging the proteins, which often leads to denaturation and loss of protein function.
Innovation Solution
A method involving the use of affinity ligands, such as methylene blue, Hoechst dyes, and cyanine dyes, which are bound to a surface to create a coupled surface-affinity ligand. This ligand is used to selectively bind and separate target macromolecules like DNA and RNA from a sample, allowing for their removal and recovery without affecting proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional chromatography media are used for purification, then separation of target proteins from contaminants is achieved, but the media are rapidly fouled by cell debris requiring pre-filtration and volume reduction steps
Solution Approach 1:
The invention segments the traditional two-step process (recovery then purification) into a single integrated step using expanded bed chromatography. The chromatography media is designed to handle both cell debris removal and target protein purification simultaneously, eliminating the need for separate pre-filtration and concentration steps.
Solution Approach 2:
The invention transitions from traditional packed bed chromatography to expanded bed chromatography, adding the dimension of fluid expansion. The media expands from a dense packed state to a fluidized expanded state, creating interparticle lumens that allow cell debris to flow through while soluble products are captured by functional groups on the beads.
2Reliability
If anion ion exchange methods are used to remove DNA and RNA, then purification to regulatory limits is achieved, but proteins may be denatured or degenerated
Solution Approach 1:
The invention changes the operating parameters of chromatography by using expanded bed conditions with large pore sizes (60-180 μm) and controlled flow rates. These parameter changes allow gentle handling of proteins while effectively removing DNA and RNA contaminants through the interparticle lumens, avoiding denaturation that occurs with high pressure or aggressive pH buffers.
Solution Approach 2:
The expanded bed chromatography media acts as an intermediary that selectively interacts with DNA and RNA contaminants while leaving proteins unaffected. The large bead chromatography media with specific functional groups captures nucleic acids in the interparticle lumens without exposing proteins to denaturing conditions.
3Productivity
If high pressure or aggressive pH-buffers are used in Fast Protein Liquid Chromatography, then protein purification is achieved, but protein function is lost due to denaturation
Solution Approach 1:
The invention introduces dynamic control of bed expansion through flow rate adjustment. The media can be expanded to different degrees depending on the purification needs, allowing optimization between speed and gentleness. The fluidized state enables rapid processing while the controlled expansion prevents protein denaturation.
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
The method effectively isolates and removes DNA and RNA from samples with high specificity, reducing the target macromolecule concentration to below regulatory limits while preserving the integrity of the proteins, thus overcoming the limitations of existing purification techniques.
Implementation Method 1
selecting an affinity ligand that will bind to the target macromolecule; binding the affinity ligand to a surface to create a coupled surface-affinity ligand
Data Source
AI summary
A method of isolating and separating a target macromolecule, such DNA (double stranded or single stranded), RNA (double stranded or single stranded), messenger RNA, or other oligonucleotide or oligonucleoside, from a sample by binding the target macromolecule to an affinity ligand that is bound to a surface is disclosed. The method may be employed in chromatography or any other of the separation sciences.