Supercoiled Plasmid DNA Separation via Convective Anion Exchange
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Solution Overview
Problem
Existing methods for purifying supercoiled plasmid DNA (pDNA) are inefficient and unsustainable, requiring lengthy processes and high consumption of buffers and chemicals.
Innovation Solution
A two-step chromatography method is introduced, involving an anion exchange chromatography material with convective flow support and a second material with a thiophilic aromatic adsorption ligand, enabling selective separation of supercoiled pDNA from open circular pDNA without additional chromatography steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional multi-step chromatography methods are used for pDNA purification, then purity can be achieved, but process time increases and productivity decreases
Solution Approach 1:
The patent combines group separation and selective separation functions into a single chromatography step using a specially designed resin with convective flow properties, eliminating the need for sequential chromatography steps while maintaining high purity levels
Solution Approach 2:
The invention segments the purification process by using a two-stage chromatography approach where the first stage captures all pDNA forms and the second stage selectively separates supercoiled from open circular forms, achieving high purity in fewer steps
2Manufacturing precision
If conventional chromatography methods are used, then pDNA can be purified, but buffer and chemical consumption increases
Solution Approach 1:
The patent changes the flow regime parameter from diffusive to convective flow, which enhances mass transfer efficiency and reduces the volume of buffers needed for effective purification while maintaining high purity outcomes
3Device complexity
If RNA removal is not performed before chromatography, then process steps are reduced, but chromatography efficiency decreases
Solution Approach 1:
The patent performs RNA removal as a preliminary step before chromatography to prevent RNA from competing for binding sites on the chromatography resin, thereby ensuring efficient capture and separation of pDNA without compromising chromatography performance
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 achieves a purity of at least 95% supercoiled pDNA in significantly reduced time (within 5 hours) and with lower ammonium sulfate consumption, enhancing productivity and sustainability.
Implementation Method 1
a first chromatography material comprising (i) an anion exchange chromatography ligand for binding to pDNA
Implementation Method 2
a second chromatography material comprising a ligand that binds to pDNA and enables selective separation of supercoiled pDNA from open circular pDNA
Implementation Method 3
a support material allowing convective flow through the first chromatography material
Data Source
AI summary
The present disclosure is directed to a method for separating supercoiled plasmid DNA (pDNA) from a liquid sample. the method comprising the steps of: (a) adding a liquid sample comprising pDNA to a first chromatography material comprising (i) an anion exchange chromatography ligand for binding to pDNA and (ii) a support material allowing convective flow through the first chromatography material, wherein the liquid sample originates from a cell culture harvest and has been subjected to a step of removing RNA before step (a): (b) eluting a liquid sample. comprising a purified mixture of supercoiled pDNA and open circular pDNA, from the first chromatography material: (c) adding the liquid sample from step (b) to a second chromatography material comprising a ligand that binds to pDNA and enables selective separation of supercoiled pDNA from open circular pDNA: (d) eluting the purified supercoiled pDNA from the second chromatography material: wherein the supercoiled pDNA eluted in step (d) has a purity degree of at least 95% without use of any further chromatography material than said first and second chromatography materials. Steps (a)-(d) and any intermediate steps can be completed within 5 hours. Further disclosed are uses of supercoiled pDNA obtained by said separation method.