mRNA Purification Using TFF After Precipitation Capture
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Solution Overview
Problem
Current methods for large-scale production of messenger RNA (mRNA) for therapeutic use are inadequate in achieving high purity and safety, as they fail to effectively remove prematurely aborted RNA sequences and enzyme reagents from the synthesis process.
Innovation Solution
A method involving precipitation of mRNA followed by membrane filtration, specifically tangential flow filtration, using reagents like guanidinium thiocyanate and ethanol, to capture and purify mRNA, followed by washing and elution, achieving high purity and integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional purification methods are used for large-scale mRNA production, then production scale can be increased, but purity and safety are compromised due to inability to effectively remove prematurely aborted RNA sequences and enzyme reagents
Solution Approach 1:
The purification process is divided into multiple sequential steps: precipitation step to concentrate mRNA, membrane filtration step to remove impurities, washing step to eliminate residual reagents, and elution step to recover pure mRNA. This segmentation allows each step to target specific impurities effectively
Solution Approach 2:
A membrane filter serves as an intermediary between the crude mRNA preparation and the final purified product. The membrane physically separates prematurely aborted RNA sequences and enzyme reagents from full-length mRNA based on size exclusion, enabling high-purity large-scale production
2Quantity of substance
If precipitation method alone is used for mRNA purification, then concentration can be achieved, but purity is insufficient due to co-precipitation of impurities
Solution Approach 1:
The invention merges precipitation method with membrane filtration in a sequential workflow. Precipitation first concentrates the mRNA from dilute solutions, then membrane filtration removes co-precipitated impurities. This combination leverages the strengths of both methods to achieve both concentration and high purity
3Manufacturing precision
If membrane filtration is used without prior precipitation, then purity can be improved, but productivity decreases due to clogging and handling difficulties
Solution Approach 1:
Precipitation is performed as a preliminary action before membrane filtration. This pre-concentration step reduces the volume and increases the viscosity of the mRNA solution, preventing membrane clogging during filtration and enabling efficient large-scale processing without compromising productivity
4Quantity of substance
If high concentration of guanidinium thiocyanate is used for precipitation, then mRNA recovery is improved, but removal of enzyme reagents becomes more difficult
Solution Approach 1:
The purification process continues with additional steps after precipitation: membrane filtration to remove enzyme reagents and washing to eliminate residual guanidinium thiocyanate. This continuous action ensures that high recovery during precipitation does not compromise final purity
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 enables the production of high-quality mRNA with less than 0.1% prematurely aborted RNA sequences and enzyme reagents, ensuring integrity greater than 95%, suitable for therapeutic use at scales up to 100 kg per batch.
Implementation Method 1
treating the impure preparation with a solution comprising a reagent selected from the group consisting of lithium chloride, sodium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, ammonium acetate and combinations thereof
Implementation Method 2
subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane
Implementation Method 3
the step of precipitating mRNA further comprises a step of treating the impure preparation with absolute ethanol
Data Source
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AI summary
The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of (a) precipitating mRNA from an impure preparation; (b) subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane; and (c) eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution. In some embodiments, a purification process involving membrane filtration suitable for the present invention is tangential flow filtration.