mRNA Purification by Tangential Flow Filtration Under Denaturing Conditions
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Solution Overview
Problem
Existing mRNA purification methods using spin columns and caustic or flammable solvents like ethanol are undesirable for therapeutic administration and large-scale production, and fail to effectively remove prematurely aborted RNA sequences (shortmers) which are highly immunostimulatory.
Innovation Solution
The use of tangential flow filtration (TFF) combined with a denaturing agent, such as urea or guanidinium thiocyanate, to purify mRNA without caustic or flammable solvents, effectively removing reactants, enzymes, and byproducts, including shortmers, while maintaining mRNA integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If spin columns and caustic or flammable solvents like ethanol are used for mRNA purification, then purification can be achieved, but the method is undesirable for therapeutic administration and large-scale production due to safety concerns and inability to remove shortmers
Solution Approach 1:
The patent changes the chemical parameters of the purification system by replacing caustic or flammable solvents with aqueous buffer solutions containing denaturing agents. This parameter change eliminates safety concerns while maintaining purification effectiveness, allowing large-scale manufacturing of therapeutic mRNA without hazardous chemicals.
Solution Approach 2:
The patent introduces an intermediary substance (aqueous buffer with denaturing agent) that mediates the purification process. Instead of using direct contact with hazardous solvents, the denaturing agent in aqueous buffer serves as a safe intermediary that achieves the same separation function through different chemical mechanisms, enabling removal of shortmers without safety risks.
2Device complexity
If traditional purification methods are used, then the process is simple, but prematurely aborted RNA sequences (shortmers) are not effectively removed which are highly immunostimulatory
Solution Approach 1:
The patent applies preliminary action by treating the mRNA sample with a denaturing agent before the filtration step. This preliminary denaturation step unfolds proteins and separates them from the mRNA, enabling the subsequent filtration to effectively remove shortmers and other impurities that would otherwise be difficult to separate. The denaturing action prepares the sample in advance for more effective purification.
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
TFF with denaturing agents achieves highly pure mRNA suitable for pharmaceutical use, substantially free of shortmers and enzyme reagents, with integrity greater than 95%, reducing immunostimulatory risks and enhancing safety.
Implementation Method 1
purifying the mRNA from the impure preparation from step (a) by tangential flow filtration
Implementation Method 2
subjecting an impure preparation comprising in vitro synthesized mRNA to a denaturing condition
Data Source
AI summary
The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of subjecting an impure preparation comprising in vitro synthesized mRNA to a denaturing condition, and purifying the mRNA from the impure preparation from step (a) by tangential flow filtration, wherein the mRNA purified from step (b) is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.