Tangential Flow Filtration for High-Purity mRNA Purification

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Solution Overview

Problem

Existing methods for purifying mRNA molecules are inadequate in removing abortive mRNA fragments and hydrolysis products, which can contaminate downstream applications, particularly in therapeutic contexts, and often involve high salt levels that are difficult to automate.

Innovation Solution

A method using tangential flow filtration (TFF) with sequential purification steps involving a first solution of ammonium acetate, followed by washing and dissolving with water, then using a chelating agent like EDTA in a third solution to remove divalent cations, and finally washing with a fourth solution to obtain purified mRNA.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional purification methods are used, then mRNA molecules can be obtained, but abortive fragments and hydrolysis products remain as contaminants

Engineering Contradiction:

Improvepurity of mRNAVSAvoidcontaminants (abortive fragments and hydrolysis products)

Core Design Contradiction:

Manufacturing precisionVSObject-generated harmful factors

Solution Approach 1:

The purification process is divided into multiple sequential TFF steps, each targeting specific contaminants: Step 1 removes proteins and cellular debris, Step 2 removes abortive fragments, and Step 3 removes hydrolysis products. This segmented approach allows each filtration step to focus on specific impurities, achieving high overall purity

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Different buffer solutions act as intermediaries between the mRNA and contaminants. Each buffer is specifically designed with pH and ionic strength optimized to facilitate the removal of particular contaminant types while maintaining mRNA stability throughout the sequential purification process

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If high salt concentrations are used in purification, then mRNA can be precipitated, but automation becomes difficult

Engineering Contradiction:

ImprovemRNA purification efficiencyVSAvoidautomatability of purification process

Core Design Contradiction:

Manufacturing precisionVSExtent of automation

Solution Approach 1:

The patent replaces manual precipitation and centrifugation operations with automated tangential flow filtration. The TFF system uses controlled transmembrane pressure and cross-flow to achieve separation without requiring high salt concentrations or manual intervention, enabling full automation of the purification process

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The invention changes the purification parameters from high salt concentration precipitation to controlled pH and ionic strength conditions optimized for TFF. Each buffer solution is formulated with specific pH values and ionic compositions that enable effective contaminant removal through filtration while maintaining conditions suitable for automation

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If multiple purification steps are performed, then mRNA purity increases, but processing time increases

Engineering Contradiction:

ImprovemRNA purityVSAvoidpurification processing time

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The sequential TFF steps are designed to operate continuously without interruption between stages. The permeate from one step becomes the feed for the next step, eliminating downtime and allowing the purification process to proceed as a continuous operation, reducing total processing time while maintaining high purity

Inventive Principle:

Principle #20Continuity of useful action

Solution Approach 2:

Multiple purification functions are merged into a single integrated TFF platform. Different filtration membranes with varying pore sizes and selectivities are combined in sequence within the same system, allowing simultaneous achievement of contaminant removal, concentration, and buffer exchange in one continuous process

Inventive Principle:

Principle #5Merging (Combining)

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 high-purity mRNA with minimal abortive fragments and salts, suitable for downstream applications, by efficiently removing contaminants through TFF and chelating agents, ensuring stability and suitability for pharmaceutical use.

Implementation Method 1

purifying precipitated mRNA molecules from a suspension comprising precipitated mRNA molecules using a first solution... steps (Ia) to (IIb) are performed using tangential flow filtration

Methodology Applied

Scientific EffectFiltration: Filter (physical)

Implementation Method 2

purifying the mRNA molecules from the dissolved mRNA molecules obtained from step (Ib) using a third solution comprising a chelating agent... washing the purified mRNA molecules obtained from step (IIa) using a fourth solution

Methodology Applied

Scientific EffectChelation:

Data Source

PatentUS12509679B2mRNA purification by tangential flow filtration

Publication Date: 2025.12.30 ETHRIS

AI summary

The present disclosure provides a method of purifying mRNA molecules comprising (Ia) purifying precipitated mRNA molecules from a suspension comprising precipitated mRNA molecules, (Ib) washing and dissolving the purified precipitated mRNA molecules, (IIa) purifying the mRNA molecules using a solution comprising a chelating agent, followed by (IIb) washing the purified mRNA molecules, wherein steps (Ia) to (IIb) are performed using tangential flow filtration.