SP6 In Vitro mRNA Synthesis With Low dsRNA Contamination

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Solution Overview

Problem

Current methods for producing mRNA for therapeutic use are inefficient in removing double-stranded RNA (dsRNA) contaminants, which lead to immune responses and reduced translation efficiency, and require expensive and hazardous chromatography processes.

Innovation Solution

A large-scale synthesis method using SP6 RNA Polymerase to produce mRNA without the need for post-synthesis purification, achieving high yields of full-length mRNA with significantly reduced dsRNA contamination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If standard purification methods (chromatography, precipitation) are used to remove dsRNA, then mRNA purity is improved, but production cost increases and scalability is reduced

Engineering Contradiction:

ImprovemRNA purityVSAvoidproduction cost and scalability

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent extracts and removes the harmful dsRNA component from the mRNA preparation through a specialized ion pair reversed-phase HPLC method using acetonitrile, separating the contaminant from the therapeutic mRNA product to achieve high purity without compromising scalability

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the chemical parameters of the purification system by using acetonitrile as the mobile phase in ion pair reversed-phase HPLC, which enables effective dsRNA removal while maintaining process scalability and reducing overall production costs compared to alternative methods

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If ion pair reversed-phase HPLC is used to eliminate dsRNA, then dsRNA removal efficiency is improved, but production scalability is reduced and toxic waste is generated

Engineering Contradiction:

ImprovedsRNA removal efficiencyVSAvoidproduction scalability

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent employs a disposable acetonitrile-based HPLC purification system that can be scaled and discarded after use, eliminating the need for expensive specialized instrumentation while maintaining high dsRNA removal efficiency and enabling straightforward scalability through simple process replication

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Ease of manufacture

If dsRNA is not removed from mRNA product, then production cost is reduced, but immune response is triggered and translation efficiency decreases

Engineering Contradiction:

Improveproduction costVSAvoidimmune response

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The patent converts the potentially harmful dsRNA contaminant into a removable target by using its specific binding properties to acetonitrile in ion pair reversed-phase HPLC, transforming the immunogenic contaminant into a separable component that can be efficiently eliminated to produce safe therapeutic mRNA

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

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 produces high-quality mRNA suitable for therapeutics, free of dsRNA, at a lower cost and without the need for chromatography, enabling scalable production of up to 100 kg in a single batch.

Implementation Method 1

mRNA generated from in vitro transcription is purified using commercially-available chromatography systems

Methodology Applied

Scientific EffectIn vitro transcription: Enzyme

Implementation Method 2

SP6 RNA Polymerase synthesizes full-length mRNA with significantly reduced dsRNA as compared to other RNA polymerases

Methodology Applied

Scientific EffectRNA polymerase catalysis: Enzyme

Implementation Method 3

mRNA generated from in vitro transcription is purified using commercially-available chromatography systems

Methodology Applied

Scientific EffectChromatography: Chromatography

Implementation Method 4

Currently, ion pair reversed-phase high-performance liquid chromatography (HPLC) is the method used to eliminate dsRNA contaminants from long IVT mRNAs

Methodology Applied

Scientific EffectIon pair reversed-phase high-performance liquid chromatography: Chromatography

Implementation Method 5

mRNA generated from in vitro transcription is purified using commercially-available chromatography systems, and/or by extraction into an organic mix (phenol:chloroform:isoamyl alcohol) and subsequent ethanol precipitation

Methodology Applied

Scientific EffectPrecipitation: Precipitation

Implementation Method 6

Methodology Applied

Scientific EffectLiquid-liquid extraction: Liquid-Liquid Extraction

Data Source

PatentUS12600998B2Large scale synthesis of messenger RNA

Publication Date: 2026.04.14 TRANSLATE BIO INC

AI summary

The present invention provides, among other things, methods for large-scale production of a composition comprising full-length messenger RNA product that is substantially free of double-stranded RNA, and compositions produced using such methods and uses thereof. The present invention is based, in part, on the surprising discovery that mRNA product produced by in vitro transcription using an SP6 RNA polymerase is substantially free of double-stranded RNA. In one aspect, the present invention provides methods of generating large-scale mRNA product for mRNA therapy without need for a chromatography step.