ssRNA Separation on Multimodal Solid Phase Using pH Gradient

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Solution Overview

Problem

Current methods for separating single-stranded RNA (ssRNA) from other nucleic acid species, such as double-stranded RNA (dsRNA) and DNA, face challenges in scalability, chemical harshness, and inefficiency at ambient conditions, leading to incomplete separation and low recovery rates, especially for mRNA production in biopharma applications.

Innovation Solution

A method utilizing a solid phase with a multimodal ligand comprising a nitrogen-containing heterocycle, employing an ascending pH gradient near or crossing the ligand's pKa value of 4.0 to 8.5, allows for high-resolution separation and recovery of ssRNA at ambient temperature and neutral pH without harmful additives.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If LiCl precipitation is used to purify RNA, then purification speed and cost-effectiveness are improved, but chemical harshness increases and scalability decreases

Engineering Contradiction:

Improvepurification speedVSAvoidchemical harshness

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The invention changes the chemical parameters of the purification system by using controlled pH gradients (pH 3-10) instead of harsh LiCl precipitation. The pH-dependent elution allows selective separation of RNA species under milder conditions, resolving the contradiction between fast purification and chemical harshness.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention replaces the mechanical/chemical precipitation process with a chromatographic separation mechanism based on pH-dependent binding and elution. This substitution eliminates the need for harsh chemicals while maintaining high purification efficiency and enabling scalable continuous processing.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Manufacturing precision

If oligo-dT affinity chromatography is used to separate mRNA, then selectivity is improved, but capacity decreases and scaling becomes difficult

Engineering Contradiction:

Improveseparation selectivityVSAvoidcolumn capacity

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The invention creates a universal chromatographic system that can separate multiple RNA species (mRNA, dsRNA, tRNA, rRNA) simultaneously through pH gradient elution, rather than requiring separate oligo-dT columns for each RNA type. This multi-functionality increases overall system capacity while maintaining selectivity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention uses pH gradient changes as the separation mechanism, allowing different RNA species to elute at different pH values based on their binding characteristics. This parameter-based separation provides both high selectivity and large capacity, enabling scale-up without the limitations of oligo-dT affinity chromatography.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If primary and secondary amine-based chromatography is used at room temperature, then separation efficiency is improved, but mRNA hydrolysis risk increases due to high pH requirements

Engineering Contradiction:

Improveseparation efficiencyVSAvoidmRNA stability

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The invention implements dynamic pH control during the chromatographic process, starting with low pH binding conditions that stabilize mRNA, then gradually increasing pH for selective elution. This dynamic approach maintains mRNA stability during binding while achieving efficient separation during the controlled elution phase, resolving the contradiction between separation efficiency and mRNA stability.

Inventive Principle:

Principle #15Dynamics

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 recovery and selective separation of ssRNA from other nucleic acids by leveraging multimodal interactions, including charge and hydrogen bonding, enabling efficient purification of mRNA and dsRNA by size under mild conditions suitable for GMP production.

Implementation Method 1

The solid phase comprises a multimodal ligand... enabling separation of ssRNA... by leveraging multimodal interactions, including charge and hydrogen bonding

Methodology Applied

Scientific EffectCharge interaction: Electrostatics

Implementation Method 2

The solid phase comprises a multimodal ligand... enabling separation of ssRNA... by leveraging multimodal interactions, including charge and hydrogen bonding

Methodology Applied

Scientific EffectHydrogen bonding: Chemical Bonding

Implementation Method 3

affinity-adsorption chromatography on cellulose based chromatography media... Chromatography on the other hand offers a selective and easily scalable approach

Methodology Applied

Scientific EffectAdsorption: Adsorption

Data Source

PatentEP4215613B1Method of separating single stranded RNA molecules from other nucleic acid species, use of solid phase therefor, and method of separating double stranded RNA molecules by size

Publication Date: 2026.03.04 SARTORIUS BIA SEPARATIONS D O O

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AI summary

The present invention relates to a method of separating single-stranded RNA (ssRNA) molecules from other nucleic acid species and to the use of a solid phase for separating ssRNA molecules from other nucleic acid species. The invention claims that ssRNA could be eluted from a solid phase media comprising multimodal ligands comprising nitrogen-containing heterocycles in an ascending pH gradient with: 1) a high product recovery at room temperature, 2) in the proximity of neutral pH, 3) without harmful additives.