Optimization of Industrial Production and Purification of Self-Amplifying mRNA

Despite achieving good results during the pandemic and showing great potential, mRNA technology still has several limitations, such as short expression times and limited protein translation. While these features can provide a certain level of safety, they become limitations for therapies like protein replacement. This requires repeated dosing to maintain protein expression levels, introducing new risks and challenges such as immunogenicity and neutralizing antibodies.

Self-amplifying RNA (saRNA) can generate the same immune response at doses hundreds or even thousands of times smaller than traditional mRNA. Lower doses mean lower production costs, and less frequent injections can reduce potential toxic side effects from the mRNA and delivery vectors. Currently, several saRNA candidates have entered clinical trials worldwide. Major companies like BioNTech are actively developing saRNA technology pipelines.

Although saRNA offers significant cost and safety advantages, its unique structure presents new production challenges. The saRNA molecule, which combines the sequence encoding RNA polymerase with the target protein sequence, is much larger (~10kb) and more complex (containing more secondary structures) than traditional mRNA. This significantly increases the difficulty of in vitro transcription reactions, reducing the yield and purity of saRNA production. Therefore, saRNA production demands higher standards in synthesis, separation, and purification processes.

 Affinity chromatography, a method used for separation and purification, has issues such as low target product yield and integrity. Combining multiple chromatographic purification methods (e.g., adding cellulose chromatography, hydrophobic reverse-phase chromatography) is cumbersome, introduces new impurities, has low recovery rates, and is costly. Additionally, the IVT reaction system used for large-scale production is optimized from systems suitable for synthesizing 100nt nucleic acid sequences, making it unsuitable for saRNA, which exceeds 10kb in size. This necessitates the optimization of existing IVT reaction systems. Currently, conventional mRNA purification methods cannot meet industrial raw liquid quality requirements. Therefore, there is an urgent need to develop and optimize a complete and efficient industrial-grade separation and purification process for self-amplifying RNA.

Optimization of Industrial Production and Purification Methods

 To develop an industrial production system for self-amplifying mRNA (saRNA), it is essential to first optimize the buffer ion types and components in the IVT cotranscription system on a small scale. This also involves refining the chromatographic buffer and sample washing ratios for downstream purification processes. These optimized conditions can then be scaled up to large-scale production to verify if the system can effectively increase production capacity and improve the quality of the raw product.

Cotranscription Cap-Adding Reaction System

In the cotranscription cap-adding reaction system, the T7 buffer components include 400mM HEPES, 20mM spermidine, 100mM DTT, and Mg2+ salts.

T7 Buffer - Magnesium Ion Salt Type

The type of magnesium ion salt used affects the yield and integrity of the target saRNA product.

1. For small-scale cotranscription synthesis of 1mg mRNA, using Mg(OAc)2 as the Mg2+ salt results in the best yield and integrity, with values of 100.5µg and 62.9%, respectively.
2. In contrast, using other magnesium ion salts such as MgCl2 and MgSO4 reduces yield by approximately 25-50% and integrity by about 10%, significantly lowering both yield and integrity.

T7 Buffer - Magnesium Ion Concentration

An optimal concentration of magnesium acetate ions (30-50mM) enhances the yield and integrity of the target saRNA, with the best results at 35mM.

1. At a magnesium acetate ion concentration of 30-50mM, the saRNA yield reaches 88.5-100.5µg, and integrity is 58.6-62.9%, showing good results.
2. At a concentration of 35mM, the yield and integrity are at their highest, reaching 100.5µg and 62.9%, respectively.

T7 Buffer - Magnesium Ion Salt Combined with Other Salts

Adding other salts to the T7 buffer can significantly improve the yield and integrity of the target product.

1. The base T7 buffer includes 400mM HEPES, 20mM spermidine, 100mM DTT, and Mg2+ salts, resulting in a yield and integrity of 100.5µg and 62.9%, respectively.
2. Adding a second salt, NaOAc, further enhances yield and integrity: yield increases by 20-110µg, and integrity improves by about 2-8%.

T7 Buffer - Concentration of Combined Salts

When the second salt component NaOAc (Na+) is at a concentration of 5-30mM, the target saRNA yield and integrity significantly improve, with the best concentration at 15mM.

1. At Na+ concentrations of 3-30mM, the saRNA yield reaches 120-210µg, and integrity is 64.4-70.2%, showing good results.
2. At a Na+ concentration of 15mM, the yield and integrity are at their highest, reaching 210µg and 70.2%, respectively.

Single Purification Methods

During small-scale (<50mg) saRNA production and purification, using different purification methods can significantly reduce dsRNA content and protein residues, ensuring high yield, capping efficiency, and product integrity. The effectiveness of the purification methods from best to worst is: affinity chromatography > lithium chloride precipitation > ultrafiltration, cellulose chromatography.

1. Affinity Chromatography

For large-scale mRNA production, chromatography is preferred. Options include reverse-phase, ion exchange, hydrophobic, and affinity chromatography, each with its pros and cons. Affinity chromatography, often used for capturing poly(A) mRNA, offers scalability and platform solutions with >90% recovery rates.

mRNA's key structural features, the 5’ cap and 3’ poly(A) tail, facilitate purification. Affinity chromatography, similar to magnetic bead purification, uses dT-linked beads to capture poly(A) tail mRNA. High salt conditions shield negative charges, allowing binding through A-T hydrogen bonds, and mRNA is eluted under mild neutral pH and low conductivity.

Affinity chromatography for RNA involves "high salt binding, low salt elution." Buffer composition, molecular size, temperature, and sample concentration significantly impact the process. Selecting the optimal salt type and concentration through experimentation is essential for efficient purification.

Purification: This method yields the highest product integrity (80.3%), the lowest dsRNA content (0.01µg/mg), the highest capping efficiency (96.4%), and the least protein residues (1.20µg/mg), with a yield of 136µg and a recovery rate of 65%, making it the best in terms of product purity and quality.

2. Other Purification Methods:

Lithium Chloride Precipitation

The principle of purifying mRNA using LiCl is that lithium can reduce electrostatic repulsion between molecules at a certain pH, enabling efficient RNA precipitation. The precipitate is then separated by centrifugation, dissolved to obtain a pure RNA sample. LiCl precipitation is simple, fast, effective for mRNAs of various sizes, and yields high-purity products. However, residual lithium ions can inhibit mRNA. It's recommended to use LiCl precipitation for solutions containing at least 400 µg/ml RNA to ensure purification efficiency. This method produces a high yield (210µg), but the product integrity is only 70.2%, significantly reducing product quality. It is not suitable for large-scale production.

Magnetic Bead Method：

Magnetic beads are small particles that move directionally under a magnetic field, typically composed of an iron oxide core and an external coating. Magnetic bead purification is a common molecular biology technique for quickly and efficiently enriching mRNA molecules from mixtures. Different functional magnetic beads have different surface functional groups (e.g., hydroxyl, carboxyl, Oligo(dT), streptavidin) for purifying various biomolecules.

Carboxylated magnetic beads can efficiently purify nucleic acids, with mRNA molecules binding through electrostatic interactions under acidic conditions. Adjusting conditions allows for the selective binding and release of mRNA. Longer mRNAs with more exposed negatively charged phosphate groups bind more easily to the beads. For shorter mRNAs, larger bead volumes may be needed. Oligo(dT)-coupled magnetic beads, similar to affinity chromatography, utilize specific binding between the poly(A) tail of mRNA and the beads' Oligo(dT).

Ultrafiltration

This method results in the lowest saRNA integrity, approximately 8% lower than affinity chromatography, with the highest protein residue (4.58µg/mg).

Cellulose Chromatography

This method achieves low dsRNA content (0.009µg/mg) and a high capping rate (96.4%). However, protein residues are slightly higher (5.30µg/mg), with a recovery rate of only 57% and a yield of 120µg, both of which are significantly lower than those achieved with affinity chromatography (by about 10% and 16µg, respectively).

Purification Process

Chromatographic Buffer Components - Addition of Reducing Agents

Adding reducing agents to the chromatographic buffer during purification can significantly improve product integrity, recovery rate, and capping efficiency, while reducing the levels of by-product dsRNA and protein residues compared to not adding reducing agents.

1. Without Reducing Agents:

    - Yield: 136µg

    - Recovery Rate: 65%

    - Integrity: 80.3%

    - dsRNA: 0.01µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 1.20µg/mg

    - Product purity and quality are good.

2. With Reducing Agents (TCPE, DTT, β-mercaptoethanol):

    - Yield increases by 10-40µg

    - Recovery Rate increases by 5-18%

    - Integrity improves by approximately 1-5%

    - Capping Efficiency: 96.4%

    - dsRNA: as low as 0.005µg/mg

    - Protein Residue: as low as 0.20µg/mg

    - Significant improvement in product purity and quality.

Chromatographic Buffer Components - Types of Reducing Agents

Different types of reducing agents added to the chromatographic buffer can further improve product integrity, recovery rate, and capping efficiency, while reducing dsRNA and protein residues. TCPE is found to be the most effective reducing agent.

- With TCPE:

    - Yield: 175µg

    - Recovery Rate: 83%

    - Integrity: 85.2%

    - dsRNA: 0.005µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 0.20µg/mg

    - Excellent product purity and quality.

Chromatographic Buffer Components - Concentration of Reducing Agents

Adding reducing agents at a concentration of 5-15mM in the chromatographic buffer can significantly improve product integrity, recovery rate, and capping efficiency, while reducing dsRNA and protein residues. The optimal concentration is 10mM.

1. Adding 5-15mM TCPE:

    - Yield: 160-178µg

    - Recovery Rate: 76-85%

    - Integrity: approximately 85%

    - dsRNA: 0.002-0.005µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 0.20-0.52µg/mg

    - Good product purity and quality.

2. With 10mM TCPE:

    - Yield: 178µg

    - Recovery Rate: 85%

    - Integrity: 85.4%

    - dsRNA: 0.002µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 0.20µg/mg

    - Optimal product purity and quality.

Washing Ratio

Controlling the ratio of chromatographic buffer to injection water in the washing process (10-25):(75-90) can significantly improve product integrity, recovery rate, and capping efficiency, while reducing dsRNA and protein residues. The optimal ratio is 15:85.

1. Ratio (10-25):(75-90):

    - Yield: 150-179µg

    - Recovery Rate: 71-85%

    - Integrity: 84-90%

    - dsRNA: 0.002-0.006µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: approximately 0.20µg/mg

    - Good product purity and quality.

2. With Ratio 15:85:

    - Yield: 179µg

    - Recovery Rate: 85%

    - Integrity: 90.0%

    - dsRNA: 0.002µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 0.20µg/mg

    - Optimal product purity and quality.

Combined Purification Methods

Using a combination of different purification methods can further reduce the levels of dsRNA and protein residues in the product, enhancing overall quality. The preferred order of purification methods is: affinity chromatography > ultrafiltration + affinity chromatography > affinity chromatography + cellulose chromatography > cellulose chromatography + ultrafiltration. Affinity chromatography alone provides the best results.

1. Affinity Chromatography Alone:

    - Yield: 179µg

    - Recovery Rate: 85%

    - Integrity: 90.0%

    - dsRNA: 0.002µg/mg

    - Capping Efficiency: 96.4%

    - Protein Residue: 0.20µg/mg

    - Highest product purity and quality.

 

2. Ultrafiltration + Affinity Chromatography:

    - Yield: 170µg (9µg less than affinity chromatography alone)

    - Recovery Rate: 81% (4% less than affinity chromatography alone)

    - Integrity: 88.5%

    - dsRNA: 0.0015µg/mg

    - Protein Residue: 0.18µg/mg

    - Slight reduction in dsRNA and protein residues compared to affinity chromatography alone, but significant reduction in yield and recovery rate. This method is more complex, doubling the purification time and increasing costs.

3. Affinity Chromatography + Cellulose Chromatography / Cellulose Chromatography + Ultrafiltration:

    - dsRNA: 0.005µg/mg lower than single methods

    - Yield: 60-100µg less

    - Recovery Rate: 30-40% lower

    - Increased steps result in higher labor and material costs.

 Large-Scale Production

In large-scale production, using affinity chromatography, affinity chromatography combined with cellulose chromatography, or ultrafiltration combined with affinity chromatography, the yield of self-amplifying mRNA significantly increases to 140-185µg, with a recovery rate of 67-88%. Product integrity is 93-94%, dsRNA content is controlled within 0.0018-0.0020µg/mg, capping efficiency reaches 96.1-96.4%, and protein residues are kept within 0.04-0.05µg/mg. This demonstrates good scalability and efficiency for large-scale production.

Reference:

[1] LiCl Precipitation for RNA Purification, Retrieved Jan 8, 2024, fromhttps://www.thermofisher.cn/cn/zh/home/references/ambion-techsupport/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html.

[2] Product Information Sheet of Lithium Chloride Precipitation Solution, Retrieved Jan 8, 2024, fromhttps://www.thermofisher.cn/document-connect/document-connect.html?url=https://assets.thermofisher.cn/TFSAssets%2FLSG%2Fmanuals%2F4386633_LithiumChloridePrecipSol_PI.pdf.

[3] Product of BeyoMag™ RNA Clean Magnetic Beads, Retrieved Jan 8, 2024, from https://www.beyotime.com/product/R0081-1ml.htm.

[4] Product of VAHTS RNA Clean Beads, Retrieved Jan 8, 2024, from https://www.vazyme.com/product/215.html.

[5] Dynabeads™-Based Solid-Phase In Vitro Transcription and RNA Purification, RetrievedJan8,2024,from https://www.thermofisher.cn/order/catalog/product/cn/zh/65020D.

[6] RNA Clean XP Performance and Data, Retrieved Jan 8, 2024, from https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/rna-and-cdna/performance.

[7] News from ThermoFisher Scientific, Retrieved Jan 8, 2024, from https://bydrug.pharmcube.com/news/detail/39287a37147e22e2d978f7dd9228df58.

[8] Industrial Production and Separation Purification Methods of Self-Amplifying mRNA Raw Liquid and Its Applications [P]. Patent: CN118048418A. 2024.05.17.

Ordering Information

Yeasen developed a novel CleaScrip™ T7 RNA Polymerase which is a top-notch enzyme for mRNA synthesis, for that the cellulose treatment is no more needed for dsRNA removal, saving both time and labor costs. The dsRNA content in mRNAs produced by CleaScript™ T7 RNA polymerase is lower than that in mRNAs produced by Wild Type T7 RNA Polymerase, both before and after the dsRNA removal step using cellulose treatment. Check out more details from the following links: