mRNA therapeutics is a newly emerging form in recent years. Its basic principle is to introduce mRNA expressing antigen targets into the body through specific delivery systems, express protein antigens in the body, stimulate the body to produce specific immune responses, thereby providing the body with immune protection. It has many advantages such as rapid preparation and low cost.
mRNA synthesized through in vitro transcription (IVT) not only contains the desired mRNA product but also includes enzymes (RNA polymerase, phosphatase), template DNA, and mRNA by-products formed during IVT (mainly including dsRNA and truncated RNA fragments), among other impurities. Ensuring both yield and high purity of mRNA is crucial for maintaining sample quality and immunogenicity.
Impurities in the mRNA reaction system include:
Plasmid DNA template: host DNA, RNA, proteins.
RNA polymerase: BSA stabilizer, DNA/histone, host proteins, etc.
Multivalent metal cations: The phosphoric acid residues of the RNA ribose backbone have high affinity for these metals.
mRNA services at BOC Sciences
| Services Name | Description |
| Custom mRNA Synthesis | BOC RNA provides customized research and GMP-grade mRNA, with a flexible production scale from milligrams to grams to meet the various needs of researchers. |
| mRNA Purification | BOC Sciences offers purification services to remove contaminants from modified nucleotide-containing mRNAs to improve processing efficiency for downstream applications. |
| mRNA Capping | BOC Sciences’ team has extensive experience in mRNA design and production and offers mRNA 5′-end capping services to meet the needs of our customers’ projects in all aspects. |
| Nucleotide Modifications | BOC Sciences offers nucleotide-modified mRNA with industry-leading project completion times, a wide range of deliverables and best-in-class quality. |
| mRNA Vaccine Delivery Technology Solution | BOC RNA provides professional support in the design, production, and evaluation of mRNA vaccines. |
Lithium chloride (LiCl) precipitation method
Principle of lithium chloride precipitation method
The principle of purifying mRNA with LiCl lies in lithium’s ability, at a certain pH, to reduce intermolecular electrostatic repulsion, achieving efficient precipitation of RNA. The precipitate is then separated by centrifugation, followed by dissolution to obtain a pure RNA sample. The LiCl precipitation of RNA is simple and rapid, showing good precipitation effects for mRNA of different sizes and yielding high-purity products. However, it’s worth noting that lithium chloride cannot effectively precipitate some small RNAs (such as tRNA), and residual lithium ions have an inhibitory effect on mRNA. To ensure purification effectiveness, it is recommended to use LiCl precipitation for solutions containing at least 400 µg/ml RNA.
Operating steps
Key steps of LiCl purification are as follows:
1.Add IVT products to a LiCl solution to achieve a final LiCl concentration of 2.5M in the mRNA solution.
2.Cool the reaction at -20℃ for 30 minutes.
3.Centrifuge at maximum speed in a microcentrifuge for 15 minutes.
4.Discard the supernatant and wash the precipitate with ice-cold 70% EtOH to remove residual salts. Remove ethanol.
5.Resuspend the mRNA precipitate in RNase-free solvent, ensuring not to let the precipitate dry completely, as it may become difficult to resuspend.
mRNA purification with magnetic beads
Principle of magnetic bead method
Magnetic beads are tiny particles capable of directional movement under the influence of a magnetic field. These particles typically consist of small iron oxide core particles surrounded by other substances. When magnetic particles are subjected to a magnetic field, they can move along the direction of the field. Upon removal of the magnetic field, these magnetic particles lose their magnetic memory and can easily separate from each other. Magnetic bead purification is a commonly used molecular biology technique used to rapidly and efficiently enrich and purify mRNA molecules from mixtures. Different functional magnetic beads have different surface-modified functional groups. Common functional groups include hydroxyl, carboxyl, Oligo(dT), and streptavidin, used for the separation and purification of different types of biomolecules.
Magnetic beads with carboxyl functional groups can efficiently purify nucleic acids. mRNA molecules carry a negative charge under acidic conditions and can electrostatically interact with the carboxyl functional groups on the surface of carboxyl magnetic beads, thereby achieving selective enrichment. By adjusting the conditions, mRNA binding to the magnetic beads and dissociation can be controlled, thereby achieving RNA purification. The longer the mRNA, the more exposed negatively charged phosphate groups on the surface, resulting in a stronger overall negative charge, making it easier to adsorb onto the magnetic beads. Conversely, when recovering shorter mRNA fragments, larger volumes of magnetic beads need to be added. Another type of magnetic bead used for mRNA purification is Oligo(dT)-coupled magnetic beads. Its basic principle is consistent with affinity chromatography, utilizing the specific binding between the coupled affinity ligand Oligo(dT) on the surface of magnetic beads and mRNA molecules with Poly(A) tails, separating the target RNA molecules from other nonspecifically bound impurities through magnetic force.
Operating steps
1.Key steps of mRNA purification by magnetic bead method：
2.Binding of the sample to the magnetic beads.
3.Separation of the magnetic beads from contaminants.
4.Washing the magnetic beads with ethanol-water solution to remove contaminants.
5.Elution of mRNA from the magnetic beads.
6.Collection and transfer of purified mRNA samples.
The magnetic bead method can be used to purify mRNA from various in vitro reaction systems and is also suitable for concentrating low-concentration mRNA. This method is more convenient and time-saving than LiCl precipitation, with recovery rates typically reaching 80-90%. When purifying mRNA using the magnetic bead method, precautions to be taken include: (1) avoiding freezing and high-speed centrifugation of magnetic beads; (2) allowing the magnetic beads to equilibrate to room temperature before use; (3) it is recommended to prepare the ethanol-water solution for washing the magnetic beads just before use to avoid affecting the recovery efficiency; (4) do not dry the magnetic beads for an extended period, as excessive drying will cause irreversible aggregation of the magnetic beads, thereby reducing elution efficiency.
Chromatography purification for mRNA
Principle of Chromatography
The aforementioned two purification methods can achieve high purity of mRNA but are suitable only for small-scale use in research laboratories. For large-scale mRNA preparation, chromatography remains the mainstream method in the industry. Based on the chemical specificity of mRNA molecules themselves, there are many available chromatography methods, including reverse phase, ion exchange, hydrophobic, and affinity chromatography, each with its own advantages and disadvantages. When selecting a specific chromatography method, factors such as scalability, platform compatibility, purification efficiency, yield, and cost-effectiveness need to be considered comprehensively. Affinity, ion exchange, and hydrophobic applications have good scalability and are currently widely used purification methods, especially affinity chromatography, which is widely used for Poly(A) mRNA capture, offering a platform solution with >90% recovery rate.
The most significant structural feature of mRNA is the presence of a 5′ cap and a 3′ Poly(A) tail. This structure provides a convenient selection marker for the purification of eukaryotic mRNA. Affinity chromatography is similar to the principle of magnetic bead purification, where the matrix surface is bonded with dT ligands via linkers to capture mRNA with poly(A) tails through A-T base pairing. During sample loading under high salt conditions, salt ions can shield the electrostatic repulsion between the negatively charged skeleton of the matrix and the negatively charged mRNA, allowing them to approach each other. mRNA containing poly(A) tails is captured through A-T hydrogen bonding. When the salt concentration is lowered, electrostatic repulsion occurs between the negatively charged matrix skeleton and poly(A), facilitating mRNA elution under mild neutral pH and low conductivity.
Optimization of process parameters
The process steps for purifying RNA by affinity chromatography can be summarized as “high salt binding, low salt elution” in eight words. However, buffer composition, molecular size, temperature, sample concentration, etc., can greatly affect the process.
The binding of affinity materials and mRNA molecules occurs under high salt conditions. Theoretically, any salt that promotes RNA precipitation can facilitate the binding of RNA molecules and materials, but different types and concentrations of salt can lead to differences in selectivity for the materials. In the purification process, neutral salts such as sodium chloride, potassium chloride, etc., are preferred. Insufficient salt concentration can lead to reduced binding capacity, while excessively high salt concentrations can cause RNA molecule conformations to become more relaxed, bringing RNA molecules closer to each other, leading to disordered hydrogen bonding. Therefore, to achieve efficient purification, the optimal salt type and concentration must be selected through experimentation.
Summary
In the purification phase, the above three methods all have certain drawbacks, and a single purification method is difficult to remove some rare mRNA products. Overall, precipitation and magnetic bead methods are simple and suitable for research-level purification and product concentration, and the purity of the mRNA products obtained is nearly equivalent to that of chromatography. However, the scalability of the above two methods is poor, and the amplification cost is high. For example, the precipitation method requires high equipment requirements (explosion-proof) and needs to match low-temperature high-speed centrifuges, with cumbersome operation, requiring multiple precipitations and washes, and LiCl solution can cause certain damage to RNA. The magnetic bead method requires special beads and racks, and the suspension and stability of the beads will affect the purification effect. Although affinity chromatography can be scaled up, the hybrid affinity between Oligo (dT) and Poly(A) tails has limitations. It cannot distinguish between ssRNA and dsRNA with Poly(A) tails, and dsRNA can trigger the body’s innate immune response, reducing the effectiveness of vaccines. There is no definite conclusion on the advantages and disadvantages of affinity chromatography for mRNA purification compared to anion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and reverse ion pair chromatography, so it is necessary to select the appropriate chromatography method based on the characteristics of the molecules. At the same time, with the increasingly mature overall column chromatography and two-step purification processes, mRNA purification is pushed to a higher level.