Synthetic messenger RNA (mRNA) is fundamental to modern biotechnological advances owing to the success of the COVID-19 vaccine. When introduced into a host cell, mRNA can be a blueprint to synthesize therapeutic or immunogenic proteins. Thus, mRNA products can contribute to the advancements in vaccine development, cancer immunotherapy, and gene therapy. Nevertheless, inadequate efficiency and adverse effects observed in mRNA applications point to the importance of rigor in mRNA manufacturing steps, especially mRNA purification.

Despite its simple design and scalability, the success of mRNA applications depends on the purity of the mRNA construct. Manufacturers must ensure that the mRNA product only contains the custom sequence and is processed accurately during in vitro transcription (IVT). The difficulty of removing byproducts with structures similar to the full-length mRNA is a critical bottleneck. Even trace amounts of contaminants like truncated transcripts, residual DNA, and double-stranded RNA (dsRNA) can reduce expression efficiency and generate adverse effects, such as unintended immune responses. Furthermore, heterogeneous mRNA constructs may draw attention from RNases, resulting in premature degradation.

Overall, high-quality purification techniques are instrumental in ensuring high-quality mRNA products.

mRNA Purification Methods: An Overview

Classical mRNA purification methods include phenol-chloroform and precipitation techniques. Despite simplicity and cost-effectiveness, they do not meet the precision standard for high-quality mRNA purifications. More sensitive methods include column and bead-based purification and chromatography techniques. When selecting the most suitable method, manufacturers must consider scalability, efficiency at removing different impurities, and compatibility with downstream manufacturing. More rigorous purification methods must be selected for clinical applications where patient safety and regulatory compliance are paramount.

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Classical Methods

One of the conventional methods is phenol-chloroform extraction, which separates mRNA from other contaminants based on solubility. The sample is mixed with a phenol-chloroform solution, which is centrifuged to generate three distinct layers. Due to its hydrophilic nature, mRNA remains in the top aqueous phase, while the interphase contains residual proteins and DNA. The bottom organic phase comprises hydrophobic contaminants, such as lipids. The aqueous phase is washed and precipitated to obtain pure mRNA.

Phenol-chloroform extraction has several limitations. Firstly, the purified mRNA is significantly more unstable than DNA. The separation method may damage the mRNA structure. Furthermore, mRNA becomes prone to degradation by RNases found ubiquitously in laboratory environments. Thus, the extraction methods are not scalable for therapeutic applications.

Precipitation techniques involve treating the sample with lithium chloride (LiCl) or guanidinium-thiocyanate to separate mRNA from the solution. While LiCl reduces mRNA solubility and induces precipitation, guanidinium thiocyanate-based precipitation (GITC) denatures proteins and inactivates RNases to preserve mRNA integrity. Nevertheless, these methods are not scalable as they involve multiple steps requiring careful handling.

Column & Bead-Based Purification

Column and bead-based methods bind mRNA constructs in a solution, which is then washed away to remove the impurities.

In the silica-based spin column, the solution is mixed with a buffer containing chaotropic agents that disrupt the hydrogen bonds in dsRNA. Then, the solution is run through a silica spin column that preferentially binds single-stranded mRNA. This method lends itself to smaller and lower throughput volumes.

Magnetic bead-based purification uses oligo(dT) beads coated with thymidine nucleotides that have a strong binding affinity towards the 3' poly-A tails of polyadenylated mRNA. Once the binding occurs, the beads are removed from the solution with a magnetic field, followed by washing.

Although these methods are more efficient in mRNA purification than the classical methods, they are limited. Silica-based columns may reduce yield when saturated with mRNA. On the other hand, magnetic bead-based columns may exhibit non-specific binding to aberrant yet polyadenylated mRNA sequences.

Chromatography Techniques for mRNA Purification

Chromatography techniques are more reliable and scalable for purifying mRNA than the previously mentioned methods. They can help separate mRNA from impurities based on several physical and chemical differences. Their scalability and ease of automation lend them to high-throughput research and therapeutic applications. By avoiding toxic reagents used in the classical methods, chromatography provides a safer environment for the mRNA construct.

Affinity chromatography has a workflow reminiscent of magnetic bead-based purification. The column contains oligo(dT) tagged beads that bind polyadenylated mRNA. Unlike magnetic bead-based purification in a solution, oligo(dT) is immobilized on the column in affinity chromatography.

Ion exchange (anion exchange) chromatography separates mRNA from impurities based on electrostatic interactions. The chromatography resin is positively charged and can bind the negative charges on the mRNA backbone. Although other negatively charged particles may attach to the column, they can be washed away using a salt gradient that selectively separates weakly bound impurities from the column. Thus, the method offers high resolution and selectivity by isolating mRNA from other charged impurities like truncated or double-stranded RNA.

Size exclusion chromatography involves a column packed with porous beads that mRNA can fit perfectly. Larger molecules are washed immediately. Full-length mRNA is eluted next, while truncated mRNA takes longer to elute as it gets stuck in the pores.

Challenges in mRNA Purification & Solutions

Despite higher resolution and applicability, chromatography-based mRNA purification workflows may face obstacles. A single chromatography method may not suffice for complete separation, as impurities with similar size, charge, or affinity profiles may copurify with the therapeutic mRNA. More importantly, samples contaminated with RNase may cause rapid mRNA degradation. Therefore, mRNA purification protocols must be optimized to achieve highly purified mRNA while minimizing yield loss.

A powerful strategy is to use multiple purification strategies sequentially. For instance, affinity chromatography can initially separate polyadenylated mRNA from proteins and DNA. In contrast, a follow-up ion exchange chromatography can distinguish between single-stranded and aberrant mRNA. Furthermore, chromatography workflows can be optimized by shifting from batch to continuous mode.¹

Additionally, mRNA purification must be monitored to confirm efficiency. Fluorescence-based assays, HPLC, ELISA, and western blotting can be leveraged to perform quality control on the final product to ensure purity and integrity. These methods enable the detection of copurified DNA and protein impurities.

Non-chromatographic methods can enhance purification efficiency. One strategy is to use specific RNase III enzymes to digest dsRNA, which has demonstrated efficiency in CAR-T cell engineering.² Optimization of the IVT process is another possibility. For instance, a workflow involving high-temperature IVT using template-coded poly(A) tailing was reported to eliminate dsRNA before purification.³

Careful environmental control and reaction setup can prevent RNase degradation. Strategies include decontaminating the benches and equipment and using RNase inhibitors in the reaction mixtures during IVT.

Trends in mRNA Purification

Automation and AI-driven purification systems can overcome many of the challenges associated with mRNA purification. Automated platforms comprise equipment that eliminates manual labor, which would otherwise introduce contamination or batch variability. Automation enables simultaneous purification of large mRNA samples necessary for manufacturing mRNA vaccines and therapeutics.

Artificial intelligence (AI)–driven tools are rising to prominence in mRNA design and synthesis workflows. These tools may predict the risk of degradation and instability by inferring mRNA secondary structures from sequences.⁴ Machine learning algorithms can also be used to deduce the efficacy and safety of vaccine products from their formulation. When designing lipid nanoparticle (LNP)-mRNA vaccines, AI tools can be leveraged to identify LNP formulations that may react with mRNA constructs, jeopardizing their safety and translational efficiency.⁵

References

1. Qu J, Nair A, Muir GW, Loveday KA, Yang Z, Nourafkan E, et al. Quality by design for mRNA platform purification based on continuous oligo-dT chromatography. Mol Ther Nucl-Acids 2024;35(4).
2. Foster JB, Choudhari N, Perazzelli J, Storm J, Hofmann TJ, Jain P, et al. Purification of mRNA encoding chimeric antigen receptor is critical for generation of a robust T-cell response. Hum Gene Ther 2019;30(2):168-178.
3. Wu MZ, Asahara H, Tzertzinis G, Roy B. Synthesis of low immunogenicity RNA with high-temperature in vitro transcription. RNA 2020;26(3):345-360.
4. Binet T, Padiolleau-Lefèvre S, Octave S, Avalle B, Maffucci I. Comparative study of single-stranded oligonucleotides secondary structure prediction tools. BMC Bioinformatics 2023;24(1):422.
5. Bae SH, Choi H, Lee J, Kang MH, Ahn SH, Lee YS, et al. Rational Design of Lipid Nanoparticles for Enhanced mRNA Vaccine Delivery via Machine Learning. Small 2025;21(8):2405618.

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