mRNA technology has emerged as a versatile platform, finding applications in preventive and therapeutic vaccines, tumor treatment, immunotherapy, protein replacement, and gene editing. However, during in vitro transcription (IVT) of mRNA, a range of impurities are generated alongside the desired product. These include reaction enzymes like T7 RNA polymerase and inorganic pyrophosphatase, nucleoside triphosphates (NTPs), DNA templates, salt ions, and by-products such as double-stranded RNA (dsRNA) and truncated RNA fragments. These impurities can interfere with accurate mRNA quantification, reduce protein translation efficiency, and trigger immunogenic responses. Therefore, purification is a critical step post-IVT to enhance the purity and integrity of the target mRNA, ensuring the safety and efficacy of the final product.

Conventional Purification Methods

Traditional mRNA purification techniques encompass lithium chloride (LiCl) precipitation, magnetic bead purification, ammonium sulfate precipitation, and affinity chromatography. LiCl precipitation and magnetic bead methods are relatively straightforward and rapid but are typically limited to small-scale applications, making them more suitable for early research and development phases. In contrast, chromatography, particularly affinity chromatography, is the predominant method in industrial settings, albeit requiring specialized purification equipment.

Affinity Chromatography: The Mainstream Method

Oligo dT affinity chromatography is the leading approach within affinity chromatography. This method utilizes an Oligo dT resin, which is based on polystyrene and is coated with numerous hydroxyl and functionalized poly(dT) groups. Under high-salt conditions, salt ions neutralize the electrostatic repulsion between the negatively charged resin and mRNA, facilitating interaction between the mRNA molecule and the Oligo dT ligand. The poly(A) tail of the mRNA forms hydrogen bonds with the functionalized poly(dT) group, capturing the mRNA. Impurities lacking a poly(A) tail, such as free nucleotides, short transcription products, and enzymes, do not bind to the Oligo dT column. When the buffer is salt-free, the negative charges on the phosphate groups of the dT ligand backbone and the poly(A) backbone create electrostatic repulsion, overcoming the hydrogen bonds and allowing for efficient elution of mRNA with a yield exceeding 90%.

A New Purification Kit

Traditional affinity chromatography necessitates specialized equipment and continuous process optimization to meet industrial demands. However, a novel lightweight affinity chromatography purification kit has been introduced, simplifying the process. This kit requires only centrifugation to purify samples within 30 minutes, making high-quality mRNA readily accessible for various downstream experiments.

Product Features

• High Efficiency and Flexibility: 

Suitable for RNA samples of varying starting quantities, capable of purifying milligram-level mRNA samples.

• Convenient and Economical: 

Eliminates the need for purification equipment or low-temperature devices; straightforward operation reduces equipment and labor costs.

• Good Reproducibility: 

Yields mRNA with high integrity, significantly removes NTPs, and completes purification within 30 minutes, catering to diverse experimental requirements.

Performance Data

Compared to the LiCl precipitation method, the affinity chromatography purification kit demonstrates superior performance in enhancing capillary electrophoresis (CE) integrity, size exclusion chromatography (SEC) purity, and NTP removal. When using the self-developed purification kit versus the LiCl precipitation method for mRNA fragments with different poly(A) lengths, the integrity of the poly(A) tail fragments is notably improved for the same mRNA fragment sequence.

Product Information

• HBP005016-1: 5T
• HBP005016-2: 10T

• HBP005016-3: 20T

References

1.LiCl Precipitation for RNA Purification, Retrieved Jan 8, 2024.
2.RNA Clean XP Performance and Data, Retrieved Jan 8, 2024.
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4.J Koubek, KF Lin, YR Chen, RP Cheng, JJT Huang, Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription, RNA 19 (2013) 1449-1459.
5.Xue Feng, Zhengjun Li, Zhiguo Su, Shiyi Che, Baiqian Dai, Yuan Cheng, Songping Zhang. Rapid and high recovery isolation of mRNA from in vitro transcription system by ammonium sulphate precipitation at room temperature. Separation and Purification Technology. Volume 336, 2024, 126331.