mRNA Analysis Using (U)HPLC Separation Methods

Ensuring mRNA purity is critical for determining its stability and protein yield during translation. Excess components (short or double-stranded RNA, DNA) can be removed by purification to improve translation efficiency.

Purification of mRNA

Purification of mRNA is most effectively accomplished using an ion-exchange column to assure higher yields. Thermo Scientific DNASwift SAX-1 Columns contain a high surface area, monolithic stationary phase with high loading capacity, which delivers high yield in analytical scale purifications, as shown in the brochure DNASwift Monolith Column for DNA and RNA Oligonucleotide Purification. DNASwift brand columns are recommended for milligram-scale separations of crude mixtures when purification is a greater priority than resolution.

 

During the enzymatic manufacturing process of mRNA therapeutics, incomplete mRNA products are generated in conjunction with other potential impurities such as double-stranded (ds) RNA. Additionally, RNA and RNA therapeutics can be degraded by exposure to heat, hydrolysis, oxidation, light, and ribonucleases throughout both manufacturing and storage. It is important to assess batch-to batch consistency, process repeatability, and the resulting mRNA quality during the production of mRNA therapeutics.

Reproducible, controlled partial digestion using immobilized RNase magnetic beads.

RNase mapping methods can be used for RNA sequence mapping to rapidly identify, characterize, and sequence mRNA therapeutics with high sequence coverage, providing important information for identity testing, sequence validation, and impurity analysis.

 

RNase digestions, possible using Thermo Scientific SMART Digest RNase Kits, are designed to induce missed cleavages in order to create longer fragments that can be uniquely matched to the original RNA sequence. Our SMART Digest RNase kits offer significant advancements in sample preparation for oligonucleotide mapping by providing a method for fast and simple mRNA digestion with high reproducibility, high sensitivity, and high quality data in a format that is compatible with automation.

Optimization chromatograms of partial RNase T1 digests of mRNA.

It is beneficial to perform a partial RNase digestion as a full digestion will produce too many small oligonucleotides. Partial digestions can be challenging, but with specifically developed Thermo Scientific SMART Digest RNase T1 Enzymes immobilized on magnetic particles, the enzymatic reaction can be controlled by simply removing the magnetic particles after a short, defined period of time. Separation is then performed on a long Thermo Scientific DNAPac RP Column to assure the necessary peak capacity for such a mapping sequence. The full setup of such a mapping sequence is described in detail in the article Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry.

HPLC Column Selection Guide

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Introducing mRNA into cytoplasm

Irrespective of their therapeutic mechanism of action, the large size of some therapeutic RNA molecules such as mRNAs, their anionic charge, and their susceptibility to RNases present in both the bloodstream and tissues make it difficult for therapeutic RNAs to enter cells efficiently and function on their own.

 

Lipid nanoparticles (LNP) are effective nonviral delivery vehicles for the in vivo delivery of nucleic acids and vaccines. LNPs have been increasingly used in the fields of gene therapy, protein replacement therapy, and mRNA-based vaccines development for the treatment of cancer and infectious diseases. The delivery of the mRNA molecule requires cellular uptake of the LNP delivery system followed by endosomal escape of the mRNA into the cytosol of the targeted cell to start the translation process. The composition of the LNP is an important aspect for function and so must be characterized in the formulation process. As such, the identification, ratio, and purity of the lipids in the formulation are regarded as critical quality attributes for safety and efficacy.

 

Thermo Scientific Accucore C30 Columns are ideal for fast, high-resolution separations of hydrophobic, long-chain compounds. An appropriate method for analyzing LNPs using an Accucore C30 column can be found in the application note Characterization of lipid nanoparticle (LNP) composition using UHPLC-CAD.

Separation of LNP formulation containing four different lipids.

For Research Use Only. Not for use in diagnostic procedures.