mRNA Purification

mRNA transcribed in vitro by T7 RNA polymerase may contain a variety of contaminants. BOC Sciences offers purification services to remove contaminants from modified nucleotide-containing mRNAs to improve processing efficiency for downstream applications.

Introduction

Related Impurities During IVT

In process production, mRNA is synthesized by in vitro IVT reaction using linearised plasmid DNA as the template and T7 RNA polymerase as the vehicle. T7 RNA polymerase is a single-subunit enzyme with high fidelity and enables precise transcription. Other essential components of the IVT reaction include free nucleotides (NTP), transcriptional buffer, polymerase cofactor magnesium ion, antioxidants, spermidine, etc. Removal of immunogenic by-products from the IVT reaction is necessary to ensure the efficiency and safety of the mRNA drug.

Template DNA

If the mRNA stock solution generated by the IVT reaction contains intact template plasmid DNA, then the intact plasmid DNA will also be wrapped in it when the mRNA is subsequently encapsulated using LNP.

Endotoxin

Endotoxin is a complex of lipopolysaccharides and proteins in the cell wall of Gram-negative bacteria, containing the highly immunogenic lipid A.

RNA polymerase

At present, the most mainstream RNA polymerase in IVT reaction is T7 RNA polymerase. In addition, T3 RNA polymerase and SP6 RNA polymerase can also be used in this reaction. RNA polymerase is produced by bacterial fermentation, so it may also contain endotoxin.

Free mononucleotides

Free nucleotides that are not added to mRNA in the IVT response may induce neuroinflammatory responses in the central nervous system and are therefore one of the impurities that need to be removed.

DNA-RNA hybrid fragments

In the IVT reaction, RNA polymerase initiates transcription from the T7 promoter region and synthesizes mRNA along the 5'-3' direction. If the template DNA is not linearised, then there will be continuous transcription, leading to the production of extra-long transcripts, so the template must be linearised by restriction endonucleases.

Partial transcripts

During the initiation of transcription in the IVT reaction, transcriptional events are frequently aborted, producing short-stranded 5-11nt RNA fragments.

Double-stranded RNA

T7 RNA polymerase has DNA-dependent RNA polymerase activity in addition to RNA polymerase activity, resulting in the formation of dsRNA (double-stranded RNA) by-products.

mRNA Purification Methods

LiCl precipitation purification method

In LiCl solutions, RNA can be effectively precipitated out, while proteins, carbohydrates or DNA are difficult to precipitate out.

Affinity chromatographic purification

By binding with the Poly(A) tail and performing affinity purification, most impurities such as NTPs, enzymes, and DNA can be removed, but dsRNA and truncated RNA cannot be distinguished.

Anion chromatography purification

According to the charge difference of various components in the IVT reaction stock solution, the target mRNA is separated from the plasmid DNA template, free nucleotides, aborted RNA short chains and T7 RNA polymerase by anion exchange chromatography.

Hydrophobic chromatographic purification

Nucleic acids are on average weakly hydrophobic and require high salt concentrations to facilitate the binding of RNA molecules to the hydrophobic chromatographic matrix. The high salt stabilizes the interaction between non-specific impurities and nucleic acids, allowing these complexes to bind to the hydrophobic column and to bind more strongly than the target RNA molecules.

Hollow fibre dwelling TFF

Tangential flow filtration (TFF), also known as staggered flow filtration, is a process whereby the feed stream is parallel to the membrane surface, part of the feed stream passes through the membrane (filtrate) and the rest of the feed stream (retentate) is recycled back to the feed vessel.

mRNA Purification