mRNA has emerged as a promising new platform for developing vaccines and therapeutics. However, producing high-quality mRNA remains a challenge. Chromatography plays a critical role in purifying mRNA and removing impurities that can impact efficacy and safety. This article explores the key chromatographic tools used to enable high-yielding mRNA production.
Reverse phase chromatography, ion exchange chromatography, and affinity chromatography are the core techniques for purifying mRNA to the highest standards required for human use.
mRNA Production Workflow
The manufacturing process for mRNA can be divided into four main steps:
pDNA Template Isolation
The template for mRNA is encoded within plasmid DNA (pDNA) propagated in E. coli bacterial cells. After fermentation, the first step is to isolate the pDNA from the bacterial biomass. Common techniques include alkaline lysis followed by anion exchange chromatography. Robust purification is required to remove bacterial genomic DNA, RNA, proteins, and endotoxins.
The pDNA isolated from bacteria exists in a circular form. Linearization of the plasmid is required to enable efficient transcription of the mRNA. This is accomplished using restriction enzymes targeting a specific sequence within the plasmid. Optimization of the linearization process is needed to maximize complete cutting while avoiding degradation.
In Vitro Transcription (IVT)
Linearized DNA serves as the template for enzyme-driven transcription to synthesize the mRNA. Components of the IVT reaction include RNA polymerase, nucleoside triphosphates (NTPs), and buffer. Reaction conditions like time, temperature, and ratios of components must be optimized. The crude IVT mixture contains the full-length mRNA product along with shorter failure sequences, double-stranded RNA, proteins, and NTPs.
The crude IVT mixture must go through extensive chromatography-based purification to isolate the full-length mRNA drug substance. As covered in the sections below, multistep chromatography leveraging reverse phase, ion exchange, and affinity techniques is employed to remove impurities and maximize recovery.
Reverse Phase Chromatography
Reverse phase chromatography with SDVB is well documented for the separation of ssRNA from truncated forms, dsRNA, and DNA.
Reverse-phase chromatography separates molecules based on hydrophobicity. The stationary phase is hydrophobic, while the mobile phase is a polar organic solvent like acetonitrile.
As hydrophobicity increases, molecules interact more strongly with the stationary phase resulting in longer retention times. mRNA is amphipathic with a hydrophilic negatively charged backbone and hydrophobic nucleobases. These properties make the reverse phase an ideal first purification step.
Key outcomes from reverse phase chromatography:
- Remove failure sequences and nucleoside triphosphates
- Reduce double-stranded RNA
- Initial purification of full-length mRNA
However, the reverse phase lacks sufficient resolution between mRNAs of different lengths. This necessitates the use of other orthogonal techniques.
Ion Exchange Chromatography
Ion exchange chromatography separates molecules based on charge. The stationary phase contains immobilized charged functional groups that interact with the charged groups of biomolecules.
mRNA carries a strong net negative charge due to its phosphate backbone. Anion exchange chromatography uses a positively charged stationary phase to bind and resolve mRNAs based on length. Shorter failure sequences elute first followed by longer mRNA.
Key outcomes from ion exchange chromatography:
- High-resolution purification of full-length mRNA
- Remove remaining double-stranded RNA
- Reduce overall impurity levels
The high selectivity of ion exchange makes it a workhorse technique for mRNA purification. However, any remaining closely related impurities will require another orthogonal method.
Affinity chromatography leverages reversible and highly specific biomolecular interactions. This technique requires immobilizing an affinity ligand that will bind the target molecule.
Affinity chromatography can help remove remaining impurities like proteins by using immobilized antibodies specific to those contaminants. mRNA itself can also be purified by using a sequence-specific oligonucleotide ligand.
Key outcomes from affinity chromatography:
- Highly selective capture of full-length mRNA
- Final polishing step to remove trace proteins
- The highest purity required for therapeutic use
Carefully designed affinity ligands enable mRNA purification while avoiding product losses. Affinity chromatography provides the final polishing step for an optimal high-yielding process.
Hybridization Affinity Chromatography
Hybridization affinity chromatography can provide highly specific purification of mRNA. This technique uses immobilized oligonucleotide probes designed to be complementary to the target mRNA sequence.
The mRNA will hybridize or bind to the complementary oligo probe under optimized conditions like temperature and salt concentration. Other impurities will not hybridize and can be washed away. Changing conditions then allows the purified mRNA to be eluted.
The selectivity of hybridization affinity chromatography depends on the oligo probe design. Probes targeting the 5’ or 3’ untranslated regions can allow purification of the full-length mRNA from shorter failure sequences.
Hydrophobic Interaction Chromatography
Hydrophobic interaction chromatography (HIC) separates molecules based on hydrophobicity like reverse phase, but uses different conditions. While the reverse phase uses organic solvents, HIC uses high salt concentrations.
Under high salt, water molecules hydrate the salt ions forcing hydrophobic regions of biomolecules to interact. As salt is decreased, these interactions weaken and molecules elute.
For mRNA, HIC is particularly useful for removing endotoxins which tend to be more hydrophobic. Endotoxins will be retained on the HIC resin under high salt while the mRNA can be collected in the flow-through.
Careful optimization of salt type and concentration is needed to balance mRNA recovery and endotoxin clearance. The orthogonality of HIC makes it a complementary tool for mRNA purification processes.
Additional Tools for Process Development
Other key chromatographic techniques for mRNA purification process development include:
- Multimodal chromatography using mixed-mode ligands
- Membrane chromatography for flow-through polishing
Frequently Asked Questions
What are the critical quality attributes for mRNA?
The purity, integrity, and potency of full-length mRNA are critical quality attributes. Key impurities to remove include proteins, failure sequences, double-stranded RNA, and nucleoside triphosphates.
Why is yield important for mRNA production?
Given the large doses required for mRNA vaccines and therapeutics, high yields are essential to manufacture the quantities needed at a reasonable cost. Maximal recovery during purification is key.
How does chromatography purity and separate mRNA?
Chromatography leverages the unique physicochemical properties of molecules. Strategically combining methods like reverse phase, ion exchange, and affinity chromatography enables the removal of impurities and the isolating of pure mRNA.
What is the difference between linear and plasmid DNA templates?
Linear DNA is easier to amplify and transcribe but less stable. Plasmid DNA is more stable but requires extra steps to linearize the circular plasmid and remove bacterial contaminants.
Are there significant equipment differences for mRNA production?
While standard biopharm equipment is used, the sensitivity of mRNA requires specialized handling including the use of low-binding plastics and RNase-free consumables as well as avoiding stainless steel.
- Sahin, U., Karikó, K. & Türeci, Ö. mRNA-based therapeutics — developing a new class of drugs. Nat Rev Drug Discov 13, 759–780 (2014).
- Pardi, N., Hogan, M.J., Porter, F.W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nature Reviews Drug Discovery 17, 261–279 (2018).