mRNA is an important intracellular molecule that carries genetic information transcribed from DNA and directs protein synthesis. In biotechnology and medical fields, especially vaccine development and gene therapy, mRNA technology occupies a pivotal position and holds enormous market potential.
1 Overall Process Flow of mRNA Vaccine
1.1 pDNA Preparation
Plasmid production, pDNA manufacturing and purification
1.2 IVT Reaction
Complete pDNA linearization and in vitro RNA transcription.
5′ capping is adopted to prevent mRNA degradation.
1.3 mRNA Downstream Processing
Concentration & Diafiltration: Conduct buffer exchange and remove impurities such as T7 RNA polymerase.
Purification: Adopt combined application of Oligo dT and IEX/HIC for mRNA purification.
Filtration: Apply TFF to complete buffer replacement and remove enzymes, DNA, nucleotides and other impurities, followed by sterile filtration.
1.4 LNP Preparation
Complete lipid nanoparticle encapsulation and implement overall quality control.
1.5 Formulation & Filling
Carry out sterile filtration, aseptic filling and subsequent cryopreservation.
2 Purification Process of Plasmid DNA (pDNA)
As the starting raw material for mRNA production, pDNA preparation adopts two mature mainstream processes, including the classical three-step method and the improved two-step method.
Three-step method: Simple operation and strong platform compatibility
Two-step method: Short process cycle, easy scale-up and low production cost
2.1 Classical Three-Step Purification Method
Step 1 Alkaline Lysis
Alkaline lysis is used to selectively precipitate chromosomal DNA and other macromolecular impurities.
Resuspension buffer: 50mM Tris-HCl, 10mM EDTA-2Na, 50mM Glucose, pH 8.0
Lysis buffer: 0.2M NaOH, 1% SDS
Neutralization buffer: 3M KAc, 5M HAc
Key Process Parameters
Core control indicators cover bacterial resuspension concentration, lysis time and neutralization solution ratio.
Excessively high resuspension concentration will lead to insufficient lysis, while excessively low concentration will increase process volume and overall production cost.
Improper lysis time causes incomplete lysis and low product yield. Excessively long lysis time will increase the proportion of open-circular plasmid and reduce the quality of supercoiled plasmid.
Parameter interaction exists in the whole process. DOE experimental design is recommended to confirm the optimal process conditions.
Step 2 Clarification
The system will form flocculent precipitate after alkaline lysis and neutralization.
Add an appropriate amount of ammonium bicarbonate or sodium bicarbonate to promote solid-liquid separation, and then complete clarification through depth filtration or centrifugation.
Step 3 UF/DF Ultrafiltration & Diafiltration
Main functions include removal of residual RNA, HCP, pigments and other impurities, reduction of process volume and reduction of load in subsequent chromatographic procedures.
Optional process media include ultrafiltration membrane cassettes and hollow fiber membranes.
Hollow fiber membranes are more suitable for fragile plasmids due to lower shear force.
Key Process Parameters
Main control indicators include ultrafiltration loading capacity, shear force, concentration factor and transmembrane pressure.
Avoid excessive concentration which may cause plasmid aggregation. Avoid excessive shear force that damages plasmid structural integrity.
Step 4 Chromatographic Polishing
(1) Size Exclusion Chromatography (SEC)
Main purpose is the removal of residual RNA.
Under optimal salt concentration conditions, pDNA with larger molecular size elutes first and RNA elutes later, so as to realize effective separation.
Equilibration buffer: 2.1M (NH₄)₂SO₄, 10mM EDTA, 100mM Tris-HCl, pH 7.5
Process parameters: Loading volume less than 0.3 CV, column height not lower than 30 cm, linear flow velocity lower than 60 cm/h
High-concentration ammonium sulfate can shrink RNA molecules, maximize the size difference between DNA and RNA, and maintain direct compatibility with subsequent thiophilic affinity chromatography.
(2) Thiophilic Affinity Chromatography
Supercoiled plasmid presents higher base exposure degree and surface charge compared with open-circular plasmid, which enables stronger binding with thiophilic media and achieves efficient separation.
Two mainstream separation strategies are available.
The first strategy works under high-salt binding conditions. Both supercoiled plasmid and open-circular plasmid bind to the medium. Gradient washing is adopted to elute open-circular plasmid, and supercoiled plasmid is collected in the final elution step.
The second strategy relies on flow-through separation. Adjust the salt concentration to allow open-circular plasmid to flow through directly, while supercoiled plasmid remains bound on the column.
Equilibration buffer: 2.1M (NH₄)₂SO₄, 10mM EDTA, 100mM Tris-HCl, pH 7.5
Elution buffer: 0.3M NaCl, 1.7M (NH₄)₂SO₄, 10mM EDTA, 100mM Tris-HCl, pH 7.5
(3) Anion Exchange Chromatography (AEX)
Main purpose is the removal of trace endotoxins and residual micro-impurities.
Equilibration buffer: 0.4M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5
Elution buffer: 1M NaCl, 10mM EDTA, 100mM Tris-HCl, pH 7.5
Step 5 Final UF/DF & Sterile Filtration
Complete product concentration and buffer exchange to reach the target concentration and solvent system.
Common storage solvent types:
TE buffer, Tris-HCl buffer, PBS, WFI, Nuclease-free water
TE buffer is suitable for long-term plasmid storage with EDTA inhibiting nuclease activity.
WFI is the preferred option for subsequent plasmid linearization and IVT reaction arrangement.
Strictly control ultrafiltration loading capacity and shear force to prevent prolonged operation time and plasmid quality deterioration.
2.2 Improved Two-Step Purification Method
Adopt calcium chloride precipitation to replace traditional SEC for RNA removal, so as to shorten process time and reduce ammonium sulfate consumption.
Core control parameter: CaCl₂ concentration
Excessively high concentration will cause co-precipitation of supercoiled plasmid and lead to low yield
Excessively low concentration results in incomplete RNA precipitation, high RNA residue and increased load of chromatographic procedures
Complete clarification and UF/DF treatment after calcium chloride precipitation. Select SEC, affinity chromatography or ion exchange chromatography. Mixed-mode media is also applicable to remove HCP, endotoxins and open-circular plasmid impurities.
Mixed-mode media integrates the comprehensive effects of size exclusion, ion exchange and hydrophobic interaction to realize synchronous removal of multiple impurities.
3 Plasmid Linearization
Use restriction endonuclease digestion to linearize supercoiled pDNA.
Adopt ion exchange chromatography to remove residual enzymes and process impurities. Configure UF/DF buffer exchange to fully meet the requirements of IVT reaction system.
4 mRNA Purification Process After IVT Reaction
Arrange two-stage purification procedure.
Primary capture adopts Oligo dT affinity chromatography to remove residual enzymes, NTPs and template DNA.
Fine polishing applies high-resolution HIC or IEX media to remove truncated fragments, dsRNA and other product-related impurities.
Application of Mixed-mode Chromatography
Mixed-mode chromatography can serve as an alternative to traditional Oligo dT process. It features higher loading capacity and can effectively remove digested pDNA, host proteins and NTPs.
Its comprehensive advantages include shorter process time, smaller required column volume and lower overall production cost.
