Insight

mRNA vaccines feature rapid production and superior process versatility, granting them unparalleled advantages in responding to public health emergencies. The successful deployment of SARS-CoV-2 mRNA vaccines has greatly accelerated the development of numerous mRNA vaccine candidates targeting infectious diseases, cancers and genetic disorders, demonstrating enormous application potential. Currently, extensive research is underway focusing on nucleotide modification and optimization of lipid nanoparticle (LNP) delivery systems, aiming to further enhance the immunogenicity, efficacy and stability of mRNA vaccines. This article briefly describes the fundamental manufacturing workflow of mRNA vaccines.

The core principle of mRNA vaccines is to deliver antigen-encoding mRNA into host cells via appropriate delivery vectors. Host cellular machinery is then utilized to translate and synthesize target antigen proteins, which trigger immune responses against pathogens or abnormal cells and ultimately achieve immune protection or therapeutic effects.

Two major categories of mRNA vaccines have attracted widespread attention: conventional mRNA vaccines and self-amplifying RNA (saRNA) vaccines. Distinct from conventional mRNA vaccines, saRNA vaccines carry an additional open reading frame (ORF) that encodes RNA-dependent RNA polymerase (RdRp). This enzyme enables self-replication of antigen-coding mRNA, leading to higher and more sustained antigen expression. Therefore, saRNA vaccines can elicit robust immune responses at much lower dosages. Nevertheless, the excessive length of saRNA sequences and the potential activation of innate immunity remain key challenges in saRNA vaccine development. Both conventional mRNA and saRNA possess conserved structural elements including the 5′ cap, untranslated regions (UTRs), ORF and poly(A) tail, all of which are indispensable for efficient translation and antigen expression. Rational optimization of these components can improve translation efficiency and reduce the effective dosage of mRNA required for immune activation.

Eukaryotic mRNA contains a 7-methylguanosine (m7G) cap at the 5′ terminus. This structure binds to eukaryotic initiation factor 4E (eIF4E) to initiate protein translation. The 5′ cap also protects mRNA from degradation by exonucleases and helps exogenous RNA evade recognition by the host innate immune system. For in vitro transcribed (IVT) mRNA, 5′ capping is commonly achieved either by using vaccinia virus-derived enzymes or co-transcription with cap analogs such as ARCA and CleanCap. Incomplete capping generates uncapped mRNA transcripts, which are recognized as viral RNA, resulting in transcript degradation and unwanted immune activation. Accordingly, enzymatic dephosphorylation is routinely performed post-IVT to eliminate uncapped mRNA and mitigate adverse immune reactions.

The 5′-UTR and 3′-UTR regulate mRNA translation efficiency. The 5′-UTR facilitates ribosome recruitment and translation initiation, whereas long sequences with high GC content or stable secondary structures inhibit this process. Aberrant start codons within the 5′-UTR may disrupt normal translation of the target ORF. The 3′-UTR modulates mRNA stability, subcellular localization and translational activity. Optimization of UTRs effectively improves mRNA stability and protein output. Common strategies include adopting 5′-UTRs derived from highly expressed human genes such as α-globin and β-globin, screening optimal 3′-UTRs via random UTR libraries and SELEX technology, and designing high-performance 5′-UTRs through in silico prediction of ribosome loading and translation efficiency. These approaches play a vital role in improving the overall performance of mRNA vaccines.

Codon optimization of the ORF enhances translation efficiency and increases protein yield. However, excessively rapid translation may lead to protein misfolding and loss of immunogenicity. Related research directions include utilizing rare codons to modulate translation kinetics, adjusting the GC content of the ORF to improve mRNA stability and translation performance, and applying artificial intelligence (AI) to optimize ORF sequences, predict structural stability, conduct molecular modification and redesign secondary structures to eliminate degradation-prone regions.

The poly(A) tail is an essential native feature of eukaryotic mRNA, which maintains mRNA stability, regulates translation efficiency and extends its half-life in vivo. It is recognized by poly(A)-binding protein (PABP), which further interacts with eIF4G to form a translation initiation complex. The length of the poly(A) tail is critical to this process. Studies have demonstrated that a poly(A) tract consisting of 120 nucleotides delivers optimal protein expression for IVT mRNA vaccines.

Two mainstream methods are applied to append the poly(A) tail to IVT mRNA:

The entire production process starts with screening and identifying the optimal antigen sequence for the targeted pathogen or disease. The ORF sequence is then codon-optimized for the host organism, followed by construction of plasmid vectors. Plasmid DNA (pDNA) is produced and purified using Escherichia coli expression systems. Subsequent procedures include linearization of pDNA, in vitro transcription and purification of mRNA. Purified mRNA is encapsulated into lipid nanoparticles (LNPs) or other delivery carriers to form mRNA-LNP complexes. Finally, the final mRNA vaccine products are obtained through formulation development and lyophilization.

After optimization and validation of the antigen-encoding ORF, the target sequence is inserted into a plasmid vector containing all essential mRNA structural elements. The integrity of the full-length mRNA expression cassette is verified via DNA sequencing and restriction enzyme digestion. Recombinant plasmids are transformed into E. coli, and positive clones are selected for large-scale cultivation and amplification. GMP-compliant pDNA templates are fundamental to high-quality in vitro mRNA synthesis. Strict quality control criteria must be established to monitor process impurities and inhibitory contaminants, which may compromise IVT efficiency and downstream mRNA purification.

In vitro transcription (IVT) refers to the enzymatic synthesis of mRNA transcripts using DNA templates (pDNA or PCR products). Plasmid DNA is the preferred template for large-scale GMP production due to its cost-effectiveness, low mutation rate and excellent scalability. Firstly, restriction endonucleases are used to cleave the plasmid downstream of the poly(A) site for complete linearization, ensuring precise transcription termination. RNA polymerases are then employed to transcribe linearized pDNA into mRNA. Rigorous quality control strategies are implemented throughout the IVT process to maximize yield and minimize by-products, particularly double-stranded RNA (dsRNA).

High-purity mRNA is a prerequisite for homogeneous and stable vaccine formulations, as well as high-yield and high-fidelity antigen translation in vivo. Post-IVT, crude mRNA must be purified to remove residual reaction components and impurities including enzymes, template DNA, truncated transcripts and free nucleotides, with special emphasis on dsRNA removal. Prior to purification, DNase I is added to the reaction mixture under controlled conditions to digest residual template DNA and reduce feed viscosity. Resulting small DNA fragments are further eliminated via ultrafiltration or chromatographic techniques. Available purification platforms include lithium chloride (LiCl) precipitation, ion-exchange chromatography, affinity chromatography, size-exclusion chromatography and tangential flow filtration (TFF).

Naked mRNA cannot efficiently cross cell membranes and is highly susceptible to degradation by ribonucleases. Functional delivery systems facilitate cellular uptake of mRNA, extend its in vivo half-life and mitigate undesired innate immune activation. A variety of delivery strategies have been developed, including lipid-based carriers, peptide vectors, dendritic cell (DC)-targeted delivery systems and polymeric nanoparticles. Among these, LNPs represent the most advanced and clinically validated delivery platform, which have been successfully applied in FDA-approved COVID-19 mRNA vaccines and other investigational products such as Iribovax COVID-19 mRNA vaccine.

mRNA encapsulation into LNPs is typically performed via microfluidic mixing, where aqueous mRNA solution is blended with lipid dissolved in organic solvent. This technology enables scalable manufacturing and uniform particle distribution of mRNA-LNP formulations.

LNPs for mRNA delivery are composed of four key components: ionizable lipids, neutral helper lipids (e.g., distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE)), polyethylene glycol (PEG) lipids and cholesterol. Ionizable lipids carry positive charges under acidic conditions and become neutral at physiological pH, ensuring good biocompatibility and minimal non-specific interaction with cell membranes. PEG lipids stabilize nanoparticles by preventing particle aggregation and reducing binding to serum proteins. Cholesterol maintains the structural integrity of lipid bilayers. A core functional feature of LNPs is the capability to facilitate endosomal escape, allowing mRNA to release into the cytoplasm for antigen protein synthesis. This process involves endosomal membrane fusion: the acidic environment inside endosomes protonates ionizable lipids, which in turn disrupt the endosomal membrane and release encapsulated mRNA.

If the concentration of purified mRNA-LNP bulk solution fails to meet formulation specifications, concentration steps are carried out via ultrafiltration using flat sheet cassettes or hollow fiber membranes. The molecular weight cutoff (MWCO) of ultrafiltration membranes is selected within the range of 50–300 kDa based on the hydrodynamic radius of mRNA-LNP complexes. Critical process indicators for ultrafiltration include particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency and overall recovery rate before and after concentration. Compared with flat sheet cassettes, hollow fiber modules feature open flow channels and low-shear internal flow, which preserve the homogeneity of LNP particles during processing and achieve low residual volume after concentration. These advantages are critical for quality assurance and high recovery, and hollow fiber ultrafiltration has been widely adopted in large-scale mRNA vaccine production. To reduce microbial load prior to sterile filtration, gamma irradiation or pre-sterilized closed fluidic systems can be utilized.

Lyophilization is an effective approach to improve the thermal stability of mRNA vaccines. Optimized formulations combined with appropriate lyoprotectants enable excellent product stability under refrigerated and freeze storage conditions. After freezing, storage and reconstitution, the rehydrated LNPs should retain their original particle size and transfection activity.