HomeVaccine ManufacturingManufacturing Strategies for mRNA Vaccines

Manufacturing Strategies for mRNA Vaccines

The rapid development and success of COVID-19 vaccines proves the power of mRNA – but the potential extends well beyond this application: mRNA technology  holds exciting promise to address unmet medical needs and offers new therapeutic options in the fight against cancer, heart disease or infectious diseases.

Delivery of mRNA into the cytosol of a patient cell can induce the production of a target protein which can function as a therapeutic or prophylactic, act as an antigen to trigger an immune response for vaccination purposes, replace a defective protein or activate an anti-tumor response.

In contrast to viral delivery systems such as adeno-associated virus (AAV) vectors which are widely used in vaccines and gene therapies, non-viral delivery systems such as mRNA technology offer multiple benefits: a better safety profile, a high degree of versatility, and simplified manufacturing using a templated process. Development scientists are dedicated to enhancing the stability, translation, and safety of mRNA by optimizing processes and delivery platforms.

View all of our products and services for mRNA development and manufacturing.

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Considerations for mRNA Manufacturing

The development and manufacturing of mRNA vaccines and therapeutics are comparatively simple, scalable and extremely rapid (Figure 1). With a compressed timeframe from development to clinic and towards approval, mRNA technology is attractive not only as a fast and effective response to infectious disease outbreaks, but also for development of novel therapeutic approaches to address diseases with unmet needs.

mRNA is produced by in-vitro synthesis through an enzymatic process. In contrast to classical in-vivo protein expression, no time-consuming cloning and amplification steps, no removal of cells and host cell proteins are needed. This simplified manufacturing process demonstrates remarkable speed and flexibility, as it uses the same reaction materials and vessels for any target which allows GMP facilities to switch to a new protein target within a very short timeframe, with minimal adaptation to process and formulation.

General process overview for mRNA manufacturing

Figure 1.General process overview for mRNA manufacturing.

There are several key considerations to make to optimize the process, yield, quality, and safety of the final product. This includes, for example, the selection of the right process chemicals and raw materials. Especially during in-vitro transcription and downstream purification, the mRNA is unprotected with a high risk for enzymatic degradation. The use of products that are tested for the absence of endonuclease activity minimizes risk of RNase-induced degradation throughout the process, from making to purification to formulation of the mRNA. To control the risk of microbial and endotoxin contaminations, it is also important to use products with specified low microbial and endotoxin levels. This is especially critical for high-risk applications such as injectables because of the increased potential for contaminating microbes to cause harm.

This webpage is dedicated to mRNA and provides in-depth information on the selection of technologies and products, helping you to navigate the mRNA process from manufacturing to final fill with confidence. To support your informed decision, we are providing detailed information about products, services and expertise in our brochures "Enabling Capabilities and Solutions for all mRNA platforms" and “Process chemicals for mRNA drug manufacturing”.


The mRNA construct is designed to ensure efficient expression of the gene of interest. Stability, gene expression and efficient protein translation depend upon several structural elements (Figure 2):

mRNA structure

Figure 2.mRNA structure.

  • Cap region at the 5’ end of the sequence: Essential for mRNA maturation, recognition by the ribosome for an efficient protein translation, and protection from nuclease digestion for improved stability.
  • Untranslated regions (UTRs) at the upstream and downstream domains of the mRNA coding region: Regulating mRNA translation, localization and stability; can be utilized to improve protein expression efficiency.
  • Open reading frame or coding sequence region: Contains the gene of interest (GOI).
  • Poly(A) tail: Crucial for protein translation and mRNA stability by preventing digestion by 3’ exonuclease.

Making mRNA

Production of mRNA-based therapeutics and vaccines begins, as shown in Figure 1, with a plasmid DNA (pDNA) template that is then linearized and transcribed into RNA:

  • pDNA production: The pDNA template contains a DNA-dependent RNA polymerase promoter and the corresponding sequence for the mRNA construct. Given the central role of the pDNA construct, its design and purity are important factors for optimizing the mRNA product. The required pDNA is amplified within bacterial cells, typically E. coli, and subsequent purification steps yield a pure, concentrated, circular pDNA. Strategies to overcome challenges related to the large size of the nucleic acid and its high viscosity, shear sensitivity and the similarities between pDNA and impurities are addressed in-depth in our publication “Designing a Plasmid DNA Downstream Purification Process”.
  • pDNA Linearization: The circular pDNA is linearized using a restriction enzyme in a reaction buffer1 to serve as a template for the RNA polymerase to transcribe the desired mRNA. Linearization is required to avoid transcriptional readthrough events that may generate undesired forms of mRNAs leading to additional impurities that would need to be removed.
  • pDNA purification: Impurities such as the restriction enzyme, bovine serum albumin (BSA), DNA fragments, endotoxins and others are removed. The majority of lab-scale processes utilize solvent extraction techniques, which are not suitable for GMP production environments. As an alternative, tangential flow filtration (TFF) and chromatography are efficient impurity removal techniques for this purification step. Endotoxins have been identified as a critical impurity from the pDNA manufacturing process with a high impact on subsequent process steps and, ultimately, patient safety. Detergents can be used for endotoxin removal, e.g. using Deviron® C16 detergent as a suitable sustainable, biodegradable and REACH compliant alternative to traditional detergents.2

Learn more about pDNA production and purification in our dedicated technical article.

  • In-vitro transcription (IVT): The linearized pDNA, serving as the DNA template, is transcribed into mRNA in an enzymatic reaction using elements of the natural transcription process. Critical components in in-vitro transcription include the RNA polymerase to transcribe the DNA sequence into an RNA sequence, nucleoside triphosphates (NTPs) as building blocks of the mRNA, inorganic pyrophosphatase (IPP) to improve mRNA yield, and RNase inhibitors to prevent RNA degradation. In the transcription buffer, typically chemicals such as dithiothreitol (DTT; a disulfide bond reducer inhibiting RNase activity, thus supporting mRNA stability) and spermidine (a component that improves transcription efficiency and nucleic acid stability) are included.
    Raman, as a powerful analytical tool, could potentially be used to monitor enzymatic reactions, such as the IVT step. To date, tedious off-line analytics are performed to monitor the IVT step, which does not allow for immediate action if reaction conditions and/or productivity targets are not met. This issue could be resolved with Raman spectroscopy, which allows for much faster measurements of CPPs & CQAs, enabling operators to make faster decisions to ensure optimal process conditions.
  • Capping: Following transcription, the final mRNA structure requires a 5’ cap structure for stability and efficient transduction in the cell. The cap can be added in two ways – either co-transcriptionally or post-transcriptionally by a two-step enzymatic process.
    Co-transcriptional capping, usually uses cap analogs and guanosine triphosphate (GTP) in the transcription mix, added at a ratio of four cap analogs for one GTP. Co-transcriptional capping is less expensive and faster than enzymatic capping as it is performed during the IVT step, in the same reactor mix. However, efficiency and yield are lower, and it can generate non-capped impurities due to wrong binding or reverse incorporation. Antireverse cap analogs (ARCA) have been developed to prevent reverse incorporation of a 5’ cap, leading to higher translation efficiency.
    Enzymatic capping (or post-translational capping) is performed after mRNA purification from the in-vitro transcription mixture. This reaction usually uses a vaccinia virus-capping enzyme to add the capping structure to the mRNA structure. While enzymatic capping has a very high capping efficiency, it is more expensive and requires an extra unit operation.

These process steps are followed by purification and formulation of the mRNA to yield the final drug product.


Following the in-vitro transcription step, mRNA is purified from the impurities and materials used in the previous steps including endotoxins, immunogenic double stranded RNA (dsRNA), residual DNA template, RNA polymerase and elemental impurities. At this stage, the mRNA will need to be in the appropriate buffer for TFF, enzymatic capping and/or chromatography steps. Several options are available for mRNA purification and removal of residual DNA:

  • Tangential flow filtration (TFF) is used for an efficient separation of mRNA from smaller impurities that are not retained by the membrane. Typically, the DNA template is degraded by the addition of DNases; the resulting small DNA fragments can then be easily separated from larger mRNA molecules using TFF. Based on the size of the mRNA, molecular weight cut-offs ranging from 30 to 300 kDa can be used. With TFF it is possible to purify, concentrate and diafilter the product within the same unit operation. However, small DNA fragments can hybridize to the mRNA, generating additional impurities, which is avoided if capture is used to remove the DNA template.

  • Chromatography techniques such as reverse-phase ion pair (IPRP),  anion exchange (AEX) and affinity chromatography (AC) using poly(dT) capture (Figure 3) provide an efficient means for DNA template removal, eliminating the need for DNA template digestion and the risk of hybridization that can occur during ultra-/diafiltration steps.1 To remove unwanted products and oligonucleotide impurities, chromatography is also used after the enzymatic capping step. However, it is more expensive and media exchange and preparation for the subsequent step still necessitates a TFF step.
Comparison of reversed-phase ion-pair, anion exchange and affinity chromatography for mRNA purification (DBC: dynamic binding capacity).<sup>4,5</sup>

Figure 3:Comparison of reversed-phase ion-pair, anion exchange and affinity chromatography for mRNA purification (DBC: dynamic binding capacity).4,5

  • Reversed-phase ion pairing (IPRP) is commonly used at small scales and allows a very efficient and rapid RNA purification and good separation of single stranded RNA (ssRNA) from DNA, double stranded RNA (dsRNA), and short transcripts. Drawbacks of this method include the use of solvents impacting suitability for GMP manufacturing, complexation of mRNA by ion-pair reagents requiring diafiltration steps for complex removal, and its sensitivity to protein and aggregate fouling making this technique better suited for polishing than for capture.

  • Anion exchange chromatography (AEX) has a high dynamic binding capacity of > 10 mg RNA/mL and is very efficient for removing immunogenic impurities such as dsRNA, uncapped RNA, RNA–DNA hybrids and other RNA structures such as hairpin mRNA. While AEX allows the use of aqueous solutions, it might require the addition of potentially toxic chaotropic agents and an operation at temperatures of up to 85 °C to desorb large mRNA molecules bound to the resin. Elution at ambient temperature is typically applicable to mRNA species < 500 bases.5

  • Affinity chromatography (AC) poly(dT) capture uses a resin to specifically capture the poly(A) tail of full-length mRNA transcripts. This process efficiently removes DNA, nucleotides, enzymes, buffer components and any other impurities not having a poly(A) tail. Same as AEX, it allows for the use of aqueous solutions, in the case of AC typically a salt gradient. Unlike IPRP and AEX, AC cannot discriminate dsRNA from ssRNA and is not efficient for removing other product-related impurities such as DNA fragments that have hybridized to the mRNA. For this reason, a common approach is to apply AC as an initial chromatography step, followed by AEX for polishing purposes.

  • A final concentration and diafiltration is performed following the chromatography step(s) to maximize product purity and transfer the mRNA into the appropriate buffer for formulation or storage. At this stage, mRNA can be further purified, concentrated and diafiltered within the same unit operation. A sterile filtration step can be performed following this TFF step; however, sterilizing grade filtration of mRNAs with a molecular weight of 5000 kDa or higher can be challenging.


After the final mRNA purification step, the next consideration is the delivery mechanism (Figure 4). Delivery tools are crucial to ensure effectiveness of mRNA vaccines and therapeutics. One of the most advanced delivery approaches is based on combinations of lipids and polymers, including complexes of oligonucleotides bound to lipids forming a lipoplex or positively charged polymers such as polyethyleneimine (PEI) forming polyplexes. Lipid nanoparticles (LNP) are the most commonly used mRNA delivery platform.

Several mRNA delivery systems are available

Figure 4.Several mRNA delivery systems are available.

Considerations for lipid selection

When choosing lipids, it is essential to consider the delivery route to ensure maximum effectiveness and optimal biodistribution. Along with lipid selection, the ratio between individual lipids is crucial for fine-tuning, directly impacting bilayer fluidity and LNP fusogenicity. Multiple critical factors come into play when choosing a lipid, including type, source, and quality, which directly influence impurity profile and properties such as particle characteristics, stability, and release profile in the final formulation. Consistent lipid quality is necessary for reproducible results, heavily reliant on the raw materials' quality used for lipid synthesis and the lipid's material characteristics. Synthetic lipids  with a consistent, high-product quality coupled with tailored support and services by a trusted partner are ideal to meet individual needs and ensure an optimal final product performance.

Each LNP consists of four different lipids allowing the mRNA to be carried in it and protected from degradation:

  • Cationic/ionizable lipids are required for encapsulating the RNA via electrostatic interactions. Delivery to hepatocytes (for boosting or silencing of protein expression) requires ionizable lipids (passive targeting, endosomal release) whereas uptake by immune cells is much easier. Strong cationic lipids also serve this purpose and are responsible for the efficient release of RNA into the cytoplasm. The structure of cationic lipids significantly affects LNP activity, toxicity, and biodistribution.

  • Polyethylene glycol (PEG) lipids provide colloidal stability and prevent protein binding to the particle, thereby shielding it from the immune system and achieving longer circulation. The length of the PEG chain and fatty acid chains determine the circulation lifetime and fusogenicity, or how well the particle can fuse with the endosomal membrane of the LNP. For extended circulation, longer fatty acid chains, like polyethylene glycoldistearoylglycerol (DSG PEG 2000), can be utilized. PEG concentration also affects particle size. However, PEG use may lead to antibody formation, potentially compromising immunization effectiveness.

  • Neutral/anionic lipids provide structural stability and play a role in defining the fusogenicity and biodistribution. 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine. For example, it was shown that LNPs containing 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE), which plays an important role in endosomal release, led to enhanced delivery of mRNA to the liver as compared to 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).5 Study results suggest that these helper lipids also assist in the stable encapsulation of the RNA.6

  • Cholesterol is used to modulate the bilayer density, fluidity and uptake (raft formation) of the LNP. While there are animal-derived and synthetic versions of cholesterol available in the market, synthetic cholesterol offers several advantages including higher purity, lack of animal derived molecules such as prions, scalability, and highly consistent quality.

The purified mRNA can be formulated into the delivery particle via different techniques. In the commonly used solvent injection technique, lipids are dissolved in a solvent such as ethanol and rapidly mixed in an aqueous, low pH buffer containing the mRNA using crossflow mixing or microfluidic mixing to create the LNPs. The low pH buffer is then diafiltered into a neutral buffer and ultrafiltration is used to concentrate the particles. The TFF step must be rapid as lipids can be hydrolyzed at low pH, leading to formation of impurities such as hydrolipids that can affect the lipid bilayer structure, stability of the formulation and drug release characteristics. Degradation of the lipids can also increase the size of the particle, resulting in aggregation.

LNPs have a very good stability, structural plasticity and enhanced gene delivery compared to other delivery systems. They increase the transfection rate compared to naked mRNA, allow for intravenous injection without the risk of being degraded by RNases present in the bloodstream and enable active targeting if specific ligands are incorporated. Disadvantages of LNPs include the fact that they may require cold chain logistics. In addition, sterile filtration is not always possible with LNPs and in such cases alternatives, such as gamma irradiation, heat sterilization, high-pressure sterilization or closed processing must be considered.

For more information on mRNA formulation, read our white paper “Considerations for Advancing a Lipid Nanoparticle Formulation to Clinical and Commercial Manufacturing”.

Scale-up Considerations

There are several considerations to keep in mind when scaling up the mRNA manufacturing process, and these should be top-of-mind during process development when working in a small scale.

  • Methods using solvent extraction and precipitation steps for mRNA purification are difficult to scale and the use of hazardous solvents are not suitable for GMP environments and can be replaced by TFF or chromatography.

  • Because mRNA can be degraded by RNases within seconds, every raw material, solution and equipment that comes into contact with the product must be free of these enzymes.

  • The appropriate delivery system contributes to the efficiency of the vaccine or therapeutic and should be selected carefully.

  • If the final product is a large mRNA complex, alternatives for the sterile filtration of the product should be evaluated.

  • Extraordinary supply chain requirements (e.g. cold chain) are a significant cost driver. Therefore, the stability of the drug should be evaluated carefully.


mRNA technology has enabled development of COVID-19 vaccine candidates with unprecedented speed and outstanding efficacy rates. Going forward, this technology will not only revolutionize the field of vaccine development by allowing a rapid response to disease outbreaks, it also has the potential to be a rapid and flexible platform for both vaccines and therapeutics which will help to address unmet medical needs.

To ensure this therapeutic approach reaches its full potential, however, innovative solutions, expertise and ingenuity will need to coalesce to establish a simple and robust platform at production scale. With our products, services, and expert support we are dedicated to streamline your decision-process, helping you to navigate your path to your successful mRNA drug product, and advance mRNA-based vaccine and therapeutics manufacturing together.

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