Messenger RNAs (mRNAs) are a fast-emerging class of biotherapeutics. While they hold considerable promise, the field is still in its infancy. mRNA therapies offer a new opportunity for targeted treatment of challenging diseases and flexible manufacturing, as demonstrated by the rapid development of mRNA vaccines against COVID-19. The clinical potential of mRNA extends beyond vaccines to other advanced therapies, such as protein replacement, gene therapy, and cancer immunotherapy. At Sartorius, our mRNA journey began with our leading purification technologies. Our comprehensive knowledge now extends to the entire process of mRNA drug substance (DS) production.
mRNAs are single-stranded nucleic acids transcribed from DNA, representing a critical component of gene expression. To exert their clinical function, mRNA molecules are delivered to the target tissue, directing the production of the coded protein inside cells. By mimicking the actions of endogenous mRNAs, therapeutic mRNAs avoid the immunogenic properties and manufacturing challenges associated with therapeutic recombinant proteins. mRNA expression is also temporary, and the stability of the mRNA molecule is directly linked to gene expression; the longer the mRNA says inside the cell, the greater the production of the encoded protein.
mRNA Production Process
From Gene of Interest to mRNA
The DNA sequence coding for the gene of interest is first inserted into a plasmid. The plasmid DNA (pDNA) is amplified in host bacteria, typically E. coli, before it is purified and linearized. mRNA is synthesized from the pDNA template, which is incubated with enzymes and nucleotides to produce the mRNA. This method is called in vitro transcription (IVT) and is a cell-free process. Further steps are required to add the mRNA cap and stabilize the molecule.
The new mRNA molecule is then isolated by removing impurities such as enzymes, remaining nucleotides, and nucleic acids. Finally, mRNA is concentrated and subject to sterile filtration before it is ready for encapsulating into LNPs.
mRNA Process Flow
The absence of standardized IVT processes restricts the development of a single mRNA purification platform. There is also no one-size-fits-all process; for example, the selection of the capture method depends on the mRNA construct.
- mRNAs are large, unstable molecules sensitive to shear stress and enzymatic degradation
- mRNA must be separated from contaminants with similar properties like double-stranded RNA (dsRNA), truncated mRNA, and DNA
- Insufficient analytical tools hinder the ability to optimize the manufacturing process
mRNA Synthesis | In Vitro Transcription and Capping
mRNA molecules are commonly synthesized from linearized pDNA by an in vitro transcription (IVT) reaction. The IVT reaction requires a DNA template, nucleotides, polymerase, and capping enzymes. The synthesis of different mRNA constructs could require unique conditions which might not align with recommended procedures. These may include intermittent purification steps to optimize productivity.
A robust IVT reaction platform supported by rapid analytics
- Well-defined raw materials
- Limited contaminants introduced into the reaction
- Efficient transcription processes yielding high concentrations of mRNA
Our Unique Solutions:
At Sartorius, we have industry-leading process development services to optimize your IVT reactions.
- Process development activities supported by a deep understanding of the IVT reaction
- Strong experience in developing analytical chromatography to characterize every step of the process, maximizing productivity and ensuring patient safety
- Rapid HPLC analytics based on CIMac PrimaS™ to monitor reagent consumption and mRNA production rate
- IVT reactions can be performed in closed bioreactor systems to scale up manufacturing. Our Biostat STR® is an industry-proven bioreactor with a unique and robust film to minimize extractables and leachables. Automated technology facilitates the integration of a wide range of process analytical technologies used in IVT applications.
- The Flexsafe® Pro Mixer technology is the only single-use mixing solution with integrated sensors for pH, conductivity, and temperature. Accommodating both low-shear and rapid mixing applications in a fully closed design, the mixer is ideally suited for low shear mixing of fragile molecules like mRNA.
During mRNA capture, mRNA is purified, and the IVT reagents (including nucleotides, pDNA, and enzymes) are removed. mRNA capture is usually carried out by chromatography, based on affinity interactions or the chemical properties of the mRNA molecule. However, the properties of mRNA, including its large size, similarity to other contaminants, and instability, can hinder the success of its isolation. Additionally, the slow adoption of scalable purification technologies also forces reliance on laboratory-scale purification methods even at large scales. Monolithic columns offer a high flow rate and achieve high removal of impurities in a bind-elute mode, facilitating efficient mRNA capture.
Efficient and scalable purification technologies capable of high-resolution separation of mRNA from impurities, including those with similar chemical compositions.
- Sufficient capacity to enhance productivity
- Plug-and-play systems with rapid performance
- Flexible solutions applicable to any production process
- High recovery of mRNA molecules
Our Unique Solutions:
Sartorius offers comprehensive mRNA capture solutions based on monolith technology, along with process development support to help optimize your mRNA recovery.
- Multiple purification options available, based on the presence of a polyA tail or absence of a polyA tail
- High dynamic binding capacity for mRNA (up to 3 and 10 mg/ml using OligodT and PrimaS, respectively)
- Low shear forces for maximum recovery with excellent contaminant removal
- Overcome manufacturing concerns such as the need for high temperatures or organic solvents
The Resolute®️ Flowdrive SU single-use chromatography system is designed for pilot, clinical, and commercial production. The flexible modular design enables monolithic column operation with isocratic or gradient set-up options.
Polishing can be used to target remaining impurities - including proteins, dsRNA, and truncated RNAs - leftover after mRNA capture. In a fast-evolving field like mRNA therapeutics, you need comprehensive tools to remove all types of contaminating material, ensuring your molecule meets the necessary quality requirements for clinical applications.
Flexible, scalable solutions that enable the removal of residual contaminants at high-resolution
- High binding capacity for mRNA
- Ability to discriminate even highly similar molecules (such as separating mRNA from dsRNA)
Our Unique Solutions:
Sartorius delivers mRNA polishing solutions through our range of monolithic technologies
- Flexible, targeted removal of residual impurities by anion exchange, reverse phase, or hydrophobic interactions
- Avoid the need for high temperatures and organic solvents, which can be a manufacturing bottleneck
- High dynamic binding capacity for mRNA and low shear procedures to maximize yield
- Superior resolution with minimal peak broadening
Concentration, Diafiltration, and Sterile Filtration
Concentration and diafiltration steps reduce the sample volume and remove small molecules in the buffer. This can be performed at various stages throughout the procedure. During the final steps, the purified mRNA is subject to sterile filtration.
Gentle filtration methods compatible with the sensitivity of mRNA to degradation and shear stress
- Maximum recovery of mRNA despite its instability
- Mitigate the risk of introducing new contaminants, such as RNase, into the final product
Our Unique Solutions:
Sartorius offers intuitive, user-friendly purification solutions for concentration and diafiltration of mRNA with features including a gamma-irradiable cassette format. Hydrosart® is a state-of-the-art cross-flow membrane designed for low adsorption of mRNA and ensures compatibility with any application.
- Membranes with molecular weight cut-offs from 2 kD to 300 kD available
- Provides low adsorption, high flow rate, and increased product recovery
- Resistant to chemicals, steam, heat, and gamma-irradiation, and can be used with up to 40% ethanol concentration, allowing a wide range of operating conditions with low fouling
- Sartocon® Self Contained Units (0.1m² to 14m²) can be delivered gamma sterile, facilitating closed, scalable, and ready-to-use processing
- Sartopore® delivers the broadest range of PES membrane combinations, perfectly adapting to all product types for the lowest filtration costs. Available with 0.2 or 0.45 µm pore sizes, these membranes allow sterile filtration with a high retention rate. Pre-designed and configurable transfer sets are delivered gamma-irradiated.
- A broad range of cross-flow systems cover needs process development to large-scale commercial production, with the option to implement functionally closed solutions with single-use and gamma-irradiated flow paths.
Drug Substance Storage and Transportation
Biotherapeutics are valuable and often sensitive products that require careful handling throughout production, storage, and movement to other facilities. Detailed design of drug substance storage and transportation methods is essential to protect the product’s integrity and ensure patient safety. Many drug products, including mRNA therapeutics, are stored in a frozen state to slow down chemical and biological degradation, extend the product’s shelf life, restrict microbial growth, and prevent excessive agitation by shear stress.
Frozen storage and transport solutions that protect sensitive mRNA molecules
- Robust and reliable solutions to limit environmental fluctuations and preserve the product
- Safe, secure, and traceable systems to track the mRNA throughout its journey
- Limited effects of cryoconcentration
Our Unique Solutions:
Our Celsius® range includes single-use, gamma-irradiated, and ready-to-use products to ensure the safe and simple preservation and transfer of frozen biological material.
Celsius® CFT – our fully integrated freeze/thaw platform
- Robust, scalable, reproducible, and easy-to-use vertical controlled plate freezer technology with an established track record
- Controlled and rapid fast freeze and thawing with agitation to mitigate the cryoconcentration effect observed for large-scale freeze and thaw processes
- High capacity (up to 100 L)
Celsius® S3 – a benchtop system that is fully scalable to commercial production.
- Same freeze path length and heat exchange technology as large-scale solutions meaning freeze/thaw protocols are transferrable
Celsius® FFTp - pre-assembled and ready to fill single-use containers to leverage existing infrastructure.
- High robustness ensured by our innovative SafecoreTM technology, Celsius® FFT/FFTp containers are fully qualified down to -80°C against worst-case condition shipping
- Celsius® FFT can be used with any conventional freezer, and Celsius® FFTp is designed explicitly for plate freezers
Lipid Nanoparticles (LNPs) encapsulate the mRNA, protecting it from degradation and promoting its delivery into the target cells. Lipids and ethanol are used in the chemical reaction, during which the where mRNA is captured in the LNP. The size of the LNP differs based on company protocol, ranging from around 5 to 100 nm. Following an optional polishing step, encased mRNA is ready for final filtration, and free mRNA is removed by concentration and diafiltration.
Simplify Your mRNA Production Process
The production of mRNA is not yet a platform approach. mRNA contains a charged phosphate backbone and hydrophobic nucleotide residues with hydrogen-bonding ability; its rich spectrum of chemical properties is the basis for a diverse selection of purification columns. Our toolbox of solutions allows you to build an mRNA production process that best fits your application:
- Multiple purification options available
- Straightforward plug-and-play approach
- Does not require significant optimization
- Expert technical support available
How do Monoliths Support Efficient mRNA Purification?
Join us as we journey into the monolith matrix to discover how convective chromatography supports the robust separation of large biomolecules.
Superior separation of mRNA from the IVT mixture means your samples are free from contaminants
Our toolbox is compatible with diverse bioprocessing activities and enables the purification of a broad spectrum of mRNA molecules
Monolithic Columns for Reliable mRNA Purification
Harness the power of monoliths for high-resolution isolation of mRNA molecules.
Our range of monolithic columns are powered by distinct but complementary chemistries to suit any application. Discover the combination that works best for your mRNA production process.
CIMmultus® Oligo dT
Hybridization Affinity Chromatography
CIMmultus® Oligo dT enables a single-step affinity capture of polyadenylated mRNA. Binding occurs through hydrogen bonding, with the Oligo dT forming a stable hybrid association with the terminal poly(A) sequence of the target mRNA. The column is available with C6 and C12 linkers.
- Convenient approach for initial purification of mRNA
- Non-RNA contaminants flow through the column during sample loading, efficiently removing proteins and DNA
- Captures all RNA species, regardless of size or strandedness
- Primarily designed for mRNA capture, but can also be used during polishing
How Does it Work?
Chromatogram of IVT mixture applied to CIMmultus® Oligo dT. mRNA (teal line, peak around 16 min) and non-RNA contaminants (teal line, peak around 2 min) are shown in a single-step affinity capture, during which the column is washed, and the mRNA is recovered. The dashed line shows conductivity.
Anion Exchange Chromatography With Hydrogen Bonding
PrimaS is a multimodal chromatography ligand that combines elements of hydrogen bonding with anion exchange chromatography. CIMmultus® PrimaS supports the purification of single-stranded RNA at ambient temperatures.
- Captures all RNAs regardless of their structure or composition
- Purifies and size selects large single-stranded mRNA (ssRNA)
- DNA and proteins elute before - and are well separated from - ssRNA
- Useful for non-polyadenylated constructs
- Can be used as a high-resolution capture method or for polishing
How Does it Work?
Chromatogram of IVT mixture applied to CIMmultus® PrimaS. The teal line shows supercoiled DNA (scDNA, 6 kb) is separated from ssRNA (5 kb) across a pH gradient (dashed line).
CIMmultus® C4 HLD
Hydrophobic Interaction Chromatography
C4 (butyl) High Ligand Density (HLD) is strongly hydrophobic. The sample is applied at high ionic strength in salts that precipitate RNA, and the RNA is eluted with a decreasing salt gradient.
- Highly effective for removing proteins from nucleic acids (pDNA, RNA)
- Best results as a polishing tool
- Separates ssRNA from DNA, dsRNA, and truncated RNA
- Strongly binds proteins, removing them from the sample
Proteins typically eluted by NaOH cleaning step
How Does it Work?
Chromatogram of IVT mixture applied to CIMmultus® C4 HLD. ssRNA (teal line, peak around 8 min.) and contaminants (teal line, peak around 1 min) are shown during polishing, where salt concentrations (dashed line) are moderated to enrich ssRNA.
CIMmultus® SDVB uses styrene-divinylbenzene (SDVB)-based reverse-phase chromatography to separate ssRNA from truncated forms, dsRNA, and DNA. Its selectivity for separation of single- and double-stranded species of similar size is influenced by the degree of base pairing.
- Best suited as a polishing method
- Also performs size separation
- Tool of choice for characterizing dsRNA and discriminating intact ssRNA from short transcripts
How Does it Work?
Chromatogram of RNA ladders (denatured at 65°C) applied to CIMmultus® SDVB. The teal line shows the size separation of ssRNA during the elution. The black line shows dsRNA.
Part – 1: Increase pDNA Purity and Yields
In part one you will learn how to achieve higher yields in the plasmid DNA by ensuring the quality and purity of the raw materials and taking other steps to optimize the production process.
Part - 2: Break Down the mRNA Bottleneck with Rapid Analytical Strategies
In part two of the 'Your mRNA process, Simplified' webinar series, we continue to explore how improved processes, analytics, and tools can help meet the unprecedented demand for mRNA.
Part - 3: Select the Right Purification Approach for Your Process
In this final webinar of the series, Sartorius purification experts demonstrate the flexibility that can be achieved with an mRNA purification toolbox featuring monolithic chromatography.