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A downstream process for GMP manufacturing of mRNA
With the successful regulatory approval of mRNA vaccines, hundreds of ongoing clinical trials globally, and continued exploration of new infectious disease targets, the vaccine and mRNA-based medicine pipeline continues to expand. To efficiently develop and manufacture these medicines, downstream processing (DSP) should be optimized. Compared to monoclonal antibody (mAb) DSP, which typically requires three chromatography steps, or adeno-associated virus (AAV) DSP, which similarly involves multiple orthogonal unit operations, mRNA DSP follows a comparatively direct path. That streamlined workflow concentrates a significant portion of the purification burden on a single chromatography step, making resin performance at that step critical to process success.1, 2
Challenges in purifying mRNA at scale
Scaling mRNA purification methods from microgram bench quantities to gram-scale clinical quantities introduces challenges. Maximizing the dynamic binding capacity (DBC) of the affinity capture resin reduces the column size requirements and overall cost. The resin must also remove in vitro transcribed (IVT) process-related impurities, including enzymes, unreacted nucleotides, abortive transcripts, and plasmid DNA (pDNA) template.
Affinity chromatography with an Oligo dT ligand addresses these challenges in a single, scalable capture step.
Single-step chromatography for mRNA purification
Affinity capture of mRNA using POROS Oligo (dT)25 resin
POROS Oligo (dT)25 Affinity Resin is designed for mRNA capture directly from crude IVT samples. The resin is comprised of a poly-dT 25-mer ligand conjugated to an average 50 µm poly(styrene-co-divinylbenzene) POROS bead. Through A-T base pairing, the ligand selectively binds poly-A-tailed mRNA while process- and product-related impurities pass through the column unretained, supporting clearance in a single step. Recovery exceeds 95% across a wide range of mRNA construct sizes, and elution requires water or a low-salt buffer.3
POROS Oligo (dT)25 can support the purification of mRNA used for vaccines, gene editing, and other applications.
Plasmid DNA purification as the IVT input step
In the mRNA manufacturing process, pDNA serves as the DNA template for the IVT reaction that produces mRNA. pDNA serves as the DNA template for the IVT reaction that produces mRNA. The purity and quality of the pDNA input directly affect IVT yield and downstream mRNA quality.
POROS anion exchange (AEX) chromatography resins are well-suited for pDNA purification. Large throughpores in the POROS bead increase accessible surface area for larger nucleic acid molecules, while the rigid poly(styrene-co-divinylbenzene) backbone tolerates the high viscosity and shear sensitivity of plasmid samples. Available POROS AEX resins include POROS XQ Strong AEX and POROS 50 HQ Strong AEX.
Research- and GMP-grade plasmid purification with POROS AEX resins also supports viral vector production and AAV purification, as well as broader gene therapy (GTx) bioprocessing workflows.
Process development tools for mRNA and pDNA purification
Before committing to larger experiments, process developers can use high-throughput screening formats in static and dynamic modes to identify optimal binding conditions.
The POROS GoPure Oligo (dT)25 Screening Plate, 96-well is designed for parallelized evaluation of mRNA-binding conditions and is compatible with liquid-handling automation. Screening results inform key column-loading parameters, including salt type, salt concentration, and pH.
Next, small scale prepacked columns, including Robocolumns™ and GoPure prepacked columns, can be used to further optimize operating conditions.
Support scalable manufacturing and supply reliability
Our global supply network enables supply continuity for commercial-scale processes. Regulatory support packages, including documentation for change control, lot release, and resin qualification, are available to support IND and BLA submissions.
Related resources
Frequently asked questions
POROS Oligo (dT)25 Affinity Resin is the chromatography tool of choice for GMP manufacturing of mRNA. The poly-dT 25-mer ligand binds poly-A-tailed mRNA through A-T base pairing, enabling selective separation from IVT process-related impurities, including enzymes, unreacted nucleotides, abortive transcripts, and pDNA template, in a single affinity capture step.
POROS Oligo (dT) 25 is available in bulk quantities, and it is suitable to use in GMP manufacturing. The resin can be easily packed in commonly used bioprocess columns, supporting flexibility and efficiency for your process.
Typically, mRNA downstream processes achieve purity targets purity targets with a single chromatography step using Oligo dT affinity capture. The formation of undesired product-related impurities is commonly prevented or minimized through optimization of the in vitro transcription including the raw material selection (e.g., DNA template quality, polymerase) as well as the reaction conditions.
References
- BioPhorum Operations Group. Overview of End-to-End mRNA Drug Substance and Drug Product Manufacturing Scale-Up Guide. May 2023.
- United States Pharmacopeia. Analytical Procedures for Quality of mRNA Vaccines and Therapeutics, Draft Guidelines: 3rd Edition. August 2024.
- Flook K, Sim B, Molinari J, Baek J, Sikka S, Becerra A. Accelerating mRNA-based therapy development with scalable purification of in vitro transcribed mRNA. 2020.
Additional bioprocessing resources
Bioprocessing resources
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use only.