The most comprehensive chromatographic toolbox for purification of mRNA - from affinity to reverse phase.
Affinity capture with Oligo dT allows purification of mRNA from in vitro transcription mRNA with poly-A tail (process overview here). Non-affinity capture using PrimaS allows direct purification from IVT for processes with post-transcriptional polyadenylation (read more). The following information provides an overview of each tool.
Sartorius BIA Separations offers a comprehensive toolbox of chromatography columns to help you achieve the desired product quality, with the speed and ease of use provided by the monolith.
CIM® technology offers a uniquely capable purification toolbox for rapid process development and scale-up to ensure:
- The product has a intact poly(A) tail.
- dsRNA and DNA template are removed.
- Process and product-related impurities, such as enzymes, DNA and RNA fragments are removed.
Messenger RNA is large for its mass. It is also sensitive to shear forces. The large channel structure of the chromatographic monolith allows free access for large particles, and laminar flow prevents shear damage. The structure enables purification of mRNA up to 10 kb or larger.
The rich spectrum of chemical properties of mRNA is the basis for a diverse selection of purification columns. Messenger RNA contains a charged phosphate backbone, and hydrophobic nucleotide residues with hydrogen-bonding ability.
Charge interactions, hydrogen bonding and other molecular forces also impose challenges. Messenger RNA acts as nucleation center and readily forms associations with other components in IVT mixtures. These heteroaggregates depress capacity, recovery, and the purification ability of every known purification method, including filtration, precipitation, and chromatography.
CIMmultus Oligo dT, available with 6-carbon linker or 12-carbon linker, provides a single-step affinity capture of mRNA. Binding occurs through hydrogen bonding, with the oligo dT forming a stable hybrid association with the terminal poly(A) sequence of the target. Non-RNA contaminants flow through the column during sample loading.
CIMmultus PrimaS purifies large single-stranded mRNA (ssRNA) under aqueous conditions at ambient temperature. It can be used for polishing or as high resolution capture method. It removes dsRNA, DNA, proteins, and endotoxins while fractionating ssRNA in order of increasing size.
CIMmultus C4 HLD is a hydrophobic interaction chromatography monolith for purification of nucleic acids. Sample is applied at high ionic strength in salts that precipitate RNA. The majority of DNA, dsRNA and short transcripts elute earlier than intact ssRNA. Proteins and aggregates are typically eluted eluted by a NaOH cleaning step.
CIMmultus SDVB is a styrenedivinylbenzene-based reverse phase chromatography monolith for purification of ssRNA. DNA, dsRNA, and ssRNA are fractionated according to length. Selectivity for separation of double stranded and single-stranded species of similar length is influenced by the degree of base pairing.
Custom ligand immobilisation
If our standard products are not within your required specification, other ligands can be coupled to the monolith. Our R&D can immobilise custom oligo sequences, proteins or enzymes. You provide the ligand, we immobilise it. Your process may benefit from:
Immobilisation of custom oligo sequence
for affinity purification of RNA. Rapid mass transfer in the monolith allows affinity capture at short residence times. This enables efficient chromatography purification of large biomolecules. Custom and proprietary oligonucleotide sequences may improve the purity of the product, and can be immobilised on the monolith matrix.
See our application note for more information:
Immobilisation of enzyme
for DNA/RNA or protein digestion, or plasmid linearisation. Immobilised enzymes can reduce enzyme use and cost, eliminate an enzyme removal step, and improve the digestion efficiency.
The following literature will provide more information on enzyme immobilisation:
- Bencina, 2007, Preparation and characterisation of ribonuclease monolithic bioreactor
- Naldi, 2017, Towards automation in protein digestion: Development of a monolithic trypsin immobilised reactor
With our customer-oriented approach we will ensure that the final product meets the needs for your development and process requirements. Get in touch through our contact form.