How to Purify mRNA – A strategy you need to consider
What is mRNA?
mRNA contains specific triplets of nucleobases (codons) that encode for different amino acids for the ribosome to follow and stitch together a peptide chain that will eventually (after some modification) become a functional protein. mRNA’s structure, as a result of evolutionary pressures, is such that it doesn’t stick around the body. It quickly degrades in the cellular environment avoiding over production of protein by quickly removing the instructions. Can you imagine if you couldn’t ever delete your voicemail, how quickly would your mailbox fill up? It is this feature, in part, that made it difficult to harness the flexibility and power of the mRNA platform for so long.
It wasn’t until advancements in stability (capping and base replacements) along with a reduction of immunogenicity could the therapeutic be utilized to produce target proteins in vivo as a therapeutic [¹, ²]. The platform of mRNA leverages the human body’s incredible capability to produce sophisticated proteins and merely gives the body the directions it needs to do so. In theory, mRNA can encode for almost any protein that is non-expressed or mis-expressed and address a wide variety of pathologies, including cancer. Most visible use is the use of mRNA-based vaccines in response to the COVID-19 pandemic [³, ⁴] but this is the just the tip of the iceberg. mRNA therapeutics will change the future. However, as the CEO of a leading pharmaceutical mRNA company has suggested the quantity and quality, i.e. consistent purity, of mRNA remain as bottlenecks for their production. Particularly, the lack of high-throughput downstream purification processes is a major challenge in the upscaling of industrial mRNA production.
How is mRNA purification accomplished?
Resin chromatography columns have been used in scalable antibody production [⁵, ⁶]. However, therapeutic mRNAs (300-20,000 nucleotides [nts]) are much larger than antibodies (~150 kDa), making the effective surface area and capacity of the resin columns for mRNA much lower than that for antibody purification [³,⁶]. Therapeutic mRNA usually possesses a 5-300 nts poly-adenylic acid (poly-A) tail, an essential element to the protein translation process [¹, ⁸-¹⁰].
Oligodeoxythymidine (oligo-dT) ligands have been recognized as an effective affinity ligand to isolate polyadenylated mRNA from feed streams via hybridization following Watson-Crick base-pairing between adenine in the poly-A tail and deoxythymidine in oligo-dT [⁵, ⁸, ¹¹⁻¹³]. The small pores of resins decrease access of larger mRNA (>4k nts) to binding sites, which further reduces capacity and yield. Customer interviews indicate there is significantly reduced binding capacity and yield associated with purification of mRNA with >4k nts. Together, the low binding capacity, long residence times, and poor purification performance for larger mRNAs afforded by resin columns result in unsatisfactory purification productivity.
mRNA purification can be done in minutes!
In contrast to resins, advective media, such as macroporous membranes, have been proven to provide at least ten times faster loading speed than resins for the purification of many other biologics [¹⁴]. Purilogics has recently developed a line of mRNA affinity membrane chromatography products (Purexa™-OdT) that utilize oligo-dT as affinity ligands. The Purexa™-OdT membrane has large pores that easily accommodate larger sizes of mRNA and purifications can be completed in minutes with high yield and purity.
The vision of Purexa™ OdT product line
Purilogics will develop Purexa™ OdT products that enable mRNA purifications in research & discovery, process development, clinical trial, and commercial manufacturing contexts. The scale of the membrane products will range from microliters to liters.
How does Purilogics Purexa™ OdT membrane chromatography product compare to magnetic beads?
Magnetic bead-based products have been used widely for biologics recovery. There are also a number of products available for mRNA purification. Magnetic bead-based tools can be useful in a number of purification scenarios, however, they face many challenges that may have resulted in hindered research progress. Due to the nature of the beads, incubation is needed to make sure mRNA and beads have sufficient contact time. In addition, to use the bead effectively, multiple resuspensions are needed during incubation because beads have tendency to aggregate or agglomerate, impacting recovery and productivity. The challenges can be more significant if you are handling a large number of samples with a deadline or if working with newly trained lab staff. Give yourself a break from the tedium. Try Purexa™OdT today!
If you are interested in our mRNA affinity membrane products, please feel free to contact us!
References:
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