mRNA purification is a critical step in the manufacturing process of mRNA vaccines and therapeutics. The purification process needs to effectively separate mRNA from both process and product-related impurity species. Today we’ll take a look at different purification methods used for mRNA in two categories: non-chromatography-based purification and chromatography-based purification.
Non-Chromatography-based purification
- DNase Treatment: Plasmid DNA (pDNA) is the template used for mRNA synthesis. It is double-stranded and contains other functional sequences apart from the complementary sequences to mRNA, such as the T7 promoter sequence; therefore, pDNA is much larger than the mRNA it’s producing. And it’s often a major impurity species that needs to be removed in the downstream process. DNase is an enzyme that digests DNA and chops it up into multiple small pieces while leaving mRNA intact. By treating the mRNA synthesis solution with DNase, the large pDNA copies are removed. The DNase digestion can be combined either with precipitation or tangential flow filtration (TFF) to further clear up the small DNA fragments after digestion.
- Precipitation: DNA clearance through a combination of DNase treatment and precipitation is widely applied for small lab-scale purification of mRNA. Precipitation of mRNA can happen with different reagents, such as ethanol, monovalent cations such as sodium, ammonium, or lithium (in the salt form of lithium chloride). LiCl, unlike other reagents, can selectively precipitate mRNA through an elevated level of lithium concentration, leaving other molecules like DNA, proteins, etc., in the supernatant (Barlow et al., 1963). This is a proven simple and effective method for purifying mRNA. The precipitated mRNA can then be collected by centrifugation and resuspension. However, due to intrinsic limitations of precipitation and centrifugation when scaling up, LiCl is only used at the lab scale.
- Tangential Flow Filtration (TFF): TFF explores the size exclusion effect of membrane filtration. With a certain molecular weight cutoff (MWCO), small molecules would be able to sieve through the membrane while large molecules remain in the feed stream. By recirculating the feed stream through the membrane, all the impurities are eventually removed. The rule of thumb is to use a membrane with MWCO 1/5 – 1/3 of the size of the product. With in vitro transcription (IVT) mix, through DNase and proteinase treatment, all the large molecules are digested into small pieces that can be easily cleared up by a TFF step. This is exactly what Pfizer is using for its Covid-19 vaccine.
- Magnetic Beads: Similar to biologics or AAV molecules, mRNA also has specific characteristics that can be explored to design an affinity-based purification step. mRNA normally has a poly(A) tail that is added to the 3′ end of the mRNA molecule during transcription, which contributes to the stability and translational efficiency of mRNA molecules. Magnetic beads coated with oligo(dT) sequences can be regulated to bind/unbind to the poly(A) tail under different salt conditions, allowing for the selective isolation of mRNA (see Figure 1).
Chromatography-based purification
- Affinity Chromatography: Utilizing the same concept as magnetic beads, the oligo dT sequence is also modified onto chromatographic beads or monolithic matrix and used as a chromatography-based purification method. This technology is widely used during process development and has linear scalability.
- Hydrophobic Interaction Chromatography (HIC): HIC separates molecules based on their different hydrophobicity. The ligand of HIC is hydrophobic. The mobile phase usually contains a high concentration of a kosmotropic salt. During the elution phase, the salt concentration is gradually reduced, resulting in the elution of less hydrophobic species first. The application of this technology to mRNA is very complicated and should be evaluated on a molecule-by-molecule basis. The reasons are: 1) mRNA is hydrophilic and doesn’t have a strong interaction with the ligand; 2) the high salt condition in the mobile phase requires preconditioning of mRNA-containing load material, and can easily cause degradation of mRNA molecules; 3) impurities like DNA and some proteins are also hydrophilic and may not be efficiently separated from mRNA with HIC.
- Ion Exchange Chromatography: This method separates molecules based on their different charge. mRNA has a strong negative charge due to its phosphate backbone and can be separated from other molecules with different charges. Anion Exchange Chromatography has resins modified with positively charged ligands, thus is particularly effective in purifying mRNA from other molecules by carefully controlling the ionic strength of the buffer. The process usually starts with buffers with low ionic strength to allow binding of mRNA to the resin. Then during the elution phase, the salt concentration is gradually increased, leading to the earlier elution of less negatively charged molecules.
It’s important to note that many of the above-mentioned methods are often used in combination to achieve the necessary level of purity for therapeutic applications. The specifics of the purification process can vary depending on the type of mRNA being produced and the requirements of the final product. For example, self-amplifying mRNA or mRNA for use in non-viral gene therapy vectors may require different purification processes than mRNA for use in lipid nanoparticles for vaccines.
Reference:
- Barlow, J. J., et al. “A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid.” Biochemical and Biophysical Research Communications 13.1 (1963): 61-66.