Part 1: Chromatography Purification for mRNA

The rise of mRNA therapeutics highlights the importance of scalable solutions for the production of new modalities. How can chromatography techniques support efficient mRNA purification?

^{This article is posted on our Science Snippets Blog}

While mRNA vaccines have been in development for a long time, the COVID-19 pandemic threw them into the spotlight. Not only did they survive the pressure, mRNA vaccines emerged as a triumph of modern R&D. Now, vaccine developers around the world are looking to mRNA for their next product, especially in the field of cancer therapy. For this to work, reliable methods for mRNA purification need to be established.  

Three Important Aspects to Bear in Mind  

1. Size
   RNA molecules are large, and the negatively charged backbone repels itself forcing the molecule to elongate. This means only matrices with relatively large pore sizes will be appropriate for the purification of RNA.  
2. Shear sensitivity
   The large size and tendency of the molecules to elongate introduces more problems when it comes to purification: RNA is highly likely to shear. Turbulence or shear forces close to the edge of a channel increase the probability that mRNA molecules will break. Clearly, this will have a negative effect on the process yield. More importantly, broken contaminating threads of mRNA could code for proteins that are different from what is expected in the therapy. This makes the contaminants potentially harmful. So, breakages must be avoided as much as possible, and any broken mRNA strands should be removed.
3. Chemistry
   As previously mentioned, the negatively charged backbone can force the mRNA molecules to elongate and change their physical characteristics. Metal ions can also bind non-specifically with mRNA molecules, changing their structure and introducing charge heterogeneity. 

Typical Impurities Found During mRNA Purification  

mRNA is produced via chemical reactions using premade plasmids, together with nucleotides, capping reagents, and enzymes. It is common to get enzyme and nucleotide carryover impurities that need to be removed. There are also usually a lot of unwanted nucleic acids. These could be plasmid DNA (pDNA) carry over from the IVT reaction to synthesize mRNA. However, perhaps the most difficult impurities to remove are other mRNA molecules. The fact that mRNA is so sensitive to shear can mean that molecules are truncated, giving rise to mRNA preparations with a range of lengths. These will have very similar, if not identical, properties to the desired mRNA target, making them challenging to separate.

The big problem here is that there must be no nucleic acid contamination in vaccines or genetic therapies. If this happens, products will not be approved for clinical use and must be scrapped. Careful and thorough separation of the mRNA product is therefore essential.  

Certain mRNA Properties Aid Separation  

The size or length of an mRNA molecule makes it unique and represents a valuable property to begin separating it from smaller impurities. As previously mentioned, the backbone of mRNA is highly negatively charged. However, this charge can be modified by the introduction of different salts. Combinations of high salt concentrations and EDTA will also help remove other cellular contaminants such as double-stranded DNA and proteins. 

One of the most effective ways to separate mRNA by affinity chromatography is using the poly-A tail, a series of adenosine residues at the 3’ end of the molecule. It represents a unique property that allows mRNA molecules to hybridize strongly to a short repeated thymidine ligand known as oligo dT. Adding salt to the mRNA preparation suppresses the repulsion charges, enabling the oligo dT to capture the mRNA molecules while impurities are washed away. Subsequently removing the salt allows ionic repulsion, resulting in the smooth elution of the mRNA and very effective purification.  

However, the affinity method does not necessarily remove truncated mRNA molecules that also have a poly-A tail. For this application, a pH gradient can be used to perform size separation. The purified mRNA molecules will elute in different fractions according to size, with the longest (the target), eluting last.  

The Matrix of Choice  

Optimizing Process Chromatography for mRNA Production  

The COVID-19 pandemic highlighted the clinical value of mRNA, which extends far beyond vaccines. Robust production methods, including reliable purification strategies, are required to unlock its full potential. Explore how chromatography methods can be optimally applied to the production of mRNA and more next-generation modalities. 

Comparing Chromatography for Advanced Modalities

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