What are the challenges in mRNA purification?

One challenge in mRNA purification is the impurity profile of mRNA varies with each project, requiring specific purification steps. mRNA molecules, which are quite large (30 to 50 nanometers), don’t interact well with traditional chromatography resins and often result in only surface adsorption. Another challenge arises when using self-amplifying mRNA. Self-amplifying mRNA includes sequences from viruses to create a replicon for producing more of the transgene protein. This type of mRNA is more complex and still in early stages of development. Additionally, modified mRNA (which uses base analogs like pseudo-uridine) also requires specific purification considerations to enhance stability. mRNA is also composed of a relatively simple chemical structure compared to more complex biomolecules. This simplicity means that differentiating between the mRNA and its by-products (which often have similar chemical properties) can be challenging. Lastly, mRNA is inherently unstable and prone to degradation due to chemical, physical, and enzymatic factors including exo- and endonucleases. Thus, maintaining the stability of mRNA is crucial during purification.