mRNA Vaccine Quality Control

mRNA vaccines possess several extraordinary advantages, including high efficacy, relatively low side effects, and low cost of implementation. These advantages have enabled them to become prevalent in preclinical and clinical trials against a variety of infectious diseases and cancers. In order to build public trust in innovative products such as mRNA vaccines, they must be of good quality, safety, and efficacy.

Common mRNA Vaccine quality control attributes

The following quality attributes need to be monitored to ensure the effectiveness of the mRNA vaccine.

Determination of the particle size of lipid nanoparticles
The particle size of lipid nanoparticles (LNPs) is the most important parameter affecting the delivery efficiency. It has an impact on biodistribution, internalization, degradation, clearance, and cellular uptake. The average particle size of LNPs is normally between 100 and 400 nm, and LNPs with particle sizes ranging from 10 to about 150 nm are desirable for systematic drug delivery via intravenous (IV) injection. Normally, particle size can be determined by dynamic light scattering (DLS) which measures the intensity differences of fluctuated light due to the motion of particles.

Determination of the surface morphology of lipid nanoparticles
Surface morphology is an important structural parameter that affects cellular uptake. In addition, it may also have an impact on the rigidity of liposomal nanomaterials. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) are often be employed to observe particle surface morphology. SEM and TEM provide the morphological information via electrons transmitted from the surface of the particles and the inner structure of the particles, respectively. In contrast, AFM allows for a 3D profile of LNPs.

Determination of length of mRNA product
The mRNA that should be present in the final formulation of the vaccine should be the exact mRNA sequence that was designed; the sequence should include the 5' cap, both the 5' and 3'UTRs, the portion that codes for the full-length spike protein, and the poly (A) tail. It is essential that there be a step which will ensure that the mRNA being produced is the full-length mRNA to ensure the highest possible translation efficiency. To accomplish this, a sample will be taken from the product of each in vitro transcription reaction, and RNA gel electrophoresis will be performed on the product using RNA gel electrophoresis kits. During gel electrophoresis, there will be an electric current present which will cause negatively-charged mRNA to move toward the anode. Shorter RNA fragments will migrate farther than longer RNA fragments, so this process can be used to determine whether or not the mRNA product from the in vitro transcription reaction is mostly full-length.

Measurement of endotoxin levels
Most regulatory agencies require that all manufactured vaccines be tested for endotoxin levels. Endotoxin, a frequent contaminant, can cause fever, inflammation, headache, nausea, and even death. Found in the cell walls of gram-negative bacteria, endotoxin has routinely been detected by the sensitive and specific Limulus amebocyte lysate (LAL) assay. In the presence of endotoxins, LAL coagulates through an enzyme-mediated cascade, which has then been traditionally quantified based on gelation and turbidity. Acceptable endotoxin levels for mRNA vaccines should be less than 10 endotoxin units (EU)/mL.