mRNA Vaccine Production Process : LNP Delivery Technology

mRNA is large in molecular weight andhydrophilic, but its own single-stranded structure is highly unstable andsusceptible to degradation.

The limited lifetime of mRNA enables cellsto rapidly change protein synthesis in response to its changing needs, but itis difficult to meet the requirements of drug-forming properties. In addition, mRNAmolecules themselves carry negative charges and have difficulty crossing cellmembranes with the same negative charges on the surface, so specialmodification or wrapping delivery systems are required to achieve intracellularexpression of mRNA drugs, therefore, delivery technology is one of the corepatented technologies of mRNA companies. The mRNA vaccine production process iscomplex.

Lipid Nanoparticle (LNP) is currently themainstream carrier delivery method. It is often used in vaccines because it ismore easily absorbed by antigen presenting cells. The three major mRNA vaccinegiants Moderna, CureVac and BioNTech are currently using LNP deliverytechnology for their COVID-19 vaccines.

We focus on the mRNA vaccine productionprocess. LNP components that are widely used at this stage include the followingfour major categories: cationic lipids, cholesterol, polyethylene glycollipids, and adjuvant lipids.

1. Cationic lipids

Cationic liposomes are the key componentsof LNP delivery systems. The specific structures of cationic lipid moleculescurrently used by mRNA companies vary, but all are cationic lipids that arepositively charged under specific conditions. Ionizable cationic lipids are thekey to the delivery function of the LNP system. mRNA is bound inside the LNP bypositive and negative attraction due to the negative electrical properties of mRNAitself, which can improve the stability of mRNA in vivo and escape thedegradation of lysosomes. after the LNP is taken up by cells, the low pHenvironment of the nuclear endosome will fuse with the LNP and release the mRNAinto the cytoplasmic colloid.

The development of cationic lipids requiresa balance between delivery efficiency and cytotoxicity. The cytotoxicity ofcationic lipids depends on the structure of their hydrophilic head groups; forexample, amphiphilic molecules containing quaternary ammonium head groups aremore toxic than amphiphilic molecules containing tertiary amines. Therefore,addressing the cytotoxicity of ionizable cationic lipids is one of the keypoints of LNP technology and the focus of patent protection. The proprietarycationic lipids ALC-0315 (Acuitas/Pfizer) and SM-102 (Moderna) have hydrophilichead groups that are both tertiary amines, which can be protonated positivelycharged in a physiological low pH environment and are safely cleared after mRNAis delivered.

2. Cholesterol

The main role of cholesterol is to mediateLNP endocytosis and to help ensure the bilayer structure and lipid mobility ofthe LNP. Auxiliary neutral lipids (e.g., various phospholipids) are also usedto build the LNP bilayer structure, as the bilayer structure of cationicliposomes is not stable.

3. Polyethylene glycol lipids

There are many issues that need to be takencare of in the mRNA vaccine production process. Liposomes modified by specificstructural polyethylene glycols can control the particle size of nanoparticlesduring nanoparticle synthesis. Due to the strong hydration of polyethyleneglycol ethoxy links, the polyethylene glycol structure can form a hydrophilicprotective layer in the aqueous phase, which can effectively preventnanoparticles from aggregating during storage, thus maintaining the spatialstability of LNP; at the same time, polyethylene glycol on the surface of LNPparticles can protect the particles from being detected by immune proteins invivo, shield plasma proteins and other components from binding to theparticles, and prevent LNP particles from being cleared in vivo. Theproprietary PEG lipid structure ALC-0159 (M-DTDAM-2000) currently selected byPfizer and M-DMG-2000 selected by Moderna are both derivatives of PEG-2000.

4. Auxiliary lipids

Auxiliary lipids are represented byphospholipids such as DOPE, DSPC and DOPC. In the preparation of cationicliposomes, auxiliary lipids have very strong synergistic effects, mainlyincluding stabilizing the bilayer membrane and reducing the toxicity ofcationic components, promoting the release of mRNA when LNP is endocytosed andassisting the cell permeation of cationic liposomes, and determining themorphology of mRNA-LNP complexes, making the complexes have good fusibility andimproving the transmembrane efficiency.

From a comprehensive perspective, domestic mRNA-relatedcompanies have the pre-consciousness of timely and accurate patent warning andavoiding patent barriers, while they are focusing on the layout of key patenttechnologies with independent intellectual property rights by virtue of theirself-research capability. The mRNA vaccine production process is technical innature. In the future global competition in mRNA field, Chinese companies areexpected to occupy a more favorable position.