Part 2: Design, Delivery and Manufacturing of mRNA Vaccines

 

Antigen design of mRNA vaccine

So far, the in vitro transcription technology of mRNA has matured, and the most commonly used method is to synthesize mRNA using T3, T7 or sp6 RNA polymerase and linear DNA (linearized plasmid DNA or PCR synthesized DNA)
.

In eukaryotic cells, some basic structural elements of mature mRNA are necessary to maintain mRNA function, including 5'cap (5' cap), 5'untranslate region (5' UTR), open reading frame (ORF) region, 3 'untranslate region (3' UTR) and poly(A) tail structure
.

Maintaining the integrity of the mRNA structure is beneficial to the stability and expression ability of the mRNA

 

Figure 1 Schematic diagram of mRNA vaccine design

 

The mRNA molecule is synthesized in vitro and has a cap structure (m7GpppNm)
.

Uridine is replaced by pseudouridine

 

Delivery of messenger RNA vaccines

Effective in vivo delivery is essential to prevent mRNA vaccines
.

In order to be converted into immunogenic proteins, foreign mRNA must pass through the barrier of the host cell membrane and enter the cytoplasm

 

Figure 2 The main delivery methods of mRNA vaccines

 

Figure 3 The main delivery methods of mRNA vaccines

 

The figure shows the commonly used delivery methods of mRNA vaccines and the diameters of typical carrier molecules and particle complexes: naked mRNA (part a); in vivo electroporated naked mRNA (part b); protamine (cationic peptide) complex mRNA ( Part c); mRNA related to positively charged oil-in-water cationic nanoemulsion (part d); mRNA is related to chemically modified dendrimers and is associated with polyethylene glycol (PEG) lipid complexes (part e); Protamine complex mRNA in polyethylene glycol lipid nanoparticles (part f); mRNA related to cationic polymers (such as polyethyleneimine (PEI)) (part g); cationic polymerization related to qualitative components Substances such as PEI and lipid mRNA (part h); mRNA (part i) associated with polysaccharides (such as chitosan) particles or gels; cationic lipid nanoparticles (such as 1,2-dioleoyloxy)- MRNA (part j) in 3-trimethylpropaneamine (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA in complex with cationic lipids and cholesterol (part k); cationic lipids, Cholesterol MRNA and PEG-lipid complex (part 1)
.

 

Table 1 Delivery system of mRNA

 

LNP is the most widely used platform and has been proven to have the best clinical effects in mRNA delivery
.

LNP is mainly composed of ionizable lipids, cholesterol, phospholipids and polyethylene glycol-lipids (Figure 4)

 

Figure 4 Schematic diagram of mRNA lipid nanocomplex

 

LNP was originally designed for the delivery of siRNA, and is now used for the delivery of mRNA.

It is the most clinically translatable non-viral delivery vector

At physiological pH, ionizable lipids remain neutral, improving stability and reducing systemic toxicity
.

Representative ionizable lipids are: actively designed Dlin-DMA, Dlin-KC2-DMA and Dlin-MC3-DMA; C12-200 and cKK-E12 are screened based on high-level combinatorial library throughput; The first generation of COVID-19 lipids, including DLN-MC3-DMA derivatives L319, C12-200 and CKK-E12 derivatives, COVID-19 vaccine lipids ALC-0315 and SM-102, TT3 and biodegradable derivatives FTT5, Vitamin-derived lipids SSPALME and VCLNP, A9, L5, A18 lipids, ATX lipids and LP01 are mostly biodegradable

 

Figure 5 Representative LNP structure and ionizable lipids in preclinical research and clinical trials

 

 

Production of mRNA vaccines

Once the pathogen is identified or the outbreak is announced, the genome of the pathogen and antigen will be determined through joint sequencing, bioinformatics and computational methods (if not already)
.

By electronically storing candidate vaccine antigen sequences, it can be used for computer design of mRNA vaccines worldwide, and then plasmid DNA templates can be constructed through molecular cloning or synthesis

 

Figure 6 Schematic diagram of mRNA vaccine production

 

Compared with traditional vaccines, one of the most important advantages of mRNA is that it is relatively simple to manufacture
.

In order to produce messenger RNA products with specific quality attributes, a series of manufacturing steps must be performed

 

Figure 7 Schematic diagram of the production and purification steps of the mRNA vaccine production process

 

The production of mRNA can be carried out by a one-step enzymatic reaction using capping analogs, or can be carried out by a two-step reaction using vaccinia capping enzyme
.
The laboratory-scale mRNA purification process includes DNase I digestion followed by LiCl precipitation
.
Using mature chromatographic strategies, combined with tangential flow filtration, a larger-scale purification can be obtained; new chromatographic methods can also be used to supplement standard purification
.

 

The existing IVT mRNA production method must be improved, commercialized, and supported by the market demand
.
Because the process output and production scale have an impact on the manufacturing cost and the cost of each agent, it is speculated that continuous processing will have a special advantage in reducing costs
.
Continuous processing has been used in the chemical and pharmaceutical industries to run flexible and cost-effective processes and ultimately provide on-demand production
.
In addition, process integration through continuous manufacturing can also shorten operating time, promote automation and process analysis technology (PAT), thereby improving productivity and product quality
.
The relative simplicity of messenger RNA manufacturing makes this process very suitable for continuous processing, especially at the microfluidic scale (Figure 8)
.

 

Figure 8 Conceptual design of the continuous production process of mRNA vaccines

 

The process includes a continuous two-step enzyme reaction, followed by a tangential flow filtration strategy and two multi-mode chromatography steps of the enzyme cycle, one is the intermediate purification in the combined elution mode, the other is the flow mode purification, and the first The three tangential flow filter module realizes the formula
.

 

Overview of the development of mRNA vaccines: part one

Overview of the development of mRNA vaccines: part two

Overview of the development of mRNA vaccines: part three

Overview of the development of mRNA vaccines: part four

Overview of the development of mRNA vaccines: Part 5

 

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