has been the biggest barrier to entry for RNA vaccines. As is widely known, the PB

and Moderna vaccines must be stored at extremely low temperatures; notably, −60

to −90°C and −15 to −25°C, respectively. This is partly due to the intrinsic in-

stability of the RNA molecule, but also due to the LNP carrier requiring low tem-

perature storage [28]. At refrigeration temperatures (~4°C), these vaccines will only

remain stable for 5 and 30 days for the PB and Moderna vaccines, respectively [25].

These storage condition requirements represent a major hurdle to the global dis-

tribution of mRNA vaccines, because they require a very complicated distribution

chain, and they may seriously prohibit access to the vaccine in areas without cold-

chain infrastructure. This is in sharp contrast to DNA vaccines, which may remain

stable at 25°C for up to 4 years [25].

There are two elements of stability: shelf-life stability and in-vivo stability (see

Chapter 8). In terms of in-vivo stability, there are several ways to overcome the

inherent instability of RNA in the body. For example, certain nucleotide modifica-

tions, as described above, can help the RNA molecule evade detection of the RNAses

and the immune system. These include adding 5′ Kozak and cap sequences, 3′ poly-A

BIOREACTOR PRODUCTION

DOWNSTREAM PROCESSING

LNP ENCAPSULATION

FILL-TO-FINISH

- DNA template production

- E. coli fermentation

- DNA linearization

- Cell-free in vitro RNA

transcription

- 5’ capping

- DNA digestion (DNAse I)

RNA in aqueous stream

Lipids in ethanol stream

- TFF concentration

- Chromatography purification

-A second TFF diafiltration

- Sterile filtration

- Controlled lipid condensation

- Use of microfluidic mixers

- Nanoparticle formation

- Sterile filtration

- Sterile dilution

- Filling, capping

-Sealing

- Quality control

- Optical check

- Labelling

- Packaging

FIGURE 12.1 Bioprocess flow diagram for mRNA vaccine manufacturing. There are four

main manufacturing blocks: mRNA production at bioreactor scale, mRNA downstrseam

processing, lipid nanoparticle (LNP) encapsulation, and fill-to-finish, each of the manu-

facturing block comprising different unitary operation modules.

COVID-19 vaccines

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