respectively [7]. While both candidates were shown to enhance neutralizing anti-

bodies, PB ended up going forward with the BNT163b2 candidate due to less severe

adverse effects [2,32].

12.4.1.2

mRNA Vaccine Manufacturing

To better understand the production process, we will use the PB vaccine as a case study

[33]. According to the European Medicines Agency assessment report, the manu-

facturing process of BNT162b2 takes place in four major manufacturing blocks

comprising different unit operation modules. These four major blocks are the pro-

duction of the mRNA molecule at bioreactor scale; the purification of the mRNA

molecule; the lipid nanoparticle (LNP) encapsulation, filtration, and formulation; and

the final fill-and-finish group of secondary manufacturing operations [34].

In the first block of primary manufacturing, the mRNA is synthesized through

cell-free in-vitro transcription of a linear DNA template that is previously produced.

The production of the template DNA is achieved at large scale in already well-

established fermentation processes using Escherichia coli (E. coli) as a production

platform. This is the only step that needs to be modified to produce a new vaccine for a

new emerging strain or for a completely different pathogen using this technology.

Simply modifying the template DNA molecule from which the RNA is synthesized

allows the rapid adaptation of the manufacturing process to the epidemiological si-

tuation, making this one of the main and most important advantages of the mRNA

technology. After being produced by E. coli, the circular DNA plasmid is then line-

arized using restriction enzymes to achieve the final DNA template. The final single-

stranded 5′-capped mRNA molecule encoding the S antigen of SARS-CoV-2 is then

produced in a bioreactor for 2 hours at 37°C using the T7 RNA polymerase enzyme.

The sequence was selected based on the isolate Wuhan-Hu-1 of SARS-CoV-2. The

RNA contains modified N1-methylpseudouridine instead of uridines to decrease the

immunogenicity of the RNA molecule itself and increase the efficiency of translation

[9,35]. The translated protein contains two proline mutations to ensure an optimal pre-

fusion confirmation of the S protein. Following the synthesis of the mRNA molecule,

DNAse I enzyme is added to the bioreactor for 15 min at 37°C to digest the remaining

DNA template prior to the next step of downstream processing of the RNA.

For the second major manufacturing block of production, the downstream pro-

cess begins with tangential flow filtration (TFF) that is used to filter the mRNA

molecule, while allowing the rest of the reaction mix to flow through. This retentate

is eluted and further purified by a chromatography step such as CaptoCore 700,

removing the remaining proteins and enzyme traces from the previous production

step. A subsequent second diafiltration TFF step is needed for buffer exchange,

where the selected solution is a formulation buffer suitable for the next step in-

volving LNP encapsulation. Prior to entering this operation unit, the final mRNA

solution in the corresponding formulation buffer is sterile filtered.

The LNP encapsulation block of the operation is considered as the main bot-

tleneck in the process. For this step, an aqueous phase containing the sterile RNA

solution is mixed with an organic phase containing the lipids, that are the building

blocks of the lipid nanoparticles, dissolved in ethanol. The lipid mixture is com-

bined with the aqueous solution at a fixed volume ratio of aqueous:ethanol phases.

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