The LNP are formed when the lipids dissolved in the ethanol stream are condensed

in the aqueous stream, trapping the RNA molecules within and forming the active

principle used for vaccination. Different factors govern the LNP formation, such as

the rate of mixing or the volumetric ratio between the aqueous and organic phases.

To maintain a reproducible method of LNP encapsulation and produce uniform

particles, microfluidic devices like scalable micromixers are used. Using larger

devices or running them in parallel are strategies used to scale up this step of the

process. Also, for a uniform LNP encapsulation, the nitrogen-to-phosphate (N/P)

ratio (nitrogen from the ionizable cationic lipid and phosphorous from the mRNA

molecule) is maintained fixed in the final aqueous solution [36–38]. Three main

types of lipids are usually used for LNP encapsulation: ionizable, PEGylated, and

helper lipids. For the PB LNP, the cationic lipid ALC-0315 was used as an ionizable

lipid, ALC-0159 as a PEGylated lipid, and DSPC and cholesterol as helper lipids

[39]. The presence of new-generation ionizable cationic lipids is the key for a

successful mRNA delivery. These lipids contain amine groups that maintain a

neutral or cationic charge at physiological pH. However, upon encountering the

acidic environment of late endosomes, the amine groups are ionized inducing

conformational changes that disrupt the endosomal internal membranes releasing

their content into the cytoplasm [38]. After LNP encapsulation, TFF is used again

for a new step of diafiltration, diluting the solution carrying the final active principle

to the desired concentration followed by a sterile filtration (Figure 12.1).

The final block of manufacturing operation takes the sterile LNP-encapsulated

mRNA solution and undergoes subsequent rounds of quality control, filling, cap-

ping, sealing, optical quality check, labeling, and packaging. The PB final vaccine

is stored at −60 to −90°C. The finished product is described as a concentrate for

dispersion for injection containing 225 μg/0.45 mL (prior to dilution) of BNT162b2

(5′ capped mRNA encoding full-length SARS-CoV-2 spike protein) [33]. A similar

manufacturing process for the Moderna vaccine is also described [40]. Neither

company mentions the use of an adjuvant in their formulation, but it is likely that

both the mRNA itself and the lipids in the LNP have adjuvanting properties.

12.4.1.3

Stability of mRNA Vaccines and Their Efficacy

Both Moderna and PB vaccines performed extremely well in clinical trials, which is

perhaps unsurprising when one considers how similar they are to each other.

Phase 3 studies demonstrated a 95% and 94.5% vaccine efficacy against COVID-19

after doses of the PB and Moderna vaccines, respectively [32,41]. There are other

mRNA vaccines in the pipeline, as well. For example, CVnCoV developed by

CureVac is another LNP-encapsulated mRNA encoding the S protein. However,

vaccine efficacy in early studies was not nearly as high as PB or Moderna, which

was thought to be partially due to the use of uracil rather than pseudouridine [2,42].

As mentioned previously, the potential for mRNA vaccine development has been

demonstrated since the 1990s. Therefore, one might wonder why it took so long to

reach clinical use. Firstly, plasmid DNA vaccines received far more attention than

their mRNA counterparts due to their increased stability. As already discussed

previously, the DNA double helix is far more stable than single-stranded RNA

molecules, which are specifically degraded by the body [18]. This notion of stability

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Bioprocessing of Viral Vaccines