Stuart R. Gallant, MD, PhD
Hopefully, you have received your Covid-19 vaccine. If you did, there’s a pretty good chance it was delivered in lipid nanoparticles. Both Moderna Spikevax and Pfizer–BioNTech Comirnaty are formulated in lipid nanoparticles. Today’s post looks at lipid nanoparticles formulations and what we know about them.
In PharmaTopo’s post on Factor VIII [1], we looked at Wood’s paper in Nature describing stable transfection of the Factor VIII gene to BHK cells. That transfection was done with calcium phosphate coprecipitation, a method introduced in the early 1970s as an improvement over an earlier method which used DEAE-dextran [2]. With the right set of reagents, nucleic acids can be brought to bind with the surface of a mammalian cells, leading to endocytosis.
Jacqueline Miller from Moderna presented this view of cellular importation of an mRNA vaccine to the CDC’s Advisory Committee on Immunization Practices (ACIP) in 2020 [3]:
The lipid nanoparticle (LNP) attaches to the cell, is endocytosed, the mRNA makes its way to the rough endoplasmic reticulum for translation into protein. The virus spike protein is then exported from the cell where it encounters immune cells leading to immune response and immunity to Covid-19.
We are going to take a deeper look at this chemistry and how it provides protection versus the ongoing pandemic.
Lipid Nano Particle Formulations
The two LNP formulations licensed for Covid-19 in the US are Modern’s excellently named “Spikevax” (one imagines Moderna pounding a volleyball down on Coronavirus) and Pfizer-BioNTech’s equally effective, but less excellently named “Comirnaty”:
| Vaccine | Formulation |
| Moderna Spikevax | Dose: Packaged in a multidose vial; 0.5 ml/dose. Each 0.5 ml dose contains: Active Ingredient: 100 mcg of nucleoside-modified messenger RNA (mRNA) encoding the pre-fusion stabilized Spike glycoprotein (S) of SARS-CoV-2 virus Lipids: 1.93 mg of a mixture of SM-102, polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG], cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC] Buffer: 0.31 mg tromethamine, 1.18 mg tromethamine hydrochloride, 0.043 mg acetic acid, 0.20 mg sodium acetate trihydrate Misc: 43.5 mg sucrose |
| Pfizer–BioNTech Comirnaty | Dose: Vial is reconstituted with 1.8 ml of sterile 0.9% sodium chloride; 0.3 ml/dose. Each 0.3 ml dose contains: Active Ingredient: 30 mcg of a nucleoside-modified messenger RNA (mRNA) encoding the viral spike (S) glycoprotein of SARS-CoV-2 Lipids: 0.43 mg ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 0.05 mg 2-(polyethylene glycol 2000)-N,N-ditetradecylacetamide, 0.09 mg 1,2-distearoyl-sn-glycero-3-phosphocholine, and 0.2 mg cholesterol Buffer and Salts: 0.01 mg potassium chloride, 0.01 mg monobasic potassium phosphate, 0.36 mg sodium chloride, 0.07 mg dibasic sodium phosphate dihydrate. Diluent contributes an additional 2.16 mg sodium chloride per dose Misc: 6 mg sucrose |
Focusing only on the lipid components, LNPs have 4 components surrounding their mRNA payload: 1) ionizable cationic lipids, 2) neutral lipid, 3) cholesterol, and 4) pegylated lipid. Interestingly, the ratios of these 4 components in the Moderna (50 : 10 : 38.5 : 1.5) and Pfizer-BioNTech (46.3 : 9.4 : 42.7 : 1.6) products are very similar, as pointed out by Schoenmaker [4]:
The 4 lipids form a protective layer around the mRNA, stabilizing it and preventing enzymatic degradation of the active ingredient before it reaches its target. Charge plays a significant role in the functioning of these LNPs. The ionizable cationic lipids are neutral until they reach their pKa:
- pKa (ALC-0315): 6.09
- pKa (SM-102): 6.68
The protonation of these ionizable groups below their pKa allows mRNA escape from the endosome:
LNPs bind with the cell surface and acquire a lipid coating from the cell as they enter. Following entry into the cell, a vesicle containing an LNP and its mRNA payload merge into an endosome. Inside the endosome, the pH drops due to the action of ATP-dependent proton pumps. The LNP would be marked for death inside the endosome (as it merged with a lysosome) except that protonation of the ionizable cationic lipids disrupts the endosome, allowing mRNA escape. The free mRNA diffuses to the endoplasmic reticulum (as depicted in the Moderna figure at the beginning of this post). Etc.
This aspect of LNP technology is so critical, that Alnylam has initiated a suit against both Pfizer-BioNTech and Moderna to protect its intellectual property [5], which Alnylam claims protects this type of ionizable cationic lipid. Presumably, Pfizer-BioNTech and Moderna’s legal strategy will focus on the development of this technology in the 1990s which could cast shade on the validity of the Alnylam patent [6].
Lipid Nano Particle Manufacturing and Characterization
With only two unit operations, the manufacturing appears quite simple in flow diagram depiction [7]:
However, complexity of the manufacturing operation belies the simplicity of the flow diagram. Before starting manufacturing, high-quality raw materials, including high-purity lipids should be selected. Process parameters such as the ratio of the individual component amounts and temperature are important. In the first step, the aqueous solution of negatively charged mRNA is mixed with the lipids in solvent solution. Having excellent control of the high-shear mixing required by this process, particularly during process scale up, is important to maintaining good lot-to-lot reproducibility.
The resulting LNPs are stabilized in the second unit operation (tangential flow filtration), removing the alcohol and increasing the pH to near neutral. Note: explosion protections are a must, working with such a high concentration of solvent. The resulting LNPs are then lyophilized (Pfizer-BioNTech product) or filled as a sterile liquid (Moderna product).
Selmin has characterized the Pfizer-BioNTech Comirnaty LPNs [7]:
Examining three separate batches, the particle size distributions fell right on top of each other. Clearly, this chemistry and manufacturing process control maintain a high degree of uniformity of the resulting drug product. Selmin also noted that vibration causes aggregation of the particles. However, under normal circumstances, that degree of vibration would not be encountered if the drug product was delivered relatively close to the time it was reconstituted. The PSD might be affected if a nurse traveled by car with pre-drawn syringes to the houses of patients to be vaccinated. However, that was not the intent of Selmin’s study; the researchers were looking at the concept of central syringe filling, for example in a hospital pharmacy.
How important is size to function? Mammalian cells are typically 10 to 100 microns in size. If for some reason the LPNs aggregated significantly, the propensity to form vesicles to import the vaccine into the target cells might be negatively affected. So, quality control is important to function. Hassett and coworkers found that a range of LPNs from 60 to 150 nm were successful in obtaining an immune response in their primate model [8].
How long does the mRNA vaccine function to produce protein? In a mouse study, Pardi and coworkers showed that protein expression, as a result of mRNA translation, at the site of injection for intramuscular, subcutaneous, and intradermal delivery was robust, lasting 10 days [9]. In addition, they showed that it was possible for LNPs to escape into central circulation and travel to the liver in intravenous, intraperitoneal, intramuscular, and intratracheal delivery. This resulted in mRNA translation in the liver for a shorter period of time (1-4 days).
Conclusions
Fortunately, LNP technology was in the right place and time to treat the Covid-19 pandemic. Presumably, it will be used in many pharmaceutical applications in the coming decades. I plan to do a post on some of those applications in a future post.
[1] pharmatopo.com/index.php/2022/03/22/pharmaceutical-design-focus-factor-viii-1-of-2/
[2] Graham, F.L. and van Der Eb, A.J. “Transformation of rat cells by DNA of human adenovirus 5,” Virology 1973 Aug;54(2):536-9.
[3] Miller, J.M. “Overview of Moderna’s COVID-19 Vaccine(mRNA-1273),” ACIP –December 20, 2020.
[4] Schoenmaker, L., et al. “mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability,” Int J Pharm 2021 May 15;601:120586.
[5] patentimages.storage.googleapis.com/db/6a/dd/e90d7e620f5349/WO2013086354A1.pdf
[6] Cross, R. “Without these lipid shells, there would be no mRNA vaccines for COVID-19,” C&E News, Volume 99, Issue 8.
[7] Selmin, F., et al. “Pre-Drawn Syringes of Comirnaty for an Efficient COVID-19 Mass Vaccination: Demonstration of Stability,” Pharmaceutics 2021 Jul 7;13(7):1029.
[8] Hassett, K.J., et al. “Impact of lipid nanoparticle size on mRNA vaccine immunogenicity,” J Control Release 2021 Jul 10;335:237-246.
[9] Pardi, N., et al. “Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes,” J Control Release 2015 Nov 10;217:345-51.
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