mRNA Technology and How High-Shear Processors Fit In

By Maja Hunter, Ph.D.

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Messenger RNA (mRNA) is a transient intermediator between genes and proteins. It was discovered in pioneering studies in 1947-1961. Investigations of its structure and function resulted in the development of in vitro transcribed mRNA in the late 1980s, utilized for proof-of-concept animal study in 1990. Recent advances in drug delivery systems enable the use of unstable and immunogenic mRNA molecules for preclinical development of mRNA therapeutics [1].

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The mRNA therapeutic potential was shown in a range of applications, such as viral vaccines, protein replacement therapies, cancer immunotherapies, cellular reprogramming, and genome editing [1]. Carefully designed mRNA molecules must reach specific target cells and produce sufficient proteins to achieve the desired therapeutic effect [1]. If small polar and hydrophobic molecules with a molecular weight of up to 100 Da can easily diffuse into the cell membrane, the 300-5000 kDa mRNA molecules surely cannot. Moreover, a dense negative charge of the mRNA molecules electrostatically repulses the anionic cell membrane [2]. Safe and effective materials of delivery systems that protect the encapsulated mRNA molecule are required that enable targeted delivery to the cell and mRNA release into the cytoplasm [1]. Until now, lipid, polymer and peptide-based vectors, hybrid vectors and exosomes are used, each having advantages and disadvantages [1].

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Lipid nanoparticles (LNPs) successfully entered the clinic for delivery of small molecules, siRNA drugs and mRNA. The first siRNA-LNP formulation, Patisiran, was approved in 2018 for treatment of hereditary transthyretin-mediated amyloidosis [3], which contributed to fast development of a drug delivery system for COVID-19 mRNA-based vaccines from Pfizer-BioNTech and Moderna [1].

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Manufacturing of Pfizer-Biontech COVID-19 Vaccine

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Pfizer´s COVID-19 vaccine contains mRNA, encoding the receptor-binding domain of the S (spike) glycoprotein of SARS-CoV-2 with an N-terminal signal peptide and a C-terminal membrane-anchoring helix [4], which is encapsulated by a delivery system made of ionizable cationic lipid nanoparticles, PEGylated lipid, neutral lipid and cholesterol [3]. The manufacturing procedure of COVID-19 vaccine by Pfizer was published in July 2021 [5].

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The first steps consist of cloning the plasmid with the antigen gene, transforming it into the E. coli bacterium, cultivating the bacteria at 37 °C overnight on a solid media to get the visible E. coli colonies and inoculating them into liquid culturing media for fermentation. After the fermentation is completed, the E. coli bacteria needs to be broken down to release the plasmid.

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In the next steps, lots of testing and quality checks are done in order to ensure the harvested plasmids are correct before the plasmid is cut by specific enzymes to separate the plasmid backbone and the antigen genes. The antigen gene DNA sequence needs to be purified and sequenced again. The confirmed antigen gene is in vitro transcribed into mRNA, requiring the building blocks and enzymes needed to support the process. Resulting mRNA is then filtered to ensure its purity and tested to confirm the genetic sequence is correct.

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Finally, the mRNA is ready for encapsulation into the delivery vehicle to form a vaccine particle. First, lipid nanoparticles are measured out and dissolved in ethanol, whereas negatively charged mRNAs are dissolved in acidic aqueous buffer. The current methods of mRNA lipid nanoparticle production utilize T-junction mixing to rapidly combine the lipid-ethanolic solution, and the mRNA aqueous solution. The rapid mixing of the two solutions is key in order to achieve the particle size of <100 nm [6]. Self-assembly and formation of an mRNA-LNPs delivery vehicle is driven by hydrophobic and electrostatic forces. The electric charge pulls the lipid and naked strands of mRNA together in a nanosecond, and mRNA is enveloped in layers of lipids. The last steps consist of buffer exchange, ethanol removal, concentration of the sample, filtering to remove impurities and final sterilization [7].

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How Does the High-Shear Homogenizer Like Dyhydromatics Shearjet® Processor Fit in the Manufacturing Process of mRNA Vaccine?

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In research labs, plasmids are usually isolated from 2 mL of E. coli bacterial culture. The plasmid isolation kits contain lysis solution, neutralization buffer, columns for plasmid adsorption, washing buffers, and an elution buffer. On the production scale, liters of bacterial cultures are more efficiently disrupted using mechanical methods, such as high-shear homogenization. The high pressure accelerates the liquid culture to pass through microchannels; shear forces disrupt bacterial cells and the content of the cell is released. Such cell disruption is gentle, fast, efficient, and requires no chemicals. After the first pass through the Reaction Chamber® module, the processed cell lysate contains large cell wall fragments (450 nm), therefore, the viscosity stays rather low [8]. After the second pass, the length of cell wall fragments is further reduced. Dyhydromatics ShearJet® processors equipped with L-type Reaction Chamber® modules are optimal for mechanical cell lysis. If you are interested in our equipment or you want to learn more about our technology, don’t hesitate to reach out to our friendly customer service. 

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Literature

[1] Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021). soi: 10.1038/s41578-021-00358-0

[2] Ibba ML, Ciccone G, Esposito CL, Catuogno S, Giangrande PH. Advances in mRNA non-viral delivery approaches. Adv. Drug Deliv. Rev. 177, 113930 (2021). doi: 10.1016/j.addr.2021.113930

[3] Xu L, Wang X, Liu Y, Yang G, Falconer RJ, Zhao CX. Lipid Nanoparticles for Drug Delivery. Advanced nanoBiomed research, 2021. 2(2);2100109. doi: https://doi.org/10.1002/anbr.202100109

[4] Lu, J., Lu, G., Tan, S. et al. A COVID-19 mRNA vaccine encoding SARS-CoV-2 virus-like particles induces a strong antiviral-like immune response in mice. Cell Res 30, 936–939 (2020). doi: 10.1038/s41422-020-00392-7

[5] Liu T, Liang Y and Huang L. Development and Delivery Systems of mRNA Vaccines (2021). Front. Bioeng. Biotechnol. 9:658. doi: 10.3389/fbioe.2021.718753

[6] Buschmann MD, Carrasco MJ, Alishetty S, Paige M, Alameh MG, Weissman D. Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines. 2021; 9(1):65. doi: 10.3390/vaccines9010065

[7] Cott, E, deBruyn E, Corum, J.  How Pfizer Makes Its Covid-19 Vaccine. New York Times [accessed on April 28, 2021]. https://www.nytimes.com/interactive/2021/health/pfizer-coronavirus-vaccine.html?searchResultPosition=2

[8] Agerkvist, Iréne and S O Enfors. “Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers.” Biotechnology and Bioengineering 36 (1990): n. pag.doi: 10.1002/bit.260361102

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Posted September 9, 2023