The Making of mRNA

Sudden demand for large-scale manufacturing of messenger RNA (mRNA) drug substances during the COVID-19 pandemic has few parallels in the history of pharmaceuticals. Some might posit penicillin production during World War II as an analog, or perhaps early insulin availability. Pandemics and epidemics such as smallpox, typhus, cholera, influenza, HIV/AIDS, or the bubonic plague, all relied more on infection control for resolution than on manufacturing capacity. New therapeutic uses for mRNA are now ramping up good manufacturing practice (GMP) demand, especially for clinical trial supply. Moderna collaborated with CARsgen Therapeutics to combine an mRNA therapeutic cancer vaccine with a solid tumor chimeric antigen receptor (CAR)-T cell therapy, aiming to treat multiple cancers (1). Pfizer partnered with Beam therapeutics to co-develop mRNA and base editing approaches (2), while BioNTech and DualityBio have a partnership to develop differentiated antibody-drug conjugates, for solid tumors (3). In terms of upstream manufacturing for novel mRNA drug products, this technical complexity forms a tidal wave of question marks.

Costs decrease while complexity rises

A prominent paper (4) examined reducing complexity by evaluating what common process could be standardized through extrapolations of clinical trial complexity, based on four published papers Phase I-II dose ranging studies. While not the amounts of product required during the COVID-19 pandemic, clinical trial doses quickly mounted up in terms of both volume and also manufacturing challenges related to techniques pursued, but also formulation delivery requirements. The authors state, “from the four studies, the average mRNA usage was 5500 μg and for the 75 trials in 2021, 412 500 μg of RNA would need to be formulated, purified, sterile filtered, and fill-finished. At concentrations of 100 to 200 μg of mRNA per mL, this would amount to 2063 to 4125 mL of drug product” (4). As an interesting foray into possible future manufacturing, the authors went on to contrast traditional mRNA with a self-amplifying method. Often, this is cited as potentially a 100 to 1 decrease in required finished product due to higher potency performances, but this study chose a picturesque perspective, saying, “assuming the entire population consists of 7.7 billion people, and each person receives two doses, a total of 15.4 billion doses or 4.5 million L of the vaccine is required. In perspective, 4.5 million L would fill two Olympic-sized swimming pools (2500 m3), whereas saRNA [self-amplifying RNA] vaccines, which we assume are 100-fold more potent, would only require 0.02 Olympic-sized swimming pools. Assuming that production costs, which account for materials, RNA, and LNP [lipid nanoparticles] production, scale linearly, vaccinating the entire population with Pfizer/BioNTech’s mRNA vaccine would cost approximately 150 billion USD. In contrast, with saRNA, it would only cost approximately 1.5 billion USD” (4).

The making of mRNA

Commercial-scale mRNA is produced through chemical, recombinant, or enzyme-based methods. For chains under 100 nucleotides, the standard chemical technique “utilizes phosphoramidite chemistry and solid-phase support promoting chain elongation from the 3′ end to the 5′ end” (2). It is amenable to automation, as is recombinant production through host cells such as Escherichia coli (E. Coli), which has some cost advantages as it can utilize legacy protein manufacturing lines, already in GMP compliance. Enzymatic processes are newer to arrive and have tradeoffs still being worked through. A study conducted by the Duke Human Vaccine Institute explored creating standardized robust manufacturing processing steps, saying they confronted the lack of established quality standards by, in part, surveying vendor operations “through questionnaire and/or audit relating to the vendors’ quality systems and operations as they pertained to manufacturing and/or testing of the materials” (5). The study adopted the Weismann–Karikó method of production, the upstream process requiring three enzymatic steps of plasmid linearization, mRNA transcription, and DNA template digestion. In the starting out phase, fundamentals the authors emphasized were that “for optimal in vitro transcription yields, it is critical to start with high quality template DNA. Some key quality attributes that are required before use include the presence of only a single band of linearized DNA, a supercoiled ratio of at least 70%, and a sequencing-verified correct poly(A) tail length” (5). They also noted that “endotoxin control was the primary raw material risk due to its variability in raw material lots and the difficulty of its removal in subsequent downstream purification steps … reaction agitation, temperature, incubation time, and reactant concentrations were also carefully examined for each reaction step in order to yield a robust upstream platform process across multiple mRNA constructs” (5).

Click here to read this article in the Innovations in mRNA eBook.

About the author

Chris Spivey is director, Industry Relations and Strategic Partnerships

Article details

Pharmaceutical Technology®
Innovations in mRNA eBook
April 2024
Pages: 17-19