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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 51 Stabilit y express a protein of interest such as for immunization purposes, as the successful mRNA-based COVID-19 vac- cines have demonstrated," he explains. mRNA instability particularly challenging DNA-based oligonucleotide drug substances, such as AAV vectors and plasmid DNA, share the relatively stable characteristics of DNA, Mochayedi notes. The stability of shorter chemically synthesized RNAs such as ASOs and siRNA can be improved through chemi- cal modifications during synthesis. Particularly long biologically synthesized mRNA molecules, however, are inherently unstable, and thus require additional management during manufacturing. All nucleic acid-based platforms, however, must be formulated using delivery technologies that protect the therapeutic from degradation. Unique solutions such as LNPs help ensure stability in the patient set- ting, API stability and, ultimately, the efficacy of the therapeutic approach. T here are severa l reasons why m RNA is in her- ently unstable. The physiochemical properties of single-stranded mRNA molecules that lead to their instability include their very large size, high negative charge, sensitivity to rapid degradation by omnipres- ent enzymes such as endonucleases, shear sensitivity, and high viscosity through the formation of secondary structures in solution, according to Mochayedi. Manufacturing strategies for managing mRNA instability One of the main culprits causing mRNA degradation is the enzyme RNase. Avoiding it is difficult, however, because this enzyme is ubiquitous. "It is very difficult for mRNA to move freely in the environment," Christian Cobaugh, CEO and founder of Vernal Biosciences, com- ments. "Extreme precautions must therefore be taken to avoid introducing this enzyme into manufacturing spaces or enclosures," he stresses. This issue can often, he notes, be a barrier to entry into the mRNA market for laboratories not experienced in mRNA production. Use of qualified processes, including sterile sin- gle-use technolog y, should be applied to mitigate contamination and carryover of endonucleases, Mo- chayedi says. Liberal use of RNase degrading chemi- cals for cleaning equipment surfaces, operator gloves, and so on is also essential, Cobaugh adds. The instability of mRNA at room temperature cre- ates further challenges, as the time available for hold- ing generated mRNA sequences at room temperature is fairly restricted, according to Cobaugh. The drug substance must be moved seamlessly to downstream purification and formulation or quickly frozen. "The choice of adopting a seamless manufacturing approach or having unit operations separated by con- trolled freezing and subsequent thawing steps must be considered early on, as the decision will impact the de- sign and operation of the mRNA manufacturing facility, and once implemented, that design will essentially be locked in," Cobaugh contends. "Ideally, it is best to limit the number of freezing and thaw steps, as they can impact the quality of the mRNA drug substance and formulated mRNA-LNP products," Cobaugh adds. Furthermore, gentle techniques must be employed. Continuous movement of the mRNA drug substance to purification and then LNP formula- tion avoids all but the final freezing step. "It is possible, though, with the right engineering solutions and valida- tion procedures to surmount the challenges presented by either approach to mRNA manufacturing," he concludes. In addition, stringent optimization of reaction condi- tions and the process parameters for key unit operations, such as the synthesis step (in vitro transcription) and filtration (via tangential-flow filtration), can overcome viscosity challenges and ensure low shear during pro- cessing to maintain the stability of the mRNA at each stage of the manufacturing process, Mochayedi remarks. "Bespoke process development and optimization are nec- essary for each unique mRNA sequence to address and overcome the highlighted challenges," he concludes. Minimizing formulation time While the concept of leveraging mRNA for the preven- tion or treatment of various diseases is straightforward, the safe and effective delivery of these fragile nucleic acids into cells where their message can be translated into proteins has proved to be a huge challenge, Mo- chayedi observes. "For the COVID-19 mRNA vaccines, the solution to this problem came through the devel- opment of LNPs. These particles, typically composed of four different lipids (ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA, pegylated lipids that help stabilize the particle, and phospholipids and cholesterol molecules that con- tribute to the particle's structure), provide a protective bubble for the delicate mRNA molecules, enabling their safe and efficient delivery into cells," he explains. The structure of the nucleotide API has a strong im- pact on the selection of the excipients for the formula- tion. The manufacturing process must, in addition, be highly controlled. "The buffer solution containing the mRNA drug substance used to produce the LNPs is of low pH (typically aqueous sodium citrate) that places additional stress on the nucleotide's stability, leading to even more rapid breakdown than occurs at neutral or slightly basic pH," Cobaugh says. A low pH of approximately 4.5 is needed to ionize the mRNA so that it forms nanoprecipitates with the lipids. The use of stabilizing process aids is, Cobaugh adds, generally avoided because they would interfere with the ionization of the mRNA and could also poten- tially end up in the product. Most molecules that would