Recent Advances and Innovations in the Preparation and Purification of In Vitro-Transcribed-mRNA-Based Molecules

Abstract

The coronavirus disease 2019 (COVID-19) pandemic poses a disruptive impact on public health and the global economy. Fortunately, the development of COVID-19 vaccines based on in vitro-transcribed messenger RNA (IVT mRNA) has been a breakthrough in medical history, benefiting billions of people with its high effectiveness, safety profile, and ease of large-scale production. This success is the result of decades of continuous RNA research, which has led to significant improvements in the stability and expression level of IVT mRNA through various approaches such as sequence optimization and improved preparation processes. IVT mRNA sequence optimization has been shown to have a positive effect on enhancing the mRNA expression level. The innovation of IVT mRNA purification technology is also indispensable, as the purity of IVT mRNA directly affects the success of downstream vaccine preparation processes and the potential for inducing unwanted side effects in therapeutic applications. Despite the progress made, challenges related to IVT mRNA sequence design and purification still require further attention to enhance the quality of IVT mRNA in the future. In this review, we discuss the latest innovative progress in IVT mRNA design and purification to further improve its clinical efficacy.

Understanding the impact of in vitro transcription byproducts and contaminants

“…In the laboratory under small scale production conditions, mRNA is typically precipitated using alcohol and sodium or ammonium salts or by using lithium chloride alone ( Walker and Lorsch, 2013 ). Large-scale mRNA production may however integrate diverse non-chromatography or chromatography-based mRNA purification methods (as previously and extensively reviewed in Baronti et al, 2018 ; Zhang et al, 2023 ), and these can be combined as well. Among the combined methods, the “sandwich” method is widely used, where a chromatography-based purification is flanked both before and after by tangential flow filtration (TFF) steps.…”

Enzymatic isolation and microfluidic electrophoresis analysis of residual dsRNA impurities in mRNA vaccines and therapeutics

“…However, double-stranded RNA (dsRNA), which can be a by-product of the in vitro transcription (IVT) of mRNA, is a notable challenge for manufacturers, as it behaves similarly to mRNA in current quality control assays and is notoriously difficult remove during purification. [12][13][14] To address this, improvements have been made in terms of enzyme technology and purification methods, 15 however, residual dsRNA at trace concentrations can remain in the final product. 16 dsRNA is a molecule of interest due to its ability to significantly increase the immune response to mRNA vaccines, 17 leading to both local and systemic inflammation in treated individuals.…”

Enhancement of SARS-CoV-2 mRNA Vaccine Efficacy through the Application of TMSB10 UTR for Superior Antigen Presentation and Immune Activation

“…Properly designed mRNA sequences can enhance the targeting and stability of mRNA vaccines, thereby improving their efficacy and durability [1]. Traditional nonreplicating mRNA vaccines typically comprise a 5′ cap structure, a 5′-untranslated region (UTR), an open reading frame (ORF) encoding the target antigen, a 3′-UTR, and a poly(A) tail structure [2]. UTRs are critical factors that regulate mRNA stability and translation efficiency, playing significant roles in cellular transcription and translation processes [3].…”