The COVID-19 pandemic highlighted the need for rapid vaccine development. Traditional vaccine manufacturing processes are slow and expensive, necessitating faster alternatives. mRNA vaccines offer a promising solution due to their speed of development and production. While mRNA vaccines have shown great success, particularly in combating COVID-19, current manufacturing processes involve multiple steps and are not fully automated. This paper addresses this gap by describing an integrated platform for automated, on-demand mRNA production. The platform aims to streamline the process, from DNA template amplification to LNP encapsulation, ensuring rapid response to emerging infectious diseases and facilitating personalized medicine applications. Existing methods for mRNA production often involve in vitro transcription (IVT) from DNA templates, requiring several steps including DNA amplification, transcription, purification, poly(A) tailing, and encapsulation into LNPs. These processes are often manual, time-consuming, and prone to errors. Existing automated systems, like digital-to-biological converters (DBCs), are often bulky and lack versatility, while microfluidic systems have limitations in reaction volume. The need for a universal, flexible, and automated platform for on-demand mRNA preparation is crucial for addressing urgent health crises and personalized treatment.

The introduction extensively reviews the advantages of mRNA vaccines over traditional vaccines, emphasizing their speed and cost-effectiveness, particularly in the context of the COVID-19 pandemic. The review highlights the successful implementation of mRNA vaccines like Moderna's mRNA-1273 and Pfizer/BioNTech's BNT162b2. The limitations of existing mRNA production methods, including manual steps, susceptibility to RNase contamination, and the lack of fully automated platforms, are discussed. The authors also review existing automated systems, such as DBCs and microfluidic systems, highlighting their limitations in terms of size, versatility, and reaction volume. This sets the stage for the proposed integrated platform as a solution to these limitations.

The researchers developed an integrated platform consisting of three main modules: a PCR module for DNA template amplification, a heating-magnet separating-mixing (HMM) module for IVT reactions, and an LNP module for mRNA encapsulation using staggered herringbone micromixer (SHM) microfluidic chips. The PCR module uses standard PCR reagents and conditions. The IVT module utilizes T7 RNA polymerase, cap analog, and poly(A) polymerase to synthesize capped and polyadenylated mRNA. Magnetic beads are used for DNA and mRNA purification. The LNP module utilizes SHM microfluidic chips for efficient encapsulation. The entire process is automated and controlled via a user-friendly interface. Enhanced green fluorescent protein (eGFP) was used as a model to develop and test the workflow. The methodology section details the specific reagents, concentrations, and conditions used for each step, including PCR, IVT (with capping and poly(A) tailing), DNase digestion, and mRNA purification. The purification steps rely on magnetic beads to separate DNA and mRNA, enhancing efficiency and reducing contamination risks. Lipid nanoparticle (LNP) encapsulation is performed using a microfluidic system for improved mixing and encapsulation efficacy. The experimental procedure also describes the cell culture and transfection methods used to evaluate the effectiveness of the produced mRNA in HEK-293T cells. Western blotting is used to analyze protein expression in cells transfected with SARS-CoV-2 RBD mRNA.

The integrated platform successfully produced eGFP mRNA with a batch production rate of 200–300 µg in 8 h. The platform also produced an mRNA vaccine encoding the SARS-CoV-2 RBD, which was confirmed by successful protein expression in HEK-293T cells as analyzed by Western blotting. The PCR module successfully amplified the DNA template, as shown via gel electrophoresis. The effectiveness of the IVT process was validated by successful mRNA production and purification. LNP encapsulation resulted in appropriately sized nanoparticles, further confirmed by transmission electron microscopy and dynamic light scattering (DLS). The results demonstrate the platform's capacity to produce functional mRNA at a reasonable rate, making it a promising tool for both research and practical applications.

The success of the integrated platform in producing functional eGFP mRNA and SARS-CoV-2 RBD mRNA highlights its potential for rapid vaccine and therapeutic development. The automated nature of the platform significantly reduces the time and labor required for mRNA production compared to traditional methods. The use of magnetic beads for purification steps minimizes contamination risk and ensures efficient mRNA yield. The integrated platform addresses the limitations of existing systems by combining the efficiency of automation with the flexibility needed for various mRNA products. The ability to produce mRNA vaccines on demand is especially valuable for responding to emerging infectious disease outbreaks.

This study presents a successful integrated platform for streamlined and on-demand preparation of mRNA products. The platform combines PCR, IVT, purification, and LNP encapsulation into an automated workflow. The production of functional eGFP mRNA and SARS-CoV-2 RBD mRNA demonstrates its potential for rapid vaccine and therapeutic development. Future work could focus on optimizing the platform for higher throughput and exploring its application for other mRNA-based products. The platform also needs further testing and validation to fully understand its reliability and scalability for large-scale manufacturing.

While the platform demonstrates the feasibility of automated mRNA production, its scalability for large-scale manufacturing needs further investigation. The current prototype's throughput may need to be increased to meet the demands of mass vaccination campaigns. Further optimization and validation are needed to ensure consistent quality and yield across multiple batches. A comprehensive cost-benefit analysis should be conducted to assess its economic feasibility compared to existing methods. The study used only eGFP and SARS-CoV-2 RBD as models; more extensive testing with diverse mRNA sequences is required to confirm its universality.