Streamlined and on-demand preparation of mRNA products on a universal integrated platform

The COVID-19 pandemic underscored the urgent need for vaccine platforms that can be rapidly designed, produced, and deployed. Conventional vaccine manufacturing is pathogen- and process-specific, making rapid responses to emerging threats costly and time-consuming. Nucleic acid vaccines, particularly mRNA, offer advantages including faster development and production timelines, strong cellular and humoral immune responses, and no risk of genomic integration. The real-world success of mRNA COVID-19 vaccines (e.g., Moderna mRNA-1273 and Pfizer/BioNTech BNT162b2) validates the platform’s potential. Standard mRNA manufacturing relies on in vitro transcription (IVT) from DNA templates, with 5′ capping, DNase digestion, and 3′ poly(A) tailing, followed by purification and lipid nanoparticle (LNP) encapsulation. Despite its suitability for automation, few integrated, automated, on-demand mRNA preparation platforms have been reported; existing solutions (e.g., the digital-to-biological converter or microfluidic systems) have limitations in bulkiness, volume, or versatility. This study aims to address this gap by developing a universal, automated, and modular “template-in vaccines-out” platform that integrates PCR amplification, IVT with capping, DNA digestion, poly(A) tailing, magnetic-bead purification, and LNP encapsulation. The platform is demonstrated using eGFP mRNA and a SARS-CoV-2 RBD mRNA vaccine as test cases to evaluate throughput and expression.

Prior work established mRNA vaccines as a versatile platform with strong immunogenicity and favorable safety relative to DNA vaccines, supported by preclinical and clinical successes, notably against COVID-19. Standard IVT workflows (T7 polymerase-mediated transcription from plasmid or PCR-amplified templates, enzymatic capping, DNase treatment, and poly(A) tailing) are robust and sequence-agnostic. Automation efforts include the digital-to-biological converter (DBC), which can synthesize diverse biologics but is bulky, and microfluidic systems that separately manage transcription and translation but are constrained by reaction volume and versatility. Consequently, there remains a need for compact, integrated, and flexible platforms that automate end-to-end mRNA preparation, minimize RNase exposure, prevent cross-contamination, and deliver LNP-formulated products suitable for rapid R&D and on-demand manufacturing.

System overview: A universal integrated platform was engineered with three core modules: (1) PCR module for target DNA template amplification; (2) HMM (heating–magnet separating–mixing) module enabling thermostable, magnetically assisted IVT operations (capping, DNase digestion, and poly(A) tailing) and magnetic-bead-based purifications; and (3) LNP module employing staggered herringbone micromixer (SHM) microfluidic chips for rapid mRNA encapsulation into LNPs. Workflows (washing, self-test, PCR, IVT with capping, DNase digestion, poly(A) tailing, purification) were user-programmable via a touchscreen interface. Device structure: The aluminum-alloy prototype (62.5 cm × 50.0 cm × 32.5 cm) includes: a back compartment with electronics and power; a middle compartment with seven peristaltic pumps, two five-way and four three-way electromagnetic valves interconnected by 1/16″ FEP tubing; a left compartment with 11 reservoirs (nine reagents, one pump-wash, one waste); and a front compartment hosting the PCR and IVT areas with a plunger pump for reagent mixing. A microcomputer/touchscreen on top controls workflows. The PCR module comprises a Peltier element, heat sink, cooling fan, insulation block, reaction tube pedestal, and a heated lid; real-time temperature tracking was implemented for liquid and pedestal. PCR: Reaction (total 200 µL) contained 10 U LA Taq, 1× LA Taq buffer II (Mg2+ Plus), dNTPs (200 nM each), 4.8 ng DNA template, primers (200 nM each), and nuclease-free water. Cycling: 95 °C 5 min; 30 cycles of 95 °C 40 s, 58 °C 45 s, 72 °C 60 s; final extension 72 °C 10 min. Products were analyzed on 1% agarose TAE gel. Bulk DNA capture used hydrophilic-coated streptavidin magnetic beads in 1 mL buffer (0.72 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA) at 30 °C for 45 min. IVT, capping, DNase digestion, and poly(A) tailing: IVT (200 µL total) included 20 µL T7 polymerase enzyme mix, 5× buffer, rNTPs (1.875 mM rATP, rCTP, rUTP; 0.225 mM rGTP), 0.375 mM cap analog, linear DNA captured on streptavidin beads, and nuclease-free water; incubated at 37 °C for 2 h. DNase treatment: add 10 U RQ1 RNase-free DNase; 37 °C for 15 min. Poly(A) tailing: 200 µL post-DNase mixture incubated with 40 U E. coli poly(A) polymerase (E-PAP), 5× E-PAP buffer, 2.5 mM MnCl2, 1 mM ATP, nuclease-free water to 400 µL; 37 °C for 1 h. Purification used oligo d(T)25-coated magnetic beads. Bead prep: wash 200 µL beads once with 100 µL binding buffer (40 mM Tris-HCl pH 7.5, 1.6 M LiCl, 2 mM EDTA, 0.5 U/µL RNase inhibitor), then resuspend. mRNA was heat-treated 65 °C for 5 min and immediately mixed with beads for 5 min at room temperature. Wash beads twice with 200 µL wash buffer (10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA, 0.5 U/µL RNase inhibitor). Elute mRNA with 200 µL nuclease-free water. RNA quality/concentration were assessed using an Agilent 2100 Bioanalyzer with RNA Nano 6000 kit. All solutions used 0.1% DEPC-treated water; consumables were autoclaved; surfaces treated with RNase Zap and 0.1% DEPC to minimize RNase. LNP encapsulation: Constant flow control employed a Flow EZ pressure controller (0–2000 mbar) and FLOW UNIT flow monitoring (0–1000 µL/min) from Fluigent. SHM microfluidic chips (Ibiochips) were used for rapid mixing of lipid and aqueous streams to form LNPs. LNP morphology was imaged by TEM (Hitachi H-7650); size distribution was measured by DLS (Nano-Lab Zeta Sizer, Malvern). In vitro transfection and expression assays: HEK-293T cells (3 × 10^5 cells/well, 12-well plates) were transfected with mRNA–LNP complexes (1 µg/well). At 24 h post-transfection, eGFP expression was visualized by fluorescence microscopy (Olympus IX71). For SARS-CoV-2 RBD mRNA, protein expression was assessed by Western blotting. mRNA–LNPs produced on a commercial Precision Nanosystems Ignite system served as a positive control. System operation and contamination control: Reagents were stored in isolated reservoirs and routed via valves/pumps to prevent cross-contamination; used storage units were disposed post-run; waste was collected in dedicated reservoirs. User-defined workflow files controlled sequential execution of PCR, IVT, enzymatic processing, bead-based purification, and LNP formulation.

• A universal, automated “template-in vaccines-out” platform integrating PCR amplification, IVT with co-transcriptional capping, DNase digestion, poly(A) tailing, magnetic-bead purification, and SHM microfluidic LNP encapsulation was successfully constructed and operated.
• Throughput: Batch production rate estimated at 200–300 µg of eGFP mRNA in 8 hours.
• Functional validation: eGFP mRNA–LNPs produced by the platform yielded robust eGFP expression in HEK-293T cells at 24 h post-transfection.
• Vaccine payload demonstration: An mRNA encoding the SARS-CoV-2 spike receptor-binding domain (RBD) was produced and confirmed to express RBD protein by Western blotting, indicating platform practicability for vaccine constructs.
• LNP formulation: TEM imaging and DLS characterization were performed on LNPs produced using SHM microfluidic chips, evidencing successful nanoparticle formation and size control (qualitatively reported).
• PCR module performance: Temperature control was characterized in real time for both liquid and pedestal, and agarose gel electrophoresis showed PCR products comparable to those from a standard machine, validating on-board amplification.
• Operational features: Separate reagent loading and dedicated waste handling minimized cross-contamination; customizable workflows enabled end-to-end automation from DNA template to LNP-formulated mRNA.

The results demonstrate that an integrated and automated platform can convert DNA templates into LNP-formulated, expression-competent mRNA in a single, programmable workflow. By consolidating PCR amplification, IVT with capping, enzymatic clean-up, polyadenylation, magnetic-bead purification, and microfluidic LNP encapsulation, the system addresses key bottlenecks in speed, manual handling, and contamination risk. The successful expression of eGFP and SARS-CoV-2 RBD in mammalian cells validates product functionality and indicates applicability to both reporter and vaccine-relevant antigens. Compared with prior solutions, the platform balances versatility and operational scale: unlike microfluidic-only approaches limited by reaction volumes, it supports larger reaction mixes; and unlike bulky general-purpose bioconverters, it is tailored to mRNA workflows with integrated purification and formulation. Collectively, these findings support the feasibility of on-demand, small-batch mRNA production for rapid response scenarios (e.g., emergent pathogens) and personalized applications (e.g., oncology), potentially reducing timelines from sequence to formulated product.

This work presents a universal integrated platform that automates the end-to-end preparation of mRNA products directly from DNA templates, including PCR, IVT with capping, DNase treatment, poly(A) tailing, magnetic-bead purification, and SHM microfluidic LNP encapsulation. The platform achieved 200–300 µg eGFP mRNA per 8-hour batch and generated functional mRNA–LNPs that expressed eGFP and SARS-CoV-2 RBD in HEK-293T cells. Its modular design, contamination control, and workflow programmability suggest broad utility for rapid mRNA R&D, on-demand vaccine prototyping, and potentially personalized therapies. Future work could focus on increasing throughput and scalability, integrating in-line quality control analytics, expanding to diverse modified nucleotides and payloads, validating immunogenicity and efficacy in vivo, enhancing sterility/GMP readiness, and optimizing LNP composition and process parameters for consistent particle attributes.

The study primarily demonstrates feasibility with two constructs (eGFP and SARS-CoV-2 RBD) and evaluates expression in vitro; no in vivo immunogenicity or efficacy data are reported. Quantitative LNP quality attributes (e.g., exact size, PDI, encapsulation efficiency) and detailed yield metrics beyond the eGFP throughput estimate are not provided in the excerpt. Process scalability to higher yields and long-term reliability, sterility controls, and regulatory considerations are not addressed here.