Development of Immobilized Restriction Enzyme Reactors on a Monolith Backbone for Streamlined Plasmid DNA Linearization.

Abstract

High-quality, properly linearized plasmid DNA (pDNA) is a decisive upstream determinant of in vitro transcription (IVT) performance, impacting mRNA yield, integrity, and batch-to-batch consistency. We developed immobilized restriction enzyme reactors (IMERs) on monolithic supports and benchmarked EcoRI, NdeI, and HindIII to evaluate suitability for IVT-grade template preparation. Across all three enzymes, IMERs quantitatively converted supercoiled and open-circular pDNA to the linear form and delivered a clean product, as verified by baseline-resolved chromatography and agarose gels. To further verify the functionality of the IMER platform, BspQI was immobilized and used to linearize a second plasmid construct. IVT initiated from all four IMER-processed templates produced mRNA with kinetics and titers indistinguishable from conventional in-solution digestion, underscoring that template quality was preserved. Long-term stability studies showed enzyme-dependent stability with HindIII-based IMER retaining ~20% activity for more than 4 months. While the normalized productivity of the IMER matched that of in-solution reactions, the possibility of reusing IMER across multiple linearization cycles significantly increased cumulative yield. Further, linearization by IMERs eliminated the need for post-reaction enzyme removal and harsh inactivation, thereby reducing degradation risk. Collectively, these results establish restriction enzyme-based IMERs as a robust, scalable, automation-ready tool for producing high-purity, IVT-ready linear pDNA, securing the template quality required by the current mRNA manufacturing process.