Process Analytical Technology (PAT) for Continuous Purification of mRNA via Precipitation and Tangential Flow Filtration.

Continuous downstream processing of messenger ribonucleic acid (mRNA) therapeutics requires purification strategies that are scalable, robust, and capable of removing product- and process-related impurities from in vitro transcription mixtures. Precipitation-based purification coupled with tangential flow filtration (TFF) has recently been demonstrated as a chromatography-free route for continuous mRNA purification. However, implementation of this approach requires real-time monitoring strategies that can distinguish the product-rich precipitated phase from the impurity-rich liquid phase, as well as phase-dependent transport behavior across the integrated process. In this work, we develop a phase-resolved process analytical technology (PAT) and residence time distribution (RTD) framework for continuous mRNA purification by PEG/NaCl precipitation and sequential TFF. Inline UV-Vis spectroscopy and LED-based optical density measurements were used concurrently to separate nucleic-acid absorbance from turbidity-driven responses associated with suspended mRNA precipitates. RTD experiments with dissolved and precipitated tracers revealed phase-dependent transport behavior in both the tubular precipitator and TFF module, which was quantified using axial dispersion and tanks-in-series models. During integrated operation, inline PAT captured startup, steady-state, and washout behavior, while stable TMP indicated operation without observable membrane fouling. This work demonstrates that precipitation-based continuous mRNA purification can be strengthened through phase-resolved monitoring and RTD-based process understanding, providing a framework for robust development of continuous downstream processes for nucleic acid therapeutics.