用于mRNA连续纯化的过程分析技术（PAT）：沉淀与切向流过滤

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.