# In-process analytics for IVT mRNA manufacturing: from process understanding to advanced process control

## TL;DR

This paper explores how in-process analytics can improve the understanding and control of mRNA manufacturing processes, from DNA preparation to final product quality.

## Contribution

The paper introduces a framework for categorizing in-process analytical methods based on their control intent in mRNA manufacturing.

## Key findings

- In-process analytics can enhance process understanding and decision-making during mRNA production.
- Current limitations include the lack of real-time quantification methods for double-stranded RNA and mature in-line sensing technologies.

## Abstract

RNA therapeutics are expanding rapidly, driving demand for manufacturing processes that can keep pace with clinical translation. Because mRNA yield and impurity profiles are jointly influenced by upstream plasmid DNA (pDNA) preparation and the in vitro transcription (IVT) reaction, in-process measurements are increasingly applied across the product lifecycle, albeit with distinct objectives in process development versus current good manufacturing practice (cGMP) production. Within the U.S. Food and Drug Administration’s Process Analytical Technology framework, we categorize in-process analytical methods by control intent: (1) measurements that build process understanding and define operating windows; (2) in-process controls (IPCs) that support predefined stop/go, forward-processing, and endpoint decisions; and (3) measurements that could enable advanced or adaptive process control (APC) through closed-loop feedback. We discuss how each category is deployed during process development and in cGMP manufacturing. Following the workflow from pDNA preparation through IVT, we highlight analytical measurements that establish template readiness—such as plasmid topology, linearization completeness, and co-purifying impurities that can propagate into transcription performance and complicate downstream processing—as well as time-resolved measurements during IVT that track reactant consumption and product formation to inform endpoint selection, feed timing, and deviation triage under predefined decision rules. We compare the strengths, implementation constraints, and validation considerations of at-line, on-line, and in-line approaches, and identify key gaps that currently limit broader adoption, including practical time-resolved quantification of double-stranded RNA and the availability of production-ready in-line sensing technologies. Collectively, these in-process analytics deliver near-term value by enabling process understanding and IPC-based decision support, while establishing the foundation required for future APC in mRNA manufacturing.

Flowchart illustrating in-process analytics across IVT mRNA manufacturing, divided into DNA template preparation, IVT reaction, and downstream processing. Stages include plasmid DNA production, reaction monitoring, chromatography, filtration, polishing, and pre-qualification, with icons indicating at-line, on-line, and in-line analytics. Color-coded arrows denote process understanding, IPC-style decision support, and APC-enabling measurement.