In vitro transcription for RNA vaccines and therapeutics: a quality-by-design perspective on manufacturability.

In vitro transcription (IVT) is the core unit operation for the synthesis of the drug substance (DS) in RNA-based vaccines and therapeutics. As RNA medicines mature into a clinically validated product class, the central challenge is shifting from clinical feasibility to manufacturability. This review reframes IVT as a coupled, interaction-driven synthesis process in which product quality and process performance are created during transcription rather than merely assessed afterward. Using a Quality by Design (QbD) framework, it analyzes how critical material attributes (CMAs) and critical process parameters (CPPs) shape product critical quality attributes (CQAs) and manufacturing key performance indicators (KPIs), and how these relationships define the IVT design space. The review takes a manufacturing-focused perspective, with emphasis on scalability and robustness. It combines comparative analysis of reported IVT compositions with transcription-phase-resolved mechanistic analysis to identify the principal CMAs and CPPs governing transcriptional performance, impurity formation, and transcript heterogeneity. It further shows how the IVT design space determines process- and product-related impurity burdens and, in turn, downstream separability and purification strategies. Finally, it discusses how computational models, process analytical technology, soft sensors, and digital twins can extend QbD toward quality by digital design (QbDD) in RNA manufacturing. Overall, this review shows that continued progress in IVT manufacturing depends on stronger mechanistic understanding, better standardization, and closer integration of synthesis, analytics, and control.