# Optimization of In Vitro Transcription by Design of Experiment to Achieve High Self-Amplifying RNA Integrity

## TL;DR

Researchers optimized in vitro transcription to improve the quality of self-amplifying RNA vaccines, enhancing their effectiveness and production.

## Contribution

A multivariate design space for in vitro transcription was developed to improve saRNA integrity and yield for vaccine manufacturing.

## Key findings

- Mg2+ concentration strongly influences saRNA integrity during in vitro transcription.
- Optimized IVT conditions achieved over 85% saRNA integrity and 600 μg/100 μL yield.
- Higher saRNA integrity significantly boosted immune responses in murine models.

## Abstract

Background: Self-amplifying mRNA (saRNA) holds promising application prospects. However, due to the inclusion of a replicase sequence, its extended length leads to premature termination during in vitro transcription (IVT), resulting in poor product integrity. This study aims to optimize the IVT process for saRNA vaccines to enhance integrity, thereby addressing the key challenge in saRNA vaccine manufacturing. Method: Guided by the Quality by Design (QbD) framework, Design of Experiment (DoE) methodology was employed to design diverse combinations of process parameters for IVT reactions. Predictive models were established to identify critical process parameters (CPPs) influencing integrity and yield. An optimized parameter set and process design space, meeting predefined yield and integrity standards, were developed. The impact of integrity on the immunogenicity of saRNA vaccines was further investigated. Results: Mg2+ concentration exerted the most pronounced effect on saRNA integrity. Under optimized IVT conditions, integrity exceeded 85%. Mathematical modeling simulations defined the IVT design space, meeting the preset criteria of ≥80% integrity and ≥600 μg/100 μL yield while accommodating longer saRNA constructs. Notably, murine model data revealed that higher saRNA integrity significantly enhanced antigen-specific antibody and T-cell responses. Conclusion: This study successfully established a multivariate IVT design space fulfilling preset integrity and yield criteria, providing critical data references for the industrialization and quality specification development of saRNA vaccines.