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Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

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Published on: April 4, 2025

Real-Time Monitoring of In Vitro Transcription for Production of mRNA Using Raman Spectroscopy.

Julia Puppin Chaves Fulber¹, Xingge Xu¹, Hadi El Radi²

¹Viral Vectors and Vaccines Bioprocessing Group, Department of Bioengineering, McGill University, Montreal, Quebec, Canada.

Biotechnology and Bioengineering

|March 24, 2026

Summary

This study introduces Raman spectroscopy for real-time monitoring of messenger RNA (mRNA) manufacturing. This innovation addresses challenges in mRNA production, enabling more efficient and cost-effective vaccine development.

Keywords:

Raman spectroscopy in vitro transcription inline monitoring mRNA manufacturing process analytical technologies

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Area of Science:

Biotechnology
Chemical Engineering
Spectroscopy

Background:

Messenger RNA (mRNA) vaccines, exemplified by COVID-19 vaccines, highlight the platform's potential but face manufacturing hurdles.
Current in vitro transcription (IVT) methods for mRNA production are costly and lack real-time monitoring capabilities.

Purpose of the Study:

To develop a simultaneous inline monitoring method for nucleotide triphosphates (ATP, CTP, GTP, UTP) and mRNA concentrations during IVT.
To demonstrate the sequence-independent prediction capability of Raman spectroscopy for mRNA manufacturing.

Main Methods:

Utilized Raman spectroscopy combined with partial-least squares regression (PLS) for inline monitoring of IVT.
Analyzed Raman spectral features related to total mRNA concentration and sequence-specific variations.

Main Results:

Achieved high model prediction performance with R² values from 0.82-0.99 and relative errors of 4%-13%.
Demonstrated sequence-independent prediction, negating the need for recalibration across different mRNA products.
Results were comparable to traditional offline assay errors (10%-12%).

Conclusions:

Raman spectroscopy offers a robust solution for real-time monitoring of mRNA IVT.
This advancement supports the transition to continuous processing, automated digital twins, and Pharma 4.0 in biomanufacturing.