Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.
Julien Camperi1, Steffen Lippold2, Luladey Ayalew1
1Cell Therapy Engineering and Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States.
Analytical Chemistry
|February 20, 2024
Summary
This study details methods for analyzing impurities in in vitro transcribed messenger RNA (mRNA), identifying common issues like varied poly(A) tail lengths and double-stranded RNA byproducts to improve therapeutic quality.
Area of Science:
- Biotechnology and Pharmaceutical Analysis
- Molecular Biology and Nucleic Acid Chemistry
Background:
- In vitro transcription (IVT) of mRNA is crucial for developing mRNA-based therapies and vaccines.
- Impurities in IVT mRNA can compromise the safety and efficacy of these critical biopharmaceuticals.
- Detailed structural characterization of mRNA impurities has been lacking.
Purpose of the Study:
- To pioneer comprehensive impurity profiling of IVT mRNA using integrated analytical technologies.
- To develop and validate robust analytical methods for assessing mRNA purity and integrity.
- To identify and characterize common impurities in commercially sourced IVT mRNA.
Main Methods:
- Development of ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods for mRNA purity determination.
- Application of microcapillary electrophoresis for high-throughput mRNA impurity analysis (<1.5 min/sample).
- Utilized mass photometry and native mass spectrometry for detailed characterization of mRNA and its impurities.
Main Results:
- Identified impurities primarily as mRNA variants with altered poly(A) tail lengths and double-stranded mRNA (dsRNA) byproducts, including the dsRNA 3'-loop back form.
- Mass photometry accurately determined nucleotide counts and detected size variants (aggregates, poly(A) tail absence/partiality), confirming mRNA identity and integrity.
- Native mass spectrometry provided insights into intact mRNA mass, heterogeneity, and poly(A) tail characteristics.
Conclusions:
- The study establishes advanced analytical methods for comprehensive IVT mRNA impurity profiling.
- Findings highlight key impurities and provide a foundation for refining mRNA production processes.
- This work contributes to improving the quality and reliability of mRNA therapeutics and vaccines.
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