Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity
Martin Gilar1, Catalin Doneanu2, Maissa M Gaye3
1Separations R&D, Waters Corporation, Milford, Massachusetts 01757, United States.
Abstract:
Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.
Insights
New analytical methods accurately measure messenger RNA (mRNA) poly(A) tail length and heterogeneity. These techniques are crucial for quality control in mRNA therapeutics development.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Messenger RNA (mRNA) represents a novel class of therapeutic agents.
- Advances in mRNA technology necessitate the development of precise analytical methodologies.
- Characterizing mRNA structure, particularly the poly(A) tail, is critical for therapeutic efficacy and safety.
Purpose of the Study:
- To develop and validate analytical methods for measuring mRNA poly(A) tail length.
- To assess the heterogeneity of mRNA poly(A) tails using advanced chromatographic techniques.
- To establish robust methods suitable for quality control in mRNA manufacturing.
Main Methods:
- Cleavage of the poly(A) tail from mRNA using RNase T1 enzyme.
- Analysis of average poly(A) tail length via size exclusion chromatography (SEC).
- Estimation of poly(A) tail size heterogeneity using high-resolution ion-pair reversed-phase liquid chromatography (IP RP LC).
- Confirmation of results using liquid chromatography-mass spectrometry (LC-MS).
Main Results:
- Developed SEC and IP RP LC methods for poly(A) tail analysis.
- IP RP LC demonstrated resolution of poly(A) tail variants up to 150 nucleotides.
- Both methods employed robust ultraviolet detection, suitable for quality control.
- LC-MS analysis confirmed the accuracy and agreement of the poly(A) tail length and heterogeneity measurements.
Conclusions:
- Established reliable analytical methods for quantifying mRNA poly(A) tail length and heterogeneity.
- The developed methods, utilizing SEC and IP RP LC, are suitable for routine mRNA quality control.
- Accurate characterization of the poly(A) tail is essential for the advancement of mRNA-based therapeutics.
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