Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity

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.