Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.

Area of Science:

• Pharmaceutical Science
• Analytical Chemistry
• Biotechnology

Background:

• Modified messenger RNA (mRNA) is a rapidly advancing therapeutic drug class.
• Robust analytical methods are essential for quality control in mRNA pharmaceutical development.

Purpose of the Study:

• To develop and optimize a stability-indicating ion-pair reversed-phase liquid chromatography (IP-RPLC) method for characterizing modified mRNA.
• To assess the method's suitability for analyzing mRNA drug products under various stress conditions relevant to pharmaceutical development.

Main Methods:

• An IP-RPLC method was optimized by studying column temperature, mobile phase flow rate, and ion-pair selection.
• The method was applied to separate and analyze a model RNA ladder and a modified eGFP mRNA compound (996 nucleotides).
• Stability-indicating capability was demonstrated by exposing eGFP mRNA to heat, hydrolysis, and ribonuclease treatment.

Main Results:

• The optimized IP-RPLC method achieved baseline separation of RNA fragments up to 1000 nucleotides.
• Using 100 mM Triethylamine as the ion-pairing agent provided the highest resolution for large RNA fragments (750/1000 nucleotides) and the eGFP mRNA.
• The method successfully monitored degradation products of eGFP mRNA under various stress conditions, enabling assessment of its relative stability.

Conclusions:

• The developed IP-RPLC method is robust and suitable for the characterization and quality control of modified mRNA drug products.
• This method provides critical insights into mRNA stability, supporting the pharmaceutical development of mRNA-based therapeutics.
• The method's ability to monitor degradation products is key for ensuring the safety and efficacy of mRNA therapeutics.