Stability Indicating Ion-Pair Reversed-Phase Liquid Chromatography Method for Modified mRNA

Abstract

Modified messenger RNA (mRNA) represents a promising new class of therapeutic drug product. Development of robust stability indicating methods, for control of product quality, are therefore critical to support successful pharmaceutical development. This paper presents the first ion pair reversed phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. The impacts of column temperature, mobile phase flow rate and ion-pair selection were studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100mM Triethylamine, enabled the highest resolution separations for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the resulting degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well visualized, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.