Physicochemical and functional assessment of messenger RNA 5′Cap-end impurities under forced degradation conditions

The 5'Cap is a modified nucleotide structure added to the 5'end of mRNA, which plays a critical role in stability and translation of the molecule. Accurate characterization of 5'Cap impurities is crucial during development of mRNA therapeutics. Here, we utilized orthogonal liquid chromatography-mass spectrometry (LC-MS) workflows to characterize Cap-1-modified mRNA. A complex mixture of uncapped and, notably, 5'Cap chemical degradation variants, was detected. We subjected Cap-1 analogs to process-related forced degradation conditions and characterized multiple degradation routes of the 5'Cap structure, including hydrolysis and depurination. 5'Cap hydrolysis was observed in a representative <i>in vitro</i> transcription (IVT) reaction buffer and was driven by factors including pH, temperature, and the presence of spermidine and MgCl₂. Method-induced artifactual degradation occurred during LC-MS data acquisition, emphasizing the importance of using reference standards and optimizing methods to reduce such artifacts. We investigated the effect of 5'Cap degradation on expression and reactogenicity <i>in vitro</i> using model mRNAs with elevated impurity levels. mRNA 5'Cap degradation impurities significantly impacted protein expression but did not trigger innate immune response pathways. 5'Cap degradation impurities should be considered as a critical quality attribute of mRNA therapeutics and should be monitored during production, purification, formulation, and storage.