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 in vitro 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 in vitro 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.