Liquid Chromatography-Mass Spectrometry-Based Qualitative Profiling of mRNA Therapeutic Reagents Using Stable Isotope-Labeled Standards Followed by the Automatic Quantitation Software Ariadne.

mRNA-based medicines are a promising modality for preventing virus-caused illnesses, including COVID-19, and treating various types of cancer and genetic diseases. To develop such medicines, methods to characterize long mRNA molecules are needed for quality control and metabolic analysis. Here, we developed an analytical platform based on isotope-dilution liquid chromatography-mass spectrometry (LC-MS) that quantitatively characterizes long, modified mRNAs by comparing them to a stable isotope-labeled reference with an identical sequence to that of the target medicine. This platform also includes database searching using the mass spectra as a query, which allowed us to confirm the primary structures of 200 to 4300 nt mRNAs including chemical modifications, with sequence coverage at 100%, to detect/identify defects in the sequences, and to define the efficiencies of the 5'-capping and integrity of the polyadenylated tail. Our findings indicated that this platform should be valuable for quantitatively characterizing mRNA vaccines and other mRNA medicines.

The transient expression of recombinant proteins in plant cell packs facilitates stable isotope labelling for NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labelled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with 15 N and 2 H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.