Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry

Large RNA including mRNA (mRNA) has emerged as an important new class of therapeutics. Recently, this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study, we have developed an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilized on magnetic particles were performed in conjunction with high-resolution liquid chromatography–mass spectrometry analysis. Sequence mapping was performed using automated oligoribo­nucleotide annotation and identifications based on MS/MS spectra. Using this approach, a >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS-CoV-2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation, can be completed within 90 min. The ability to rapidly identify, characterize, and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation, and impurity analysis.

包含信使核糖核酸（mRNA）的大型核糖核酸（large RNA）已成为一类重要的新型治疗剂。近期，两款基于编码改良型严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白的mRNA序列的高效疫苗，已验证了该类治疗剂的应用潜力。当前，针对包括mRNA治疗剂在内的大型核糖核酸的表征分析，亟需开发新型且更优化的分析方法。本研究开发了一套自动化、高通量的工作流程，可用于大型核糖核酸与mRNA治疗剂的快速表征及直接序列图谱分析。该流程采用固定于磁珠（magnetic particles）的核糖核酸酶T1（RNase T1）进行部分酶解，并结合高分辨率液相色谱-质谱分析。序列图谱分析通过自动化寡核糖核苷酸（oligoribonucleotide）注释及基于串联质谱（MS/MS）谱图的鉴定完成。采用该方法，单次分析即可实现包括SARS-CoV-2刺突蛋白编码mRNA在内的多款大型核糖核酸与mRNA治疗剂的序列覆盖度超过80%。整套分析工作流程（含自动化样品前处理）可在90分钟内完成。该方法可快速鉴定、表征大型mRNA治疗剂并实现高序列覆盖度的序列图谱分析，为药物的身份验证、序列确证及杂质分析提供关键信息。