A review of analytical techniques for characterization of mRNA drug substance in therapeutics and their suitability as a multi-attribute method

Abstract Messenger RNA (mRNA) therapeutics have emerged as a versatile class of prophylactic due to their high efficacy, scalable manufacturing, and favorable safety profiles. However, development and manufacturing remain constrained by the complexity and cost of quality assessment. Ensuring mRNA quality requires characterization of numerous critical quality attributes (CQAs), including identity, length, concentration, intactness, and purity. To have a successful therapeutic, mRNA manufacturers must demonstrate they have met specifications on the required CQAs. Currently, most analytical techniques evaluate only a single CQA per experiment, necessitating the use of many different methods that increase analysis time, instrumentation requirements, and operational cost. In this review, we survey analytical techniques commonly used to characterize mRNA drug substance CQAs and critically assess their suitability for integration into a multi-attribute method (MAM) analytical platform capable of reporting multiple CQAs in a single experiment. We compare electrophoretic, chromatographic, sequencing-based, and mass spectrometry-based approaches with respect to CQA, throughput, preprocessing requirements, and total analysis time. By drawing parallels to the successful adoption of MAMs in protein therapeutics, this review highlights opportunities and challenges in adapting similar strategies for mRNA. We propose that continued development of robust mRNA-specific MAM platforms will be critical for streamlining quality control workflows, reducing manufacturing costs, and supporting broader clinical translation of mRNA therapeutics.