Characterisation and analysis of mRNA critical quality attributes using liquid chromatography based methods

• We provide a comprehensive review of a wide range of HPLC methods including IP-RP HPLC, AEX, SEC and LC MS methods for the characterisation and analysis of mRNA critical quality attributes. • We discuss the challenges associated with separation of large RNA/mRNAs and associated impurities using HPLC and describe recent developments to overcome these challenges. • We provide a comprehensive review of the applications of LC MS/MS methods for the analysis of mRNA identity, 5′ capping efficiency and poly(A) tail length and heterogeneity. • Advantages and limitations of a wide range of LC UV and LC MS methods for the characterisation of mRNA are described. • Future opportunities and challenges focussing on improved analytical methods for the characterisation and analysis of mRNA are discussed. mRNA technology has been successfully deployed to rapidly develop and mass-manufacture vaccines. Beyond vaccines, RNA-based therapeutics have potential for treatments for infectious diseases, cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. mRNA based vaccines and therapeutics work by translating exogenous mRNA into the target protein. Analytical methods for mRNA characterisation, lot release and stability testing of mRNA drug substance and drug product must be developed and performed to monitor critical quality attributes (CQAs). mRNA is a highly polar molecule due to its extensive negatively charged phosphodiester backbone. Its single stranded nature forms dynamic alternative secondary structures that can generate potential sample heterogeneity, creating challenges for the analysis and characterisation of this large biomolecule. In this review, we describe current analytical methods, focussing on high performance liquid chromatography in conjunction with both UV detection and mass spectrometry for the analysis and characterisation of mRNA. In particular, we describe recent developments covering a wide range of methods centred on liquid chromatography for the analysis of important CQAs including mRNA identity, mRNA integrity, 5′ capping efficiency and poly(A) tail length and heterogeneity.