Abstract: The present invention describes methods for the characterization of mRNA molecules during mRNA production. Characterizing mRNA includes processes such as oligonucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and detection of RNA impurities. Oligonucleotide mapping includes using an RNase to digest antisense duplexes from an RNA transcript, and then subjecting the digested RNA to reverse phase HPLC, anion exchange HPLC, and/or mass spectrometry analysis. Reverse transcriptase sequencing involves reverse transcription of an RNA transcript followed by DNA sequencing. Charge distribution analysis can comprise procedures such as anion exchange HPLC, or capillary electrophoresis. Detection of impurities includes detecting short mRNA transcripts, RNA-RNA hybrids, and RNA-DNA hybrids.

Abstract: Reversed phase-High Performance (High Pressure) Liquid Chromatography (RP-HPLC) and Size Exclusion Chromatography (SEC) methods have been developed for monitoring structural and size heterogeneity as well as stability of large RNA transcripts, including lengths of up to at least 10,000 nucleotides. The methods are designed for significantly larger mRNAs that could be monitored in the past, including lengths of up to at least 10,000 nucleotides, and including chemically modified RNA transcripts. SEC techniques are also used in the preparative purification of large RNA transcripts to remove impurities, including hybridized nucleic acid impurities and multimeric RNA species. All of these techniques are also beneficial in that they can be used for large scale manufacturing of therapeutics.