RNA vaccines and CRISPR-based gene editing are two breakthroughs used to fight SARS CoV-2. The safety and efficacy of CRISPR-based gene editing products depends on the purity of the RNA final product. IP-RP chromatography is the method of choice for oligonucleotides, but conventional methods are not often sufficient for long RNAs. Here, BRAC1-sgRNA and Cas9-mRNA were used as model systems and enzymatically digested to be analyzed by IP-RP chromatography coupled to HRMS using C18 columns of 2.1 and 0.150 mm in inner diameter (ID). Here, we show how both, analytical and low flow columns can be used to obtain sequence identification and chromatographic resolution of impurities of sgRNA and Cas9-mRNA while highlighting the benefits and challenges of each column.
hRNAse 4 digestion resulted mainly in oligonucleotide lengths of 5 to 40nt, which are well suited for MS/MS analysis and consequently oligonucleotide mapping or identification. By using a combination of DIA (Data Independent Acquisition) and Intact Mode mass spectrometry workflows; accurate oligonucleotide mapping of sgRNA and mRNA was achieved. Besides sequence coverage, impurity profiling of sgRNA and Cas9-mRNA was also accomplished. Moreover, both column IDs showed sgRNA PS diastereomers resolution capabilities. mRNA 5’Cap and Poly-A tail species were also well characterized. Total ion chromatogram comparison of low-flow micro and analytical flow showed about 50% increase in ion sensitivity. Moreover, oligonucleotide sequence coverage via low flow chromatography was achieved by using a fraction of what was needed for analytical flow studies. Overall, this study shows a reliable low flow IP-RP-MS/MS workflow for sgRNA and mRNA characterization using 1/25th of the sample amount used in analytical flow setting.