Characterization of Impurities in Therapeutic RNAs at the Single Nucleotide Level

Abstract

The chemistry of guide RNA (gRNA) affects the performance of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing technique. However, the literature is very scarce about the study of gRNA degradation and in particular at the single nucleotide level. In this work, we developed a workflow to characterize the impurities of large RNAs at the single nucleotide level and identified the residues prone to degradation. Our strategy involves (i) the reduction of RNA length, (ii) a chromatographic mode able to capture subtle changes in impurity polarity, and (iii) a streamlined data treatment. To illustrate the approach, stressed gRNA samples were analyzed by coupling an immobilized ribonuclease T1 cartridge to a hydrophilic interaction liquid chromatography (HILIC) column hyphenated with tandem mass spectrometry (MS/MS). Critical findings were made possible by the presented technology. In particular, the desulfurization of phosphorothioate (PS) linkages was the major degradation observed at the single nucleotide level while no change in purity profile could be observed when using conventional ion-pairing reversed-phase (IPRP) liquid chromatography. To our knowledge, this is the first time that several impurity types are screened for a large RNA molecule using an automated online digestion analysis approach.

Sample transformation in online separations: how chemical conversion advances analytical technology

“…In their follow-up study, impurities of large therapeutics RNAs at the nucleotide level were identified by using the RNase T1 IMER. 67 The digestion time was here under three minutes. These two studies showed that there are opportunities for faster enzymatic digestion and minimizing the introduction of modifications.…”

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…In a subsequent study, the same researchers automated the investigation of sgRNA degradation at the single‐nucleotide level using an mD‐LC set‐up. The objective was to localize and quantify impurities having the same length as their RNA parent molecule but exhibiting slight differences in polarity [83]. The methodology involved i) reducing RNA length through an online digestion procedure, ii) employing a HILIC method with a BEH amide column capable of detecting subtle changes in impurity polarity, and iii) employing a straightforward MS/MS data treatment.…”

“…(B) Zoomed‐in view (time window 23–31 min) of HILIC‐UV profiles obtained for sgRNA A after oxidative stress (0, 6, 12, 18, and 24 h). Adapted [83], with permission.…”

Online Nucleotide Mapping of mRNAs

“…12 Additionally, the online setup limits the contamination of the LC-MS system and column from the free RNase present in solution with conventional approaches. 13,14 The digestionanalysis platform made it possible for online coupling to forced degradation for impurity analysis at the single nucleotide level 15 and analysis of fractions collected from reference purity methods. 12 In this study, we evaluated an online digestion-analysis platform utilizing immobilized RNase T1 and A cartridges and 2D-LC-MS analysis for the online nucleotide mapping of mRNAs.…”

“…In comparison to in-solution approaches, the required amount of RNA is significantly reduced (>2-fold) and the digestion time is also significantly shortened (>5-fold) . Additionally, the online setup limits the contamination of the LC-MS system and column from the free RNase present in solution with conventional approaches. , The digestion-analysis platform made it possible for online coupling to forced degradation for impurity analysis at the single nucleotide level and analysis of fractions collected from reference purity methods …”