Light-Scattering and LC-MS Tools for Advancing Oligonucleotide Analysis

Monitor RNA Sequence Ben Wilkes first tackled Waters’ RapiZyme series of RNases for oligonucleotide digestion and mapping. Waters have created two new digestion enzymes to tackle the problem of non-specific short overlapping digestion fragments: RapiZyme MC1 and RapiZyme Cusativin. These two enzymes enable larger, less overlapping digestion products which are able to be uniquely identified by LC-MS. Waters have developed an end-to-end workflow for RNA mapping. Starting with their digestion enzymes, they conduct LC-MS analysis of the digested fragments using BioAccord and Xevo G3. In parallel, software is used to generate in silico digestion products which can be fed into the analysis software, this can be compared to the experimental digestion analysis. Wilkes highlighted their MAP Sequence software for this task. Sequence coverage can then be generated using further in silico tools. Routinely Track mRNA CQAs Using a similar digestion strategy, Waters have built a workflow using LC-MS and their INTACT Mass software to monitor the critical quality attributes of mRNA samples. This has been applied particularly to 5’ capping and 3’ Poly(A) tails. Impurities often form when generating a 5’ cap. This can make analysis difficult when analysing sequences of thousands of base-pairs. Waters’ workflow allows them to digest the product, separate it chromatographically and analyse it using mass spectrometry to generate a graph which displays the 5’ cap and uncapped precursors. The software allows the user to generate a simple tabulated view of the percentage purity of their intended 5’ cap product. Furthermore, the after LC-MS sequence map is generated, it can be difficult to interpret the heterogeneous signal from the Poly(A) tail. However, Waters’ INTACT Mass software can deconvolute the signal into an easy to interpret result. Optimise Formulation Efficiency Next Wilkes discussed Waters’ workflows for characterising and monitoring lipid nanoparticles (LNPs). Using LC-MSE data, Waters apply their discovery tools to identify unknown impurities. This information can then be incorporated into a monitoring and quantification method. Using the same approach, Waters are able to conduct targeted screening and consultation of LNP products, as well as their raw materials and associated impurities, and generate comprehensive reports. All these processes are performed under GMP-compliant software, ensuring validation and reliability. With a single injection, they can achieve impurity identification, as well as monitoring and quantitation of targeted products. Light Scattering Solutions Wilkes then passed over to Kevin Jackson to present on multi-angle light scattering (MALS) for determining molecular weight and hydrodynamic radius in RNA analysis. Here he emphasised the tool’s utility in providing absolute calibration without the need for mass calibration. Automated techniques can be applied using MALS for assessing critical quality attributes in adeno-associated viruses (AAVs), allowing for rapid analysis of particle concentration and molecular weight. Jackson explained how light scattering can be used to monitor aggregation in biomolecules, providing insights into the stability and integrity of therapeutic proteins. Furthermore, the technology can be used for in-line monitoring of production processes, enabling real-time feedback to improve batch reliability and reduce failures. Waters have developed specialised software, such as MAP Sequence, for oligonucleotide mapping and sequencing, which integrates digestion data with LC-MS results for comprehensive analysis.