Mark J. Dickman

The rapid expansion of mRNA-based medicines has driven demand for robust, high-resolution analytical methods capable of characterising critical quality attributes including sequence identity, 5’ capping efficiency, and poly(A) tail length and heterogeneity. Here, we present two complementary liquid chromatography-mass spectrometry based workflows for mRNA characterisation, both based on site-specific digestion with the endoribonuclease MazF from Escherichia coli . A “middle-up” approach, optimised via specific “5’-ACA” cleavage enables rapid characterisation via oligoribonucleotide mass mapping providing complete sequence coverage and simultaneous assessment of 5-capping efficiency and 3’-poly(A) tail length and heterogeneity. This workflow was successfully applied to three different mRNA constructs, NLuc, eGFP and FLuc mRNAs, achieving sequence coverages of 100%, 87%, and 74%, respectively. A complementary “bottom-up” approach, employing less specific “5’-ACX” cleavage enables tandem mass spectrometry-based sequencing of shorter oligoribonucleotides and detailed characterisation via mRNA sequence mapping. Applied to SARS-CoV-2 Spike Protein mRNA, this method yielded 50% sequence coverage based on unique oligoribonucleotides with MazF alone, extended to 90% by combining MazF, partial RNase T1, and partial RNase U2 digests. Together, these workflows provide a flexible, orthogonal platform for both high-throughput quality assessment and in-depth primary sequence characterisation of mRNA vaccines and therapeutics.

Ion-pair reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) is widely employed for the characterisation of oligonucleotide (OGN) therapeutics. Mobile phases utilising alkylamines and hexafluoro-2-propanol (HFIP) provide high sensitivity but are limited by high cost, low chemical stability, and environmental toxicity. This study evaluates HFIP-free mobile phases utilising low concentrations of dibutylamine (DBA) with propan-2-ol (IPA) and 2,4-dimethylpentan-3-ol (2,4-DMP) as organic modifiers. Due to the high pH of HFIP-free eluents, separation was performed on alkali-stable polymeric and hybrid silica-based stationary phases. Chromatographic resolution and electrospray ionisation (ESI)-MS sensitivity were evaluated against a benchmark DBA/HFIP system using a variety of OGN standards, antisense oligonucleotides (ASOs), and small interfering RNA (siRNA). The HFIP-free systems demonstrated comparable resolution for ssDNA standards (8–40 mer) and only marginal peak broadening for ASOs relative to DBA/HFIP. The high pH and reduced ion-pair reagent concentration resulted in a shift toward higher charge states and a significant reduction in alkylamine and metal ion adduction. Impurity profiling revealed no significant difference in the percentage fractional abundance of OGN impurities between mobile phase systems. These HFIP-free mobile phases offer a sustainable alternative for the LC-MS analysis of OGNs, enhancing signal intensity and reducing adduction while eliminating hazardous fluorinated solvents.

MRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3′-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3′-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

A simple pH signal unlocks real-time, extensive monitoring of mRNA production via mechanistic and semi-empirical models. This aids continuous quantification of RNA yield, NTP depletion and ∼40 IVT components for monitoring and advanced control.

Mass spectrometry-based approaches have emerged as powerful tools for the analysis of a wide range of critical quality attributes of mRNA medicines, including sequence identity, 5' capping efficiency, and 3' poly(A) tail length and heterogeneity. These critical quality attributes can impact the quality, safety, and efficacy of mRNA medicines. In this study, we have utilized online partial RNase T1 digests in conjunction with two-dimensional liquid chromatography mass spectrometry (2D LC-MS) for the direct sequence mapping of mRNA. Automated online partial RNase T1 digests are performed in conjunction with ion-pair reversed-phase liquid chromatography. No sample or solvent manipulation is required following online RNase digestions, demonstrating the simplicity of the method. High-resolution tandem mass spectrometry was used to identify the corresponding oligoribonucleotides and generate mRNA sequence maps. High sequence coverage (93-99%) for eGFP mRNA was obtained in <60 min based only on unique oligoribonucleotide identifications. Moreover, the online partial RNase T1 digests result in controlled, fully automated and reproducible mRNA digests, enabling high-throughput, direct mRNA sequence mapping studies. The online partial RNase digests offer significant advantages over existing methods for rapid, automated mRNA identity testing. Furthermore, precise control of the digest conditions via flow rate and temperature of the online RNase T1 digest, enables multiattribute monitoring of 5' capping efficiency, mRNA sequence mapping, and 3' poly(A) tail length and heterogeneity in a fully automated 2D LC-MS workflow.

MRNA-based technology has emerged as a new class of medicines with a wide range of applications, including viral vaccines, cancer vaccines, and therapeutics for the treatment of metabolic diseases and cardiovascular conditions. Impurities, including double-stranded RNA (dsRNA), mRNA fragments, and mRNA multimers (aggregates) that result from the manufacturing of mRNA, as well as from subsequent purification, formulation, and storage, can potentially impact the safety and efficacy of mRNA medicines. mRNA higher-order structures and mRNA multimers (aggregates) can affect translational efficiency and also impact the efficiency of formulation into lipid nanoparticles. mRNA purity is typically analysed using denaturing or partially denaturing methods, precluding the detection of mRNA multimers (aggregates). In this study, we developed and utilized high-resolution ion-pair reversed phase HPLC (IP-RP HPLC) under non-denaturing conditions to analyse mRNA multimers. The inclusion of 1 mM Mg²⁺ in the mobile phase stabilizes mRNA higher-order structures, RNA:RNA interactions, and the formation of mRNA dimers/multimers, which can be readily separated from the mRNA monomers. The ability to resolve mRNA monomers from mRNA dimers and multimers was demonstrated for a range of mRNA sequences and lengths and was shown to be concentration dependent. Validation of mRNA multimers was performed using mass photometry analysis following the purification of mRNA monomer and dimer/multimer peaks using IP-RP HPLC. The ability to resolve mRNA monomers from mRNA dimers with short chromatographic run times, perform analysis across a wide range of temperatures and concentrations of mRNA combined with the ability to collect the mRNA monomers and dimers/multimers for further downstream analysis demonstrates significant advantages over current approaches for the analysis of mRNA multimers (aggregates). The high-throughput, temperature-dependent profiling of mRNA multimerisation using IP-RP HPLC will enable further comparative studies on the stability of mRNA multimers and provide important insights into potential factors influencing mRNA multimerisation.

Double-stranded RNA plays a key role in various biological processes. The discovery of RNA interference, a gene-silencing mechanism, revolutionised the study of gene function. dsRNA has since been used in novel therapeutics and as an agricultural biocontrol alternative to chemical pesticides. Microbial production typically involves expression systems with convergent T7 promoters. However, convergent transcription from DNA-dependent RNA polymerases can lead to transcriptional interference. In this study, we designed multiple plasmid DNA constructs to investigate the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo in E. coli, prior to dsRNA yield quantification and analysis of product quality. We demonstrate that higher yields of larger dsRNA are typically obtained using convergent promoters during in vivo production. A typical fold increase of 2.1 was obtained for dsRNA > 400 bp. However, production of smaller dsRNA (< 250 bp) by divergent promoters resulted in increased yields (2.2 fold). Furthermore, our data demonstrates that in vitro transcription production of dsRNA using divergent T7 promoters results in significantly higher yields of dsRNA, with a maximum fold increase of 6.46. Finally, independent of size, we demonstrate that dsRNA synthesised from DNA templates with multiple transcriptional terminators, compared to run-off transcription, improved the quality and purity of dsRNA due to decreased formation of dsRNA multimers or aggregates. This study highlights alternative optimal strategies for the production of a wide range of different sized dsRNA both in vitro and in microbial systems

Double-stranded RNA plays a key role in various biological processes. The discovery of RNA interference, a gene-silencing mechanism, revolutionised the study of gene function. dsRNA has since been used in novel therapeutics and as an agricultural biocontrol alternative to chemical pesticides. Microbial production typically involves expression systems with convergent T7 promoters. However, convergent transcription from DNA-dependent RNA polymerases can lead to transcriptional interference. In this study, we designed multiple plasmid DNA constructs to investigate the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo in E. coli , prior to dsRNA yield quantification and analysis of product quality. We demonstrate that higher yields of larger dsRNA are typically obtained using convergent promoters during in vivo production. A typical fold increase of 2.1 was obtained for dsRNA > 400 bp. However, production of smaller dsRNA (< 250 bp) by divergent promoters resulted in increased yields (2.2 fold). Furthermore, our data demonstrates that in vitro transcription production of dsRNA using divergent T7 promoters results in significantly higher yields of dsRNA, with a maximum fold increase of 6.46. Finally, independent of size, we demonstrate that dsRNA synthesised from DNA templates with multiple transcriptional terminators, improved the quality and purity of dsRNA due to decreased formation of dsRNA multimers or aggregates, compared to run-off transcription. This study highlights alternative optimal strategies for the production of a wide range of different sized dsRNA both in vitro and in microbial systems.