Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques

Abstract

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3′-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns

“…Camperi et al performed a comparison of CGE, mCE, and SEC for the evaluation of covalent and non-covalent aggregates of mRNA under native and denatured conditions. Their results, obtained with a commercial ultra-wide pore SEC column packed with 1000 Å particles, confirm the possible application of SEC to assess aggregation of three different mRNA samples (i.e., eGFP, Fluc, and beta gal mRNA) from two different suppliers [ 13 ]. More recently, De Vos et al investigated the capabilities and limitations of SEC for the characterization of IVT mRNA samples of different sizes [ 18 ].…”

“…However, capillary electrophoresis (CE)-based RNA integrity assays require relatively long run times and can sometimes show relatively high quantitation variability [ 8 ]. Microchip capillary electrophoresis (mCE) has recently been proposed as a quicker alternative for RNA samples consisting of up to 6000 nt [ 12 , 13 ]. Alternatively, ion-pairing reversed-phase liquid chromatography (IP-RPLC) has been widely used for the analysis of small oligonucleotides (ONs), but its application to larger ONs such as mRNA drugs is much more limited.…”

Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides

“…In the current report, we demonstrated that selective enzymatic digestion of RNA fragments (lacking a poly(A) tail), using minimal resources and no excessive heating, results in an unambiguous readout: any signal higher than the background luminescence is due to the presence of untailed mRNA fragments, which are all unwanted byproducts. However, byproducts such as 3' loop-back dsRNA and transcripts with shorter poly(A) tails are unlikely to be detected by the PNPase assay [ 9 , 17 ]. It is also important to note that the PNPase assay requires incubation at 37°C and hence also introduces some intra-assay cleavage events.…”

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

“…Fragmented mRNA is another common impurity that must be taken into account [ 26 , 27 ]. Only full-length mRNA transcripts containing both a 5′-cap and a 3′-poly(A) structure are viable for in vivo expression.…”