Synthetic mRNA is a novel type of biotherapeutic that has recently played a crucial role in the development of SARS-CoV-2 vaccines. Synthetic mRNA can translate proteins in the cytoplasm and exhibit different protein expression patterns depending on the engineering strategies. Synthetic mRNA also has the advantages of rapid, scalable, and cost-effective production. The analysis of synthetic mRNA is essential for ensuring its quality, safety, and efficacy. However, the analysis of synthetic mRNA also poses significant challenges due to its complex structure, diverse modifications, and susceptibility to degradation. Therefore, the development of reliable and sensitive analytical methods for synthetic mRNA is an important and active research area.
Synthetic mRNA has a complex structure that consists of several elements, such as the 5' cap, the 3' poly (A) tail, the coding region, and the untranslated regions. The 5' cap is a modified nucleotide that protects the mRNA from degradation by exonucleases and facilitates its recognition by the translation machinery. The 3' poly (A) tail is a long stretch of adenine nucleotides that enhances the stability and translatability of the mRNA. The coding region contains the nucleotide sequence that encodes the protein of interest, and the untranslated regions regulate mRNA localization, stability, and translation.
Synthetic mRNA can also be modified by replacing some of the canonical nucleosides with naturally modified nucleosides or synthetic nucleoside analogues. These modifications can modulate the properties of synthetic mRNA, such as its stability, immunogenicity, and translational efficiency. For example, pseudouridine and 2'-O-methyl modifications can reduce the immunogenicity of synthetic mRNA by decreasing its recognition by innate immune sensors. N1-methylpseudouridine and 5-methylcytidine can increase the stability of synthetic mRNA by enhancing its resistance to degradation by nucleases. N6-methyladenosine and 5-methoxyuridine can improve the translational efficiency of synthetic mRNA by increasing its affinity for the ribosome.
The structure and modification of synthetic mRNA are important factors that determine its performance as a therapeutic agent. Therefore, the development of optimal synthetic mRNA designs and analytical methods to characterize them is an essential and active area of research.
The current state of the art of synthetic mRNA analysis involves various methods, such as reverse phase liquid chromatography (RPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR), which can provide information on the purity, identity, content, structure, conformation, and modification of synthetic mRNA and its impurities and degradation products. These methods have their own advantages and limitations and can be used alone or in combination to achieve optimal results.
RPLC is a separation technique that can be used to measure the purity, identity, and content of synthetic mRNA. RPLC can separate synthetic mRNA from impurities such as residual plasmid DNA, inorganic salts, and organic solvents. RPLC can also provide information on the 5' cap structure, the 3' poly (A) tail length, and the nucleoside modifications of synthetic mRNA by comparing the retention time and peak area with reference standards. RPLC can be coupled with mass spectrometry (MS) or ultraviolet (UV) detection for enhanced sensitivity and specificity. RPLC is a fast, robust, and reproducible method that can be easily scaled up and automated for routine analysis. RPLC can also be integrated with other analytical techniques, such as MS and UV, for enhanced performance. However, RPLC may not be able to resolve some closely related impurities and degradation products, such as isomeric forms of modified nucleosides. RPLC may also require extensive sample preparation and optimization of the chromatographic conditions to achieve optimal separation and detection.
MS is a detection technique that can be used to identify and quantify synthetic mRNA, its impurities, and degradation products. MS can provide information on the molecular weight, sequence, and modifications of synthetic mRNA by ionizing and fragmenting the molecules and measuring their mass-to-charge ratios. MS can also detect low-level impurities and degradation products such as truncated mRNA, oxidized mRNA, and cyclic mRNA. MS can be coupled with RPLC or other separation techniques for improved resolution and accuracy. MS is a sensitive, specific, and versatile method that can provide comprehensive information on synthetic mRNA and its impurities and degradation products. MS can also be used for qualitative and quantitative analysis, as well as for structural elucidation and the identification of unknown compounds. However, MS may require high-quality samples and sophisticated instruments and software for data acquisition and analysis. MS may also suffer from ion suppression and matrix effects, which can affect the accuracy and precision of the measurements.
NMR is a spectroscopic technique that can be used to characterize the structure and conformation of synthetic mRNA and its modifications. NMR can provide information on the chemical shifts, coupling constants, and NOE interactions of the nucleosides and nucleotides in synthetic mRNA, which reflect their electronic environment and spatial arrangement. NMR can also distinguish different types of 5' cap structures and nucleoside modifications by their distinct NMR signals. NMR can be performed in solution or in a solid state, depending on the sample preparation and the desired information. NMR is a powerful and non-destructive method that can provide detailed information on the structure and conformation of synthetic mRNA and its modifications. NMR can also be used to study the interactions of synthetic mRNA with other molecules, such as proteins and lipids. However, NMR may require large amounts of sample and long acquisition times for high-resolution spectra. NMR may also be affected by the solubility and stability of synthetic mRNA, as well as by the spectral complexity and overlap of the signals.
| Method | Aim | Advantages | Disadvantages |
|---|---|---|---|
| Reverse phase liquid chromatography (RPLC) | Separates synthetic mRNA from impurities and measures its features by comparing it with standards. | Fast, reliable, scalable, compatible with other techniques. | May not separate some similar impurities and degradation products, may need a lot of sample preparation and optimization. |
| Mass spectrometry (MS) | Identifies and quantifies synthetic mRNA and its impurities and degradation products by breaking and weighing the molecules. | Sensitive, specific, versatile, comprehensive, qualitative, quantitative, structural. | May need high-quality samples and advanced instruments and software, may be affected by interference and background noise. |
| Nuclear magnetic resonance (NMR) | Characterizes the structure and shape of synthetic mRNA and its modifications by measuring the magnetic properties of the nucleosides and nucleotides. | Powerful, non-destructive, detailed, interactive. | May need large amounts of sample and long time to get clear signals, may be affected by the solubility and stability of synthetic mRNA and the complexity and overlap of the signals. |
Table 1. Synthetic mRNA quality control and characterization methods
The future prospects of synthetic mRNA analysis include the development of new and improved methods, such as capillary electrophoresis (CE), ion mobility spectrometry (IMS), and single-molecule fluorescence (SMF), which can offer higher resolution, sensitivity, and specificity, as well as the possibility of real-time and in situ analysis. Moreover, the development of standardized protocols, reference materials, and quality criteria for synthetic mRNA analysis is also needed to ensure the comparability and reproducibility of the results.
