Issue link: https://www.e-digitaleditions.com/i/1497323
42 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com AnAly tics drug delivery). Despite these challenges, the global mRNA therapeutic market is expected to reach a value of $101 billion by the year 2026 according to recent mar‑ ket reports, with further growth expected (6). A simple search using the keyword "mRNA" on clinicaltrials.gov returns over 2000 clinical studies, which further high‑ lights the growing interest in this therapeutic modality. With growing interest comes the requirement for accurate and effective analytical techniques to char‑ acterize critical quality attributes (CQAs) of both the drug substance (DS) and drug product (DP). As with other biopharmaceuticals, mRNA therapeutics will be subjected to multiple compendial and non‑compen‑ dial methods to evaluate identity, appearance, efficacy, toxicology, safety, concentration, pH, osmolality, pro‑ cess‑/product‑related impurities, sterility, and formu‑ lation (7). Although there is a wide range of techniques available to analyze mRNA CQAs (Table I), chroma‑ tography and other separation techniques sit at the forefront of drug characterization and quantification. Synthesis Unlike smaller oligonucleotide therapeutics that are chemically manufactured using solid phase synthesis, mRNA therapeutics are synthesized using in vitro tran‑ scription (IVT). This is because solid phase synthesis is limited to producing oligonucleotides smaller than 100 nucleotides, whereas mRNA therapeutics are typically larger than 1000 nucleotides. IVT is performed first by creating a plasmid DNA template that is linearized and transcribed into a mRNA sequence using RNA poly‑ merase and ribonucleoside triphosphates (rNTPs). The DNA template is designed to code for cis‑acting struc‑ tural regions: a 5' cap structure, 5' and 3' untranslated regions (UTR) sandwiching an open reading frame (ORF) containing the protein‑encoding sequence of interest, and a poly A tail at the 3' terminus (Figure 2). The 5' cap and poly A tail structures assist with splicing, translation mechanisms, and stabilization of the mRNA against degradation, and can be manufactured during the IVT process or enzymatically added post synthe‑ sis. mRNA therapeutics are synthesized using a large range of modified nucleosides that help protect against degradation in vivo and reduce the immunotoxicity of the mRNA. Such nucleosides include pseudouridine, N1‑methylpseudouridine, 5‑methylcytidine, 2‑thiouri‑ dine, and N6‑methyladenosine (4,8). IVT is challenged by the requirement for a promoter sequence to signal the initiation of transcription by RNA polymerases, which limits the ability to design modifications to the 5' terminus. In addition, non‑spe‑ cific run‑off caused by RNA polymerases can lead to terminal heterogeneity. Despite these challenges, IVT synthesis is robust and reproducible regardless of the sequence of the ORF, thereby allowing rapid alter‑ ations to the ORF (and consequently the mRNA prod‑ uct). Rapid alterations to the ORF do not result in dras‑ tic changes to the mRNA chemistry, thus giving IVT synthesis a modular, high‑throughput capability (1). Although regulatory guidelines are lacking in regard to mRNA therapeutics, they are classed as advanced TABLE I. Analytical techniques for mRNA therapeutic analysis. Analytical Technique Quality Attribute Sequencing of mRNA transcript or DNA template Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Liquid chromatography– mass spectrometry (LC–MS) (IP-RP, HILIC) Identity UV spectrophotometry qRT-PCR Gel/capillary electrophoresis LC or LC–MS (AEC, SEC, IP-RP, HILIC) Western blot (dsRNA) Quantity Gel/capillary electrophoresis LC or LC–MS (AEC, IP-RP, affinity) UV spectrophotometry Fluorescence-based assays Purity LC–MS or LC (SEC, AEC, IP-RP, RP, HILIC) Gel/capillary electrophoresis Field-flow fractionation Characterization of mRNA structure (Cap, polyA tail), nanoparticle lipid profile, encapsulation, DS/DP stability, nanoparticle size distribution, aggregation, and impurity analysis Cell expression systems LC–MS Fluorescence-based assays Efficacy and product characterization Dynamic light scattering Nanoparticle size and polydispersity Electron microscopy Atomic force microscopy X-ray diffraction Differential scanning calorimetry Field-flow fractionation Nanoparticle physicochemical characterization Endotoxin analysis Sterility and safety Microbiological assays Compendial methods pH, osmolality, CCI, elemental impurities, and formulation i) Adapted from references 35, 7, and 10. ii) IP-RP = ion-pair reversed-phase chromatography, AEC = anion exchange chromatography, HILIC = hydrophilic interaction liquid chromatography, SEC = size-exclusion chromatography, RP = reversed phase chromatography.