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PharmTech.com The Real Message Behind Commercial mRNA Products April eBook 2023 Pharmaceutical Technology ® 37 AnAly tics st abi l it y a nd del iver y. O ver ma ny yea rs, consid- erable ef for ts have been d i rec ted at at tempts to resolve bot h t hese i ss ues, lead i ng to some c re - ative solutions which, in turn, has necessitated the application of a broad range of analytics. However, one tech n ique, na mely mass spect romet r y (MS), is pa r t ic u la rly notable for: its abi l it y to prov ide ana lysis of a l l RNA t y pes, regard less of size and modification; the fact that it can be used for qualitative as well as quantitate investigations; the technology's abi l it y to be appl ied i n bot h resea rch a nd qua l- it y control (QC); and its applicabi lit y in t he area of delivery systems. Since the late 1970s, when both therapeutic bio- polymers and analytical MS began a decades-long connection, there have been some considerable ad- vancements in both therapeutic development and analytics. Most early biopolymer MS was applied to peptides and proteins, and, today, this technique has become a rg uably t he si ngle most i mpor ta nt ana ly tica l tool in t his f ield. Indeed, severa l labo- ratories within SGS, for example, were founded by one of the early innovators, H.R. Morris, whose con- tributions included the first combination of high- field magnet technology with soft ionization (5) and some of the first protein-mapping studies (6). These laboratories and others continue to encourage the adoption of such technological innovations, which have resulted in the application of advanced ana- lytical ser vices in support of the most challenging biopolymer studies. Adapting advances in MS Ma ny of t he adva nces made i n protei n MS have been adapted to make the technique appliable to t he st udy of ol igonucleot ides, where at t r ibutes such as sequence, modification, and quantitation are critical. In it iated by McLuckey et al.'s repor t i n 1992 (7), there has been increased attention given to the ap- plication of MS-based technologies in the analysis of RNA, including top-down methods that provide data from intact RNA species. In 2012, Taucher and Breu ker (8) were a mongs t t he f i rs t to repor t se- quence coverage of full-length transfer RNA (tRNA) using Fourier transform ion cycloctron resonance (F T-ICR), combi n i ng d at a f rom elec t ron det ac h- ment d issociat ion (EDD) a nd col l ision-ac t ivated dissociation (CAD) experiments. However, while such MS-ba sed st ud ies cont i nue to be ex plored, they remain constrained by several somewhat re- lated factors, such as the large molecular size, the inabilit y to distinguish different species with the same mass, the high degree of purity required, and the limited availability of software to support top- down RNA analysis. Perhaps to a lesser extent, these limitations also apply to top-down protein studies, although in this field these constraints have largely been overcome by mappi ng tech n iques. T h is approach i nvolves controlled and specific digestion of the protein to yield a peptide mixture that can then be analyzed b y a v a r ie t y of M S -re l ate d me t ho dolog ie s , t he most familiar being liquid chromatography–mass spectrometr y (LC–MS). It was inevitable that such a n approach wou ld evolve i n t he R NA f ield, a nd this has now been successf ully applied to species such as the mRNA coding for the SARS-Cov-2 spike protein (9). Such methods rely on digestion (partial and complete) wit h RNAses such as T1, RNAse A, and MazF prior to analysis of the products by LC– MS/ MS. T his is a ver y ef fective strateg y because it can be automated wit h t he use of immobi lized enz y mes (9), prov ides sequence con f i r mat ion as wel l a s t he abi l it y to conduc t de novo i nvest iga- tions, is able to identif y and locate post-transcrip- t ion a l a nd pr o ces s mo d i f ic at ion s, c a n b e u s e d qualitatively and quantitatively, and may be applied to bioanalytical applications. In contrast to polymerase chain reaction (PCR) met hodolog ies, MS has t he abi l it y to detec t a nd locate the more than 150 post-transcriptional RNA modifications that have been described to date. Im- portantly, MS is also able to identif y the presence of u nex pec ted nucleot ide a lterat ion s. For ma ny years, mass spectroscopists have taken advantage of t he met hod's abi l it y to detec t st able i sotopic forms of elements such as hydrogen, nitrogen, car- bon, oxygen, and sulfur. Nucleic acid isotope label- ing combined with MS (NAIL–MS) has been used in a variet y of applications to study the dynamics of RNA modification (10). Some reports have addressed the use of MS for the investigation of dimer formation and higher-order structures, but here great care should be taken in data i nter pretat ion a nd ext rapolat ion g iven t he tendency for non-covalent molecular association within the mass spectrometer and the f undamen- tal differences between the solution-phase environ- ment and the environment within the mass spec- trometric process. Ion-mobilit y MS has been used to investigate the gas-phase str uctures of nucleic acids such as duplexes, triplexes, and quadruplexes to tr y to determine if these str uctures are indeed similar to the structures in solution (11). Today, the use of MS for the analysis of RNA-based therapeu- tics has become widespread due largely to the tech- nique's agnostic ability to manage a wide diversity of molecular structures. From the development of Macugen (pegaptanib sodium) in the early 2000s, l aborator ies s uc h a s SGS a nd ot her s h ave been h ig h ly ac t ive i n a n a ly t ic a l s uppor t f or nuc leic