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44 Pharmaceutical Technology ® The Real Message Behind Commercial mRNA Products April eBook 2023 PharmTech.com AnAly tics be as low as 50% (18). Although "anti‑reverse" cap an‑ alogues have been developed to overcome this chal‑ lenge (19), heterogeneity of the 5' cap has led to the re‑ quirement for accurate 5' cap characterization. This can be performed by first enzymatically digesting the mRNA and identifying forward and reverse cap struc‑ tures based upon chromatographic retention time using AEC and radiolabelling the terminal phosphate. To reduce the complexity of capping analysis, non‑la‑ beled approaches can be used. One label‑free approach utilizes a biotin‑tagged RNase H cleavage probe to hy‑ bridize with the mRNA 5' terminus, which was then di‑ gested with RNase H. Following sample clean‑up, the 5' cap structures were analyzed using IP reversed‑phase liquid chromatography–mass spectrometry (LC–MS); specifically this was demonstrated using a C18 column and a mobile phase consisting of 8.15 mM triethylamine, 200 mM hexaf luoroisopropanol (HFIP) (pH 7.9), and methanol as organic solvent (18). Analysis of the cap structures using negative ionization MS facilitated 5' cap characterization of a range of structures. Another structural element that can lead to DS het‑ erogeneity is the 3' poly‑A tail, which can be encoded into the template DNA or by enzymatic addition. Poly A fragments can be enzymatically isolated and analyzed using denaturing polyacrylamide gel electrophoretic separation; however, length determination is reliant on accurate comparison to the relative migration of refer‑ ence ladders within the sample (20). Poly A tail length can also be determined using LC–MS approaches (21,22). RNase T1 was used to cleave the tail and oligo dT mag‑ netic beads were employed to remove the poly A tail fragments from the reaction mixture. The analytes were resolved with IP reversed‑phase chromatography on a polymeric reverse‑phase column, using a mobile phase of 15 mM dibutylamine and 25 mM HFIP (with methanol as organic solvent) (22). The mRNA base sequence is a key quality attribute because it defines the code for protein translation. Er‑ rors could lead to erroneous protein production and reduction in therapeutic efficacy. For many years, tra‑ ditional RNA sequencing approaches were more widely used (23). These approaches were based on the creation of complementary DNA (to the original RNA sequence) libraries and sequencing cDNA fragments using fluo‑ rescently labeled nucleotides and next‑generation se‑ quencing instruments. However, LC–MS and LC–MS/ MS approaches are becoming more common within the bioanalytical laboratory to sequence RNA polynu‑ cleotides. The mRNA is enzymatically degraded using RNases and the fragments are identified by their mass using MS. There is a range of available enzymes and often multiple are required to gain higher sequence cov‑ erage. One approach has been to use RNase T1, colicin E5, and MazF to generate mRNA fragments for orthogonal mass mapping of mRNAs (24). The challenge with single enzyme digestion is the creation of a large amount of short, isobaric RNA fragments with the same sequence, as these are hard to map to specific points in the RNA sequence. Multiple enzyme digestion overcomes this challenge by creating a wider range of isomeric and unique fragments by digesting at more RNA positions. The mRNA fragments were resolved using 0.1% diiso‑ propylethylamine, 1% HFIP (acetonitrile organic solvent for elution) on an ultrahigh‑pressure liquid chromatog‑ raphy (UHPLC) oligonucleotide C18 column. The UHPLC separation enabled sufficient resolution of all of the mRNA fragments and sequential MS/MS sequencing of collisionally activated dissociation fragments. Recent work at the University of Sheffield also applied a mass mapping approach to mRNA sequencing (25). Within this work, mRNA was partially enzymatically digested with RNase T1 to induce missed cleavages and there‑ fore create larger mRNA fragments. The larger, more unique fragments were separated using 0.2% triethyl‑ amine, 50 mM HFIP (acetonitrile solvent) on a polymeric reverse‑phase column coupled with a high resolution orbital trap mass spectrometer. Figure 3 demonstrates the ability to tune enzymatic digestion with controlled RNase immobilized enzyme on magnetic particles. As fragmentation is not driven to completion, longer RNA fragments remain, which appear at later retention times using IP reversed‑phase chromatography. A key tool in the ability to rapidly sequence the mRNA fragments in the digest is the use of oligonucleotide‑appropriated data processing software that has the capacity to se‑ quence modified oligonucleotides from MS data. An‑ other key objective in any approach to sequence mRNA by mass mapping is to optimize the chromatographic separation prior to MS detection to reduce ionization inhibition and increase sensitivity. IP reversed‑phase chromatography can be challenging to couple with MS, as the ion‑pair reagent decreases sensitivity by suppress‑ ing electrospray ionization efficiency (26). Optimiza‑ tion of sensitivity may be achieved by optimizing the mobile phase pH and selecting an appropriate ion‑pair reagent, or with the addition of an acidic fluoroalcohol (such as HFIP) to reduce the pH of the mobile phase. These approaches can narrow the oligonucleotide frag‑ ment MS charge state distribution and increase oligonu‑ cleotide fragment resolution. An alternative chromato‑ graphic approach is to employ hydrophilic interaction liquid chromatography (HILIC) to resolve ribonucleotide fragments after enzymatic degradation. This has been demonstrated using a quaternary ammonium‑bonded polymeric column and a mobile phase containing 15 mM ammonium acetate, pH 5.5 (using acetonitrile as organic solvent and water for elution) (27). The benefits of this approach are the absence of an ion‑pair reagent that would reduce MS sensitivity and the maintenance of high chromatographic selectivity for isomeric frag‑ ments containing structurally similar nucleobases, such