5.3  Many Different Methods

5.3.1  Full characterization of both mRNA drug substances and formulated mRNA–LNP drug products requires the use of many diverse and complementary methods. Established techniques from the analysis of biologics, small molecule drugs, polymeric materials, and molecular biology research are employed, along with new methods developed specifically for this application.

5.3.2  Common methods include HPLC — often combined with mass spectrometry — as well as other detection techniques, such as ultraviolet or multi-angle light scattering and capillary electrophoresis (CE). Additional electrophoretic methods include capillary isoelectric focusing (CIEF), capillary zone electrophoresis (CZE), CE-SDS, CE-ultraviolet, and CE-laser induced fluorescence (CE-LIF). Other analyses may involve particle-size measurement, cryo-electron microscopy, PCR, Sanger sequencing, and functional cell-based assays. Many of these methods find use in early development stages, with simpler versions applied in the quality control (QC) environment.^[61]

5.4  Potency Assays for mRNA

5.4.1  Potency tests must reflect the biological activity and mechanism of action of the therapeutic and should ideally correlate with the clinical response. The potency of an mRNA therapy depends on both the uptake of the mRNA into the cells (determined largely by the LNP) and the translation within the cell (determined by the mRNA payload). As with other advanced therapies, a suitable potency assay is difficult to define. Eteplirsen was approved by the FDA in 2016, but only after the agency had delayed the decision once and over the objections of the advisory board. Although the drug appeared safe, its efficacy was called into question.^[74] Although the regulatory debate will continue on this case, at heart is the question of potency and how to provide a representative measure of it in vitro: the basic molecular activity of the therapy could be demonstrated, but did that accurately predict the clinical efficacy?

5.4.2  While the potency for a vaccine (protein- or nucleotide-based) is established, multivalent vaccines also present a challenge. In a single-valent vaccine, the in vitro assay typically measures the expression of the target antigen from infected cultured cells. This dose response is correlated with whole-animal immunity to the pathogen challenge and is performed relatively consistently in mice because well-established models are available. In a multivalent vaccine, each of the mRNAs in the formulation must induce expression of its antigen at the same level it does in the absence of the other mRNAs. This is difficult to measure. It is also difficult to design and measure antigens so that they do not interfere with others in the formulation.^[75] Nevertheless, trials are ongoing for multivalent vaccines to multiple variants of COVID-19 (BioNTech), COVID-19 + influenza (BioNTech), and COVID-19 + influenza + RSV (Moderna). For a review on the topic of potency assays for advanced therapies, including mRNA, the reader is directed to Salmikangas et al.^[76]

5.5  Desire for Simplicity and Speed without Sacrificing Performance

5.5.1  There is a fundamental desire to reduce the time required for the various assays that must be completed during mRNA product development. For many current cell-based functional assays, for instance, it can take days to receive results, leaving developers to choose between longer timelines or greater process risk. New intuitive, high-throughput solutions must still provide the sensitivity, accuracy, reliability, and robustness required for any pharmaceutical method.

5.6  The Need for Flexible Platform Solutions

5.6.1  Advances in analytical capabilities are needed not only due to the significant differences between mRNA-based products and more traditional biologics but also because the field is rapidly evolving, creating a need for flexible methods that can be used with both current and future mRNA technologies.^[77] The ultimate goal is to establish standard, platform analytical technologies that are applicable across development phases and can evolve alongside advances in mRNA technology.^[61]