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24 BioPharm International ® Emerging Therapies 2022 eBook www.biopharminternational.com analytical methods, including those described in this article, to guide process development and control man- ufacturing processes. Such efforts allowed the company to confirm batch-to-batch consistency and long-term sta- bility of its liposomal and LNP formulations (Figure 2). MADLS and NTA also offer the opportunity to de- termine particle concentration, which provides quick information on process yield and any potential losses from process steps. Recently, researchers applied these methods to liposome samples and showed that the acces- sible concentration range for MADLS (10 8 to 10 12 particles/ mL) overlapped with and extended that for NTA (10 7 to 10 9 ) (Figure 3). MADLS offered a similar range when the method was applied to LNPs. Taking charge As pH can differ significantly between production and storage conditions as well as physiological environ- ments, it is vital to monitor surface charge throughout the product's life cycle. Electrophoretic light scattering (ELS) is a powerful tool that can determine the apparent charge of a particle in different conditions; for example, mimicking a pH range from slightly alkaline blood to the acidic endosome. The obtained surface charge measure- ments allow developers to balance the needs of product development and long-term storage with the require- ments for clinical efficacy, optimizing cellular delivery while minimizing toxicity risks. The heat of the moment Production, storage, and application add temperature stresses to LNPs, which can be investigated using DLS thermal ramp experiments or differential scanning calorimetry (DSC). Thermal ramps use DLS to monitor particle stability over a temperature range, while DSC measures heat uptake or release linked to structural transitions or changes in molecular interactions. Drug developers can use these methods to examine the thermal stability of both the LNP delivery vehicle and its mRNA cargo. Such information is critical during LNP formulation as developers optimize ingredient combinations to improve stability without negatively impacting efficacy. Furthermore, as the DSC thermo- gram is also a fingerprint of mRNA and LNP's higher-or- der structure, companies can use it to inform the selec- tion of mRNA sequence variants during design stages and to compare different formulations and batches of the LNP product. This detailed characterization helps support intellectual property protection. A rapid look at composition A method commonly used to monitor particle size and polydispersity is size-exclusion chromatography (SEC) coupled to static light scattering (SLS) or another de- tection system. But beyond questions of particle size, developers continue to evolve the applications of SEC– SLS to help answer questions about LNP composition, monitoring not only the lipid complexes but also the mRNA cargo. For example, researchers used SEC–SLS to perform compositional analysis of two mRNA– LNPs (4). Monitoring the concentrations of the mRNA and LNPs across the entire particle distribution, the re- searchers could determine the relative weight fractions of the components—a measure of therapeutic payload. In both mRNA–LNPs tested, the scientists observed variation in mRNA loading across the population. SEC–SLS offers analytical insights in minutes, ac- celerating throughput across development and man- ufacturing. The analysis also eliminates the need for multiple analytical steps and f luorescent reagents and reduces operator impact on measurement con- sistency. Although still relatively new to LNP man- ufacture, compositional analysis has proven vital to other medical applications, such as studies of viral FIGURE 2. Analysis showing the size distribution of a lipid nanoparticle preparation to monitor product stability over time.