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www.biopharminternational.com Emerging Therapies 2022 eBook BioPharm International ® 23 Particle size is critical because it has implications for drug function. Particles that are too small may be cleared f rom the bloodstream by the k idneys before they can take effect, whereas larger or aggre- gated particles may fail to penetrate cells or could in- duce unwanted immune responses. Thus, drug develop- ers need to ensure that the LNPs they are producing are the optimal size and that the size distribution (polydis- persity) falls within acceptable parameters. Unexpected changes in size or polydispersity could signal problems in manufacturing processes or product degradation. Another factor crucial to the success of an LNP is stability. Solvents used during production may need to be removed before administering the product to patients. Drug developers, therefore, need to be sure that the product created remains stable in the sol- vent's absence. They also need to know that the prod- uct holds its structure during transport and storage. Drug developers must also ensure that LNPs main- tain their structure and successf ully protect their mRNA cargo until the therapeutic is delivered to the target site in the patient's body. As such, scientists must analyze LNP performance under conditions that mimic the bloodstream or target tissues as well as inter- and intracellular environments. The surface charge is another critical factor that dic- tates how well an LNP functions within the body. For example, while LNPs with a positive charge have been investigated, they can trigger inflammatory responses, inducing problems such as hepatotoxicity (1). Negative- ly-charged LNPs avoid these toxicity issues; however, this is not the only place where the charge is important. LNPs need to respond to changes in the endosomal pH, often by changing charge, which can influence the en- dosomal escape of their mRNA payload. Expanding the use of LNPs from intramuscular injections for vaccines to other applications such as cancer vaccines or gene therapies, in which the LNPs need to enter other cell types, the surface charge can impact the uptake of the LNPs to different cell types (2). Thus, formulators need to monitor surface charge to identify LNPs that can access the target cells. It can also be informative to understand how this surface charge varies across relevant pH ranges. Techniques proven to meet the challenge With such a complex range of attributes to navigate, developers must use complementary analytical tech- nologies to provide a f ull understanding of LNPs. Fortunately, they don't need to develop these meth- ods de novo. Rather, LNP specialists can tap into the well-established field of lipid-based drug character- ization, where proven analytical technologies have long helped ensure product quality and function. Size matters To mon itor L NP size a nd sa mple polyd ispersit y, LNP specialists can rely on dynamic light scatter- i ng (DLS)—whet her si ng le-a ng le or mu lt i-a ng le (MADLS)—and nanoparticle tracking analysis (NTA). Using these methods, LNPs in suspension scatter laser light as they diffuse through a sample. Changes in the scattering pattern are translated to particle size and size distribution, with larger particles diffusing more slowly than smaller particles. DLS offers the lowest resolution of particle size de- termination but gives a good indication of the size of LNPs in a sample. Using back, side, and forward de- tection angles, MADLS offers a higher resolution than DLS, allowing it to identify additional populations of LNPs that DLS might miss. NTA offers even higher resolution but often requires sample dilution, which can perturb LNP stability. The choice between these complementary techniques depends on factors, such as LNP size and polydispersity, sample heterogeneity, and the questions being asked. W he n P f i z e r –BioN Te c h cont r a c t e d Poly mu n Scientific to scale up the process of producing LNPs for the Comirnaty program, Polymun needed to develop robust manufacturing processes to support large- scale production (3). Key to this development were FIGURE 1. Schematic of a messenger RNA (mRNA)–lipid nanoparticle (LNP) complex (4). DSPC is distearoylphosphatidylcholine. Analytics ALL FIGURES ARE COURTESY OF THE AUTHORS.