Formulation development of lipid nanoparticles as a non-viral vector for vaccine and cell and gene therapy

Lipid nanoparticles (LNPs) have emerged as the one of the most effective technologies in vaccine, cancer, and cell and gene therapy. LNPs are lipid-based nanocarriers engineered to encapsulate nucleic acids (mRNA, siRNA, sgRNA, and DNA).^1,2 Like viral vectors, LNPs deliver nucleic acids to the target cells and protect their sequence from degradation before and after administration. The main challenge in the industry is developing novel LNPs that ensure both safety and stability as well as robust and scalable preparation method(s) to allow the transition of these new therapies for clinical and commercial use.

Unlike viral vectors, LNPs are very versatile and tuneable. Although this is a key advantage in developing efficient drug products, it introduces the need for screening multiple formulation and processing parameters. Based on the type of therapy, the nature of RNA, potential complementary ingredient (for example, small molecules and peptides), and the route of administration (intramuscular or inhalation), the LNP formulation must be tailored to encapsulate the therapeutic nucleic acid while keeping the critical quality attributes (CQAs) such as particle size, encapsulation efficiency, stability, and potency within the regulatory limits.^2,3

To accomplish this, rational design of LNP formulation involves selecting every building block (functional lipids) of LNPs and their percentage in the final particle structure to formulate the most suitable LNPs to load the nucleic acid. This process requires screening several LNP formulations and developing an array of assays to determine the CQAs. Figure 1 demonstrates an example of two different optimized formulations for just mRNA (Formulation 1) and mRNA co-encapsulated with active adjuvants (Formulation 2).

Figure1. Schematic illustration of LNPs with an example of two different LNP formulations.

Studies and applications

At Labcorp, the Biopharmaceutical Chemistry, Manufacturing, and Controls Good Manufacturing Practices Research and Development (BiopharmCMC GMP R&D) team has performed several studies developing LNP formulations for various applications. In a key study for developing LNP formulations to encapsulate self-amplifying mRNA (saRNA), three different toll-like receptor agonists (TLR 4, TLR 7, and TLR9) were incorporated into the different formulations to investigate the impact on formulation properties and performance.⁴ The physiochemical characterization showed a significant difference in particle properties and stability (Figure 2). This data, alongside cellular uptake and animal testing, helps us select the optimum formulation for saRNA.

Figure 2. (A) The size distribution measured by dynamic light scattering. (B) The zeta potential (ZP) distribution of the formulations of LNPs in pH 5.5. (C) The particle size and polydispersity index (PDI) of the formulation after buffer exchange. (D) Stability by particle size for 45 days at 4°C.

In another study, the Labcorp BiopharmCMC GMP R&D team also developed assays to investigate the potency of mRNA delivered by LNPs using flow cytometry (Figure 3). CY5-labeled RNA, encoding green fluorescent protein (GFP), was encapsulated into LNPs and incubated with HeLa cells at different doses and tested at multiple timepoints. The assay can track the amount of CY5 RNA and the expression of GFP reporter.

Figure 3. Flow cytometry protein expression of LNP-mRNA in HeLa cells. 

The CRISPR system is one of the most promising technologies used for the development of gene-editing therapies.^3,5 Labcorp scientists developed LNPs to deliver Cas9-mRNA (encoding Cas9 endonuclease) into cells. One of the key assays in this type of therapy is to measure the amount of Cas9 protein generated by Cas9-mRNA delivery. The ELISA results show that the LNPs successfully delivered Cas9-mRNA. and the protein concentration generated from mRNA-LNPs was significantly higher than naked mRNA (Figure 4).

Figure 4. Cas9 protein expression level measured in BT-20 cells.

Conclusion

LNPs have become a vital, adaptable platform for drug delivery, offering key advantages over viral vectors. At Labcorp, we fine-tuned formulation and analytical methods by systematically varying parameters to optimize LNPs for different types of RNA with applications in vaccine or cell and gene therapy. Through detailed physicochemical characterizations, measuring particle size, charge, and encapsulation efficiency alongside potency assays, such as flow cytometry and ELISA, we enable each formulation to meet critical quality standards for safe and effective therapies.

Want to learn more about LNP assays at Labcorp? Read our white paper: 
mRNA LNPs: QC release testing and characterization