Nucleic acid therapeutics have emerged as new drug class under development to target and treat various diseases. With continued interest in discovering appropriate technologies to formulate nucleic acids into appropriate and bioavailable dosage forms, especially messenger RNA (mRNA), the industry is open to adapt novel excipients to deliver those molecules.1
Introduction of life-saving COVID-19 vaccines and the launch of small interfering RNA (siRNA) as a polyneuropathy drug - among others – has called for renewed attention in the next generation of ionizable lipids in lipid nanoparticle (LNP) technology.
LNPs are critical in this new area of drug development because they solve multiple biological and practical delivery challenges associated with nucleic acids. To meet the stringent pipelines associated with these products, biopharma teams are turning to CDMOs. The key is to select a partner that has the expertise and technologies to leverage LNPs.
Understanding Nucleic Acids
Nucleic acid therapeutics are medicines that use DNA, RNA, or chemically-modified oligonucleotides to alter, replace, suppress, or modulate gene expression or protein production. They expand the area for the active ingredient in a product by acting directly on genetic information.
Benefits of nucleic acid therapeutics are numerous, including high specificity, rapid design and modularity, and potentially lower dosage frequency. They have the ability to treat a number of diseased conditions, as outlined in Table 1..
Structure of LNPs with Nucleic Acids
Success of nucleic acid therapeutics is contingent on solving delivery, immunity, safety, and cost challenges. That is where LNPs come in. Complex structures, LNPs are used to encapsulate negatively charged nucleic acids are typically comprised of four components. Most ingredients used in marketed drugs are also listed in FDA inactive ingredient database.
It is evident that the negatively charged nucleic acid is complexed with positively charged ionizable lipids (lipoplex) and is entrapped within the LNPs. Designed with helper phospholipids, such as di-stearoyl phosphocholine and cholesterol, these lipids are distributed asymmetrically to create an outer stable monolayer boundary, while the pegylated lipids are situated outside for providing steric stability of core surface.
Figure 1 shows the LNP interior, which is comprised of electrostatically neutral inverted micelles. Nucleic acid is surrounded by the ionizable cationic lipid and other lipid components. The surface of the LNP is composed of a hydrophilic shell containing the PEG‐lipid.
Ionizable lipid nanoparticles (iLNPs) are comprised of cationic lipids with amino moiety as the head group, PEGylated lipid, and helper lipids, including phospholipid and cholesterol. All of these elements provide the stability of the outer layer core. The amino head group of the ionizable lipids are typically tertiary amine with pKa 6.2-6.9. Commercially available ionizable lipids are MC3 and ALC-0315 in Comirnaty vaccine (Pfizer/BioNTech) and SM-102 in Spikevax vaccine (Moderna).
Manufacturing of LNPs
These amphiphilic lipids can spontaneously aggregate into LNPs in an aqueous solution. The process requires directly mixing lipid and nucleic acids by agitation to encapsulate negatively charged mRNA. Hydrophobic fatty acid chains and polar headgroup help create the lipid assemblies that can further be sized into desired particle sizes.
Top-down and bottom-up approaches are common for generating these particles. Let’s briefly explain both:
- Top-down requires high shear and high energy where the lipid (dried film) is homogenized in aqueous buffer to yield the desired particle size. It begins with larger lipid structures or bulk mixtures and breaks them down into nanoscale particles. It is practical and established but can stress fragile nucleic-acid payloads and requires careful control of process conditions and equipment to ensure reproducible, high-quality LNPs.
- A bottom-up approach, such as nanoprecipitation requires, injects ethanol to form nanoparticles. Shepherd et al. (2023) demonstrated the formation of LNPs (comprised of ionic lipid D-Lin-MC3-DMA:DSPC:Cholesterol:DMG-PEG 2000; 50:10:38.5:1.5) in high throughout production with comparable and uniform particle size (ca. 70 nm) with low polydispersity index (ca. 0.004), high encapsulation efficiency (ca. 88%).2
Scale up manufacturing requires a T- or Y-mixer for rapid mixing of lamellar flow liquids into a turbulent flow at the mixing point. Changing the lipid composition, volume, or flow rate ratio between the organic and aqueous phases can control LNP size.
For typical mRNA encapsulation, one part of the lipid solution is mixed with three parts of the aqueous phase. Downstream tangential flow filtration (TFF) is used to concentrate the LNPs, as well as to remove ethanol, any residual lipids, and non-encapsulated nucleic acids. It also neutralizes the formulation to the target pH of 7.4. The authors of the paper demonstrated that for LNPs generated by the continuous T- or Y- mixing modes, those formulations meet the quality critical attributes of mRNA vaccines with particle size of 71 nm and EE of 88%. This continuous process can potentially save time and reduce costs.
Your Nucleic Acid CDMO Partner
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Ascendia Pharmaceutical Solutions has developed its LipidSol® (Figure 2) with LNPs in mind. One of four proprietary platform technologies, LipidSol leverages different process methods to make LNPs carrying various therapeutic modalities with hydrophilic and lipophilic properties.3
LipidSol uses fatty acids and polar headgroup entities to encapsulate drugs in LNPs. Coupled with lab-scale screening and cGMP sterile manufacturing capabilities in its U.S.-based modern facility, Ascendia Pharmaceutical Solutions is leading the way in the design, development, and manufacture of LNPs for novel therapeutics for oncology, infectious diseases, and many rare conditions.
Conclusion
The encapsulation and delivery of nucleic acids in efficient and effective manners creates challenges and opportunities for drug development teams. For instance, lipid assemblies are proven to be ideal vehicles for intracellular delivery of mRNA- due to their inherent abilities to protect the delicate nucleic acids from degradation under aqueous and nuclease environment.4 The encapsulation of mRNA into LNPs, however, warrants closer scrutiny since every components of LNPs, especially the cationic lipids, plays a crucial role in protecting, delivery and stability of these large macromolecules.-
Working with a CDMO with a track record of success in these areas alleviates concerns and helps maintain timelines to bring drugs to market on schedule and in the desired dosage form. To learn more about our LNP expertise, contact us.
References
- Nature Editorial, Let’s talk about lipid nanoparticles, Nature Reviews – Materials, 2021, 6, 99.
- S. J. Shepherd, X. Han, A. J. Mukalel, R. El-Mayta, A. S. Thatte, J. Wu, M. S. Padilla, M. G. Alameh, N. Sirkumar, D. Lee, D. Weissman, D. Issadore, and M. J. Mitchell, PNAS, 2023, 120, 1-12.
- J. Huang and S. Ali, LipidSol® - a lipid nanoparticle technology for drug delivery, Drug Development & Delivery, 2023.
- S. Guan and J Rosenecker, Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems, Gene Ther., 2017, 24, 133-143.