February 7, 2023 PAO-02-23-CL-01
Lipid nanoparticles (LNPs) have long been the carrier vehicle of choice for nucleic acids. The first nucleic acid–based drug formulated as an LNP received U.S. Food and Drug Administration (FDA) approval in 2018. ONPATTRO® (patisiran, Alnylam Pharmaceuticals) is an RNA-interference (RNAi) therapeutic for the treatment of the polyneuropathy of hereditary transthyretin-mediated (hATTR) amyloidosis in adults. However, the LNP sector truly moved into the spotlight upon FDA approval of the two mRNA vaccines against COVID-19 from Pfizer/BioNTech and Moderna, which clearly demonstrated the safety, efficacy, and potential of these novel products and jump-started a surge of interest in both the potential of mRNA and the ability of LNPs to help realize that potential.
Today, there is tremendous excitement across the industry (and beyond) about the potential of not only mRNA and RNAi therapeutics and vaccines but those based on small interfering RNA (siRNA), single-stranded DNA (ssDNA) and guide RNA (gRNA), among other related approaches. An abundance of different types of nucleic acids are presently under clinical investigation as new therapeutics and new vaccines, as are CRISPR-based gene-editing technologies that may serve as possible future transformative treatments. What is most tantalizing about these pipeline candidates is the fact that they may truly enable the biopharma industry to transition from therapeutic to curative approaches to medicine, a longstanding goal that has never been closer to being achieved.
LNPs have their physical and historical roots in liposomes, which were first used as delivery vehicles in the 1990s.1 Liposomes were first described in 1962,2 and the first nano-sized liposomal drug formulation — Doxil® (doxorubicin HCl liposome, Baxter Healthcare) for the treatment of Kaposi's sarcoma in AIDS patients — was approved by the FDA in 1995.3
Investigations focused on improving the properties of liposomes for drug delivery led to the development of different types of cationic lipids. While these lipids afforded positively-charged liposomes with an enhanced cellular uptake, they were found to exhibit cytotoxic effects and low encapsulation efficiency. Lipid nanoparticles share major attributes with liposomes and have a lipid matrix as part of the particle core. This lipid matrix may include water–lipid–mRNA and appears as a denser structure. These particles were found to have very attractive properties for nucleic acid delivery. Further advances have been made as different ionizable lipids were investigated to fine tune and optimize the LNPs.
Research into ionizable lipids has become a significant focus for the industry, as the properties of the drug substance dictate the optimum chemistry for the ionizable lipid. The other components of LNPs — cholesterol, a polyethylene glycol (PEG)–lipid conjugate, and typically a phosphocholine-based lipid — provide structure to the nanoparticle.
It is the cationic lipid that binds to the negatively-charged nucleic acid and thus has a major influence on the performance of LNP formulation, which in turn impacts the efficacy of the therapeutic or vaccine. Many companies are now designing novel cationic lipids that are not covered by existing patents specifically to stabilize their active pharmaceutical ingredients (APIs). It is worth noting that equal efforts are directed at modifying the structures of nucleic acid, such as 5' caps for mRNA and nucleotide swapping, to increase stability and immune response.
Ultimately, however, any nucleic acid API must be packaged into a delivery vehicle, and LNPs are the optimal choice. Remarkably, the ionizable lipids available today enable high encapsulation efficiencies above 90%.
While ionizable lipids appear to be the most critical component for unlocking the abilities of LNPs to deliver different nucleic acid payloads to target tissues, the overall lipid composition within LNPs can impact many aspects of LNP performance, including targeting and efficacy of cellular/tissue uptake. Using current LNP technology, targeting is passive, with certain formulations ending up in particular tissues or cells without any active drivers for that specificity. In the future, however, it is likely that targeting moieties will be conjugated to the surfaces of LNPs to enable active targeting, making targeting direct and customizable, which should both increase efficacy and reduce off-target localization, which can lead to undesirable side effects.
High targeting specificity may not be necessary for some novel nucleic acid–based therapeutics and vaccines, particularly CRISPR-based candidates. However, some nucleic acid drug substances are being developed to be highly specific for certain cell types. They do not perform their function if taken up by the wrong cells, but, as long as a small percentage reach the target cells, a therapeutic effect can be observed.
While the efficacy of a nucleic acid–LNP formulation should be the top priority, there are other concerns that often come into play when developing LNP-based products. Ionizable lipids that have been used in approved products tend to be attractive to many developers because they have been validated within the regulatory process. For some other ionizable lipids, companies that first used them in LNP formulations have patented their use, preventing other developers from accessing them for their own formulations.
The successful formulation and production of nucleic acid–LNP particles depends on many factors. A crucial component is the nucleic acid itself, particularly its quality and purity. The choice of lipids and their ratios, as well as the nucleic acid to ionizable lipid ratio (nitrogen to phosphate (N:P)) will impact particle stability and thus the ability to achieve high processing volumes. It is essential to develop a stable mixing process that yields uniform particles, which can be challenging as scale increases. Solvent (typically ethanol) removal and concentration of the LNPs comes next, potentially followed by surface modification and purification to ensure that a high percentage of the LNPs encapsulate the drug substance. All these steps must lead to a particle size with an acceptable polydispersity; otherwise, particles can block filters during bioburden reduction.
Throughout the entire process, it is necessary to monitor critical process parameters. These products are rather expensive formulations: the lipid is expensive, as is the mRNA or nucleic acid drug substance. Thoroughly understanding the entire process and assessing real-time data from start to finish is highly important, particularly at large production scales. As the process progresses from one step to the next, the nanoparticles undergo subtle changes, which we refer to as intermediates. Having the ability to monitor these intermediates and then relate them to the final product is highly important to ensure that the particles are stabilized throughout the entire process.
The obvious solution for achieving robust, scalable nucleic acid–LNP production is to develop a continuous process including LNP formulation through fill/finish for a true end-to-end manufacturing process in a single unit. For lipid-based products, there are major benefits that can be realized even at lower volumes. All of the steps can be implemented on low- or high-volume equipment, allowing ready up- or down-scaling in response to changes in market demand.
Minimal human intervention reduces risk of error and contamination, as would containment in a closed system. There is also minimal holding time (seconds) between steps, reducing the risk of destabilization and degradation. As a result, the LNPs are generated with a desirable particle distribution and high encapsulation efficiency.
In general, the industry has been reluctant to switch from batch to continuous processing, with most drugmakers electing to stay with validated methods and procedures previously developed for other products. They know how these systems perform and prefer to avoid introducing risks, as well as the time and costs associated with retraining, writing new standard operating procedures, and so on.
However, this perception is slowly changing. The U.S. Food and Drug Administration (FDA) and other international regulatory agencies have groups focused on identifying innovative manufacturing solutions that can lead to increased efficiencies and quality while lowering cost. Continuous processing has been identified as an approach that offers these benefits, as has been observed in other industries, including petrochemicals and paper. As a result, a transition in the pharma space –– particularly with regard to products and processes where the benefits of continuous processing are most apparent –– can be expected in the near future.
LNPs will transform the nanomedicine market. They will play a key role in transitioning the industry from a focus on developing treatments to developing cures — something that has not previously been possible. There are countless diseases for which no therapy is available. For many of these illnesses, it may be possible to advance from having no effective treatments to offering real cures. The same could be true for diseases for which therapies are available but that have limited effectiveness. The potential is enormous –– nucleic acids delivered using LNPs can tackle basically any diseases that are tied to faults in the human genome or its processing mechanisms.
There are no other nanoparticle technologies that will displace LNPs anytime soon. Largely comprising natural lipids that are safely metabolized and recycled by humans, LNPs function via natural processes, protecting and delivering nucleic acid actives into target cells. They provide advantages over synthetic polymeric nanoparticles from biological compatibility and safety perspectives and are attractive in comparison to viral delivery approaches as well.
Advances will continue to be made, of course. A fifth or sixth lipid component is already sometimes added to afford enhanced versions of the nanoparticles. The improvements in the stability of the lipid nanoparticles already achieved will be further expanded –– including being designed and modified to enable selective targeting of cells and tissues. Manufacturing solutions will be introduced that enable robust, efficient, cost-effective scalable production of nucleic acid-LNP products.
An efficient and mature LNP sector is inevitable, which will not only be critical to ensuring the realization of the promise of mRNA therapeutics and vaccines, but will likely drive further interest in LNPs as vehicles for many types of nucleic acid, as traditional drugs and other advanced modalities on the horizon.
Antonio Costa, Ph.D. is chief executive officer at DIANT Pharma Inc., an early-stage company that provides continuous manufacturing technology, and an assistant research professor at UConn in the Pharmaceutical Sciences department. His work focuses on continuous manufacturing approaches, downstream processing of nanoparticle formulations, physicochemical characterization, process analytical technology integration and systems engineering. He is an author on 12 peer-reviewed publications and inventor on two granted patents and six patent applications.