LNP synthesis: Overview of the manufacturing methods
Numerous methods are available for the synthesis of Lipid nanoparticles (LNP) and more generally for drug-loaded liposomes. The selected LNP formulation technique will depend on the sought characteristics of the synthesized drug (size, encapsulation efficiency, zeta potential), route of administration, development stage of the molecule…
The LNP manufacturing methods can be categorized into 3 different classes: high-energy methods, such as high-pressure homogenization, low-energy methods where nanoparticles will precipitate from homogeneous systems, and solvent-based methods, such as microfluidics.
This article aims at providing an introduction to all those formulation methods, from the historical ones, to the state of the art.
Drug development process steps and requirements
To start with, it is important to bear in mind that the lipid nanoparticle manufacturing requirements vary throughout the drug development process, as described in figure 1.
Whether it is for mRNA vaccine for infectious diseases or cancer, gene therapy or mRNA therapeutics, the initial steps of the process require the test of many different RNA LNP complexes (up to several 100s) with different RNA, lipids mix, and synthesis conditions… to find the most optimal one. It has indeed been proven that optimizing the nanoparticle delivery vessel can lead to a 100-fold improvement of the final mRNA transcription.
Due to the very high cost of lipids and RNA, working with a very low volume (100s of µL) of RNA-LNP is required at that stage. Depending on the chosen formulation technology, the manufacturing time can also be lengthy, which can also make the tests of hundreds of LNPs very painful.
As the process moves forward, the number of lipid nanoparticle formulations shrinks down (generally 10s of the in-vitro tests and 1 for the Mass production), while the volume requirements greatly increase, up to 100s of L. The ideal LNP manufacturing process should thus allow for the seamless transition from the initial screening phase at low volumes, all the way to the drug mass production process by keeping the RNA LNP complex as identical as possible at every stage of the development process.
Finally, as the development process progresses closer to the human trials and production, it is required to take into account current good practice manufacturing (cGMP manufacturing or GMP manufacturing) to ensure meeting the pharmaceutical industry guidelines and allow for a quick and smooth transition to market of the drug product.
Low energy methods for LNP synthesis
In those methods, particle size reduction is triggered by some spontaneous process rather than the energy brought to the system. Generally based on the modification of the curvature of the surfactant modules, those synthesis methods can be classified into 2 categories: thermal and isothermal.
Low energy methods are convenient methods as they don’t require harsh synthesis conditions, however, their main limitation lies in the high amount of surfactant used and poor control of the parameters of the synthesized LNPs.
Note that those processes can sometimes be followed by an extrusion process where the solution is forced through filters with a given pore size for a finer size control, and that they are not suitable to make LNP for RNA encapsulation, but can be used for other types of lipid-based nanoparticles (depending on their chemistry)
Microemulsion or nanoemulsion methods can be used for the synthesis of most polymeric and lipid nanoparticles. Water-in-oil (w/o) or Oil-in water (o/w) microemulsions are prepared by mixing two non-miscible liquids in presence of surfactants until a stable dispersion is formed. The nucleation and growth of nanoparticles in microemulsions is a complicated process. The most important factor is the water/surfactant ratio to control the phase separation. To better control the synthesis conditions, and thus the final nanoparticle characteristics, microfluidics mixing methods are generally used: They consist of the exact same system as described in the organic solvent method section.
When not formed with microfluidics, submitting the mixture to ultrasound can also be required to provide sufficient energy in the system. Temperature control can also be used to optimize the synthesis condition: cooling help facilitate the rapid crystallization of the lipids, while heating helps with the suspension generation.
However, those microemulsion technics are limited to the production of a few types of organic nanoparticles with a method called microemulsion polymerization, where hydrophobic monomers are polymerized by the addition of an oil-soluble initiator.
Supercritical Fluid Methods (SCFs)
SCFs methods have been developed to overcome the limitations in toxicity of the standard methods.
Several methods are available, the most popular one being the Supercritical Anti-solvent Method (SAS). A supercritical fluid is passed through the pressurized chamber, while the drug is sprayed on the SCF using a nozzle. LNPs are generated when hydrated with the aqueous solution. The supercritical CO2 is miscible with the organic solvent and acts as an antisolvent to the solute.
However, when put in practice, those methods suffer from major drawbacks: First, they can only process principles that are soluble in scCO2, which greatly limits them. Also, the high pressure/high temperature generally involved leads to risks for the API. Finally, those methods require very high investment cost and high energy consumption, which makes them hard to implements at large scale.
Nanoprecipitation-based methods for LNP synthesis
Methods based on self-assembly/nanoprecipitation constitute the primary approach employed in the development of the LNPs involved in the Covid-19 vaccine.: The underlying principle is mixing an aqueous phase, containing the hydrophilic API or oligonucleotide to be encapsulated, with a water-miscible solvent, such as ethanol, containing the lipids.
The decrease in the partial concentration of the solvent when mixed with the aqueous phase will lead to a drop in solubility of the lipids, which will then triggers its self-assembly and grow to create nanoparticles.
The self-assembly process is divided into 4 steps supersaturation, nucleation, growth, and stabilization or maturation. As introduced in our lipid nanoparticles formation review, the physicochemical characteristics of the formulated LNP are highly dependent on the synthesis conditions.
The most important factors are:
- The faster the mixing, the smaller the synthesized LNP
- Increasing the concentration of stabilizing agents decreases the size of the manufactured LNP
- Higher concentration of solute leads to a larger number of nucleic, hence an increase in coalescence events and final size of the LNP
- Viscosity and temperature play a role but their influence on the manufactured LNP
The main limitation of the solvent-based methods is the solvent potentially remaining in the final solution. However, the regulatory framework is relatively flexible in terms of ethanol content (up to 0,5% for the FDA), the remaining excess solvent can easily be extracted using centrifugation, Tangential flow filtration, Dialysis…
Solvent evaporation method
Solvent evaporation methods are very well adapted for the generation of lipid nanoparticle-trapping molecules with low miscibility in water. Those 2 processes are the original methods used for the synthesis of LNP and are still widely in use due to their ease of use. However, due to the poor control of their synthesis/mixing condition, the physicochemical properties of the LNP manufactured are very poor with a very low batch-to-batch reproducibility, and very high sample size dispersity, and near no control of the size ...
Ethanol injection methods
Lipid nanoparticles are prepared by dissolving the lipids in an organic solvent before injecting them into an aqueous solution. After emulsification of the organic phase into the aqueous one, the solvent is evaporated to obtain the nanoparticle.
Thin film hydration/Bangham methods
Thin film hydration was the first method used by Bangham for the synthesis of the first liposome. Similar to solvent evaporation, the lipids are dissolved into an organic solvent which is removed by evaporation under a vacuum. The film is then hydrated by an aqueous media containing the API, where it swells to self-produce nanoparticles.
Nevertheless, due to the poor quality of the nanoparticles produced using those methods, they are generally combined with a secondary methods such as extrusion or sonication to improve their quality.
Impingement jet mixing/T-junction
As a first step to enhance nanoprecipitation,T-mixers, such as impingement jet mixers, have been developed. Those systems are based on flash nanoprecipitation, where the solvent and anti solvent phase are mixed at very high speed and mix nearly instantly. While being efficient for large batches - such as used in the covid 19 vaccine - those methods require large volumes of liquids (at least several mL) and are thus not suitable at early stage of the drug development process. Additionally, they combine some of the drawbacks of high energy methods due to the large pressure involved, which can lead to API degradation.
State of the art: LNP synthesis using microfluidics
Considering the importance of finely controlling the mixing parameters to obtain homogenous LNP populations and good batch-to-batch reproducibility, the use of microfluidic quickly appeared as an evident solution to synthesize LNP.
Microfluidics is the technology of manipulating small volumes of liquids, down through nL, through micro-channels. At the micrometer scale, fluids behave differently than in everyday life and show laminar flow condition, which permits excellent control of their flow behavior all throughout the synthesis process, as well as provide an increased volume/surface ratio.
At that scale, thanks to the laminarity of the fluidic flow, the mixing process of the solvent and aqueous phase, triggering the self-assembly of the nanoparticle, offers the main advantage of being extremely well controllable and reproducible.
Multiple mixing methods using microfluidics are available. The most popular ones are the diffusive method (such as flow focusing), the chaotic methods (herringbone or the baffle mixer). They all provide different achievable flow rate, efficiency, repeatability, encapsulation efficiency but overall offer better trade-offs than all the previously mentioned LNP synthesis process, and are currently considered the state-of-the-art LNP formulation method.
Note that a post process purification step is usually required to ensure solvent removal for a better stability and lowered toxicity.
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High energy methods for LNP synthesis
High energy methods are top to bottom methods that rely on the generation of high shear stress forces by putting the lipids into harsh conditions (high pressure, high temperature) to achieve LNP size reduction.
The goal of these technics is to provide sufficient energy to the particles in the system to fragment them. The higher the surface tension the more energy should be supplied to the system to break them. These approaches be used either by themselves to produce nanoparticles from bulk material (such as illustrated below) or in combination with another low-quality method, to refine and improve the quality of nanoparticle populations. A typical combination is the use of thin film hydration methods together with an extruder.
Overall, those methods offer the advantage of being already available at a large scale. However, the high energy involved in the system can lead to lipid or nucleic acid alteration, thus not making is sustainable for biological application. On top of this, they offer poor encapsulation efficiency.
High-pressure homogenization (HPH)
High-pressure homogenization (HPH) permits the creation of an emulsion of molten lipids (generally 5-10%), water, and surfactants using a high-speed stirrer. The solution is pushed at high speed through small orifices using high pressures (500 to 5000 bar). The high velocities reached by the fluid are accompanied by turbulence, high shear forces, and cavitation which allows the formation of organic NPs even at high concentrations.
Two main approaches are available for LNP synthesis with HPH: hot and cold homogenization.
In hot homogenization, a drug and lipid mix is brought to a temperature above their melting point. The mix is then dispersed into a hot surfactant while maintaining the temperature. A hot pre-emulsion is formed, which is then homogenized before being cooled down, allowing the formation of the nanoparticles.
For cold homogenization, the drug-lipid melt is cooled down to form microparticles. The particles are then dispersed in a cold surfactant to generate a presuspension. The presuspension is then homogenized to break down the particles and form the nanoparticles. This approach permits the work with thermosensitive drugs by minimizing the diffusion of hydrophilic drugs into the water phase.
LNP can also be manufactured using ultrasounds. With this method, ultrasound waves will create cavitation phenomena, leading to the formation and collapse of bubbles, and thus the synthesis of LNP. In addition to this,the energy brought to the system helps breaking the nanoparticles, achieving size reduction. While offering size control, this method offers very low encapsulation efficiency and can lead to lipid degradation as well as metal pollution. 
Characterization of the manufactured LNP
Lipid nanoparticles have physicochemical attributes that need to be precisely controlled all throughout their synthesis process in order to reach the targeted area and achieve the desired effects in the body,
There is no strict regulatory framework for nanomedicines and no quantitative targets are set by the competent authorities, namely the European Medicine’s Agency (EMA), the Food and Drug Administration (FDA), and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH).
However, guidelines exist and require a careful characterization of the physicochemical attributes of the nanomaterials used in a drug formulation.
These attributes include the size of the NPs, the polydispersity index (PDI) associated with their size distribution, their morphology, and internal structure as well as their surface charge. Every batch of NPs needs to be properly characterized before proceeding to in vitro or in vivo assays. Quality checks are also needed in large-scale production of drugs or vaccines based on LNPs. Go deeper on the topic in our LNP and liposomes characterization review.
Size and distribution characterization following LNP synthesis
The lipid nanoparticle size is crucial to determine its in vivo release.
For optimal mRNA delivery, a compromise should be found between decreasing the particle size, which allows for easier absorption to the cell, and the LNP toxicity which increases when the particle size gets too small. Most ionizable lipid nanoparticles used are currently in the 50 to 200 nm range, with an optimal around 100 nm for LNP.
The particle size distribution characterization, or “polydispersity index” (PDI), defines the size range of lipid nanoparticles. This index is dimensionless and is scaled so that PDI <0.05 are nearly homogeneous, and PDI >0.5 are totally heterogeneous.
Typically, PDI below 0.2 is sought for the population of lipid nanoparticles and can easily be obtained using microfluidics methods.
It is worth noting that counterintuitively, Lipid nanoparticle size is mostly driven by the lipid mix and the manufacturing condition rather than the size of the RNA cargo.
The synthesized LNPs are generally characterized using optical methods such as dynamic light scattering (DLS) or laser diffraction (LD).
LNP surface charge – Zeta potential characterization
The Zeta potential provides information on the charge at the surface of the LNP. It helps predict the long-term stability of the formation. In order to avoid aggregation, it is necessary to have a zeta potential value as high as possible in absolute value (|ζ| ≫ 30mV). Indeed, when the latter tends towards 0 (-10 mV ≤ ζ ≤ 10 mV, the inter-particle forces decrease, and the attractive Van der Walls forces become preponderant. Generally, zeta potential values in the order of -30 mV are reported to both avoid the aggregation and decrease toxicity.
Zeta potential can also be characterized using specific features of a DLS, or laser Doppler electrophoresis.
LNP surface morphology characterization
Surface morphology is generally characterized using microscopy methods. Most often Scanning electron microscopy (SEM), Transmission electron microscopy (TEM) or Atomic Force Microscopy (AFM) are used.
Each of the methods offers a specific compromise. TEM provides a 2D image of the sample and gives access to the internal structure of the LNP with sub nm resolution. AFM instead permits a 3D visualization of the sample, including its surface with no specific sample preparation.
Load capacity (DL) and Encapsulation Efficiency (EE%)
Both parameters are related to the LNPs ability to efficiently encapsulate the API.
The DL (%) corresponds to the amount of trapped API over the total LNP weight. Instead, the encapsulation efficiency measures the percentage of drug that was successfully encapsulated, thus corresponds to the amount of trapped API over the initial amount in the formulation.
Encapsulation efficiency greatly varies with the synthesis method and can reach above 90% using the most efficient ones, such as microfluidics
Conclusion on LNP manufacturing
Lipid nanoparticles (LNP) are a promising new non viral delivery system for nucleic acid, such as mRNA, siRNA and DNA. Indeed, LNP are able to safely and effectively carry out the intracellular delivery of those nucleic acids, making them an ideal candidate for the development of new vaccines and gene therapies.
However, the physicochemical parameters (size, PDI, EE%...) of the LNP are critical to their efficient delivery. The choice of LNP manufacturing method should therefore take those requirements into account. For this reason, manufacturing methods providing good control over these critical parameters should be prefered. Thanks to its unique ability to finely control final LNP characteristics, microfluidics is considered the state of the art manufacturing method.
Nevertheless, the manufacturing of LNP is a rapidly developing field, and new and improved methods for manufacturing LNP are being developed all the time, so stay tuned!
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 Andra, Veera Venkata Satya Naga Lakshmi et al. “A Comprehensive Review on Novel Liposomal Methodologies, Commercial Formulations, Clinical Trials and Patents.” BioNanoScience vol. 12,1 (2022): 274-291. doi:10.1007/s12668-022-00941-x
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