WO2022175366A1 - Method for producing lipid nanoparticles and lipid nanoparticles resulting therefrom - Google Patents

Method for producing lipid nanoparticles and lipid nanoparticles resulting therefrom Download PDF

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Publication number
WO2022175366A1
WO2022175366A1 PCT/EP2022/053895 EP2022053895W WO2022175366A1 WO 2022175366 A1 WO2022175366 A1 WO 2022175366A1 EP 2022053895 W EP2022053895 W EP 2022053895W WO 2022175366 A1 WO2022175366 A1 WO 2022175366A1
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Prior art keywords
aqueous solution
liquid
lipid nanoparticles
solvent
weight
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PCT/EP2022/053895
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French (fr)
Inventor
Adrien DEWANDRE
Olivier ANDRÉ
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Secoya Technologies
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Publication of WO2022175366A1 publication Critical patent/WO2022175366A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • the present invention is related to a method for producing lipid nanoparticles.
  • the invention is further related to lipid nanoparticles obtained or obtainable with the method of the present invention, and to the use of such lipid nanoparticles, in particular for drug delivery applications and drug administration.
  • nanotechnology for drug delivery has been developed rapidly because of its suitability and feasibility in delivering both low weight drug molecules as macromolecules.
  • molecules that can be delivered are proteins, peptides, polypeptides, proteins or genes.
  • Drug delivery by nanotechnology further allows localized or targeted delivery to the cells or the tissue of interest.
  • oligonucleotides which include RNA, mRNA, siRNA and even DNA- based molecules.
  • oligonucleotides can trigger an effect at the genetic level to combat diseases.
  • mRNA RNA
  • mRNA RNA
  • siRNA DNA-based molecules.
  • oligonucleotides can trigger an effect at the genetic level to combat diseases.
  • One example is the delivery of mRNA to a cell to provide the expression of therapeutic proteins.
  • the disadvantage of oligonucleotides is that they are susceptible to degradation in the body.
  • LNPs lipid nanoparticles
  • complex therapeutic agents such as oligonucleotides (nucleic acid polymers) and (poly)peptides.
  • LNPs have proven their capacity to provide a stable matrix for drug molecules and therapeutic agents, allowing their delivery to the targeted location, e.g. cells or tissue.
  • LNPs can be seen as a new generation of liposomes, which are known for drug delivery since the 1970s.
  • LNPs tend to have a more complex internal lipid architecture with low or minimal internal aqueous presence, rendering the LNPs suited for stable and efficient encapsulation of different complex therapeutic agents and molecules.
  • WO 2019/016819 describes the production of LNPs by means of nanoprecipitation.
  • a solution comprising a lipid dissolved in a non-aqueous solvent is contracted with an anti-solvent, such as heptane, wherein the lipid is insoluble in the anti- solvent, and the non-aqueous solvent is miscible with the anti-solvent.
  • the anti-solvent can further comprise water-soluble particles.
  • the method can be used to introduce an agent, such as a therapeutically active agent, for example a peptide, a polypeptide, in the LNPs, for use in drug delivery and drug administration.
  • LNPs having an average size between 200 nm and 600 nm can be obtained, and can be used for nasal drug administration.
  • WO 2019/094405 discloses a method to produce a LNP for encapsulation and sustained release of therapeutic agents.
  • a cationic agent, a therapeutic agent and a first water-immiscible solvent is combined with a first aqueous solution.
  • the mixture comprising a complex comprising the cationic agent and the therapeutic agent is then combined with a second water-immiscible solvent, wherein an aqueous phase and an organic phase is obtained.
  • the organic phase, comprising the complex is separated from the aqueous phase, and combined with a sterol and a first water-miscible solvent.
  • the complex is then dispersed in a second aqueous solution to form the LNPs.
  • WO 2013/093 648 discloses a method for preparing a LNP encapsulating a double stranded RNA molecule.
  • a lipid solution comprising a cationic lipid, a helper lipid, a sterol and a PEG lipid dissolved in a water-miscible organic solvent is injected in a first aqueous solution, comprising the RNA molecule, at continuous stirring of the first aqueous solution.
  • the organic solvent is removed from the mixture by diafiltration against a second aqueous buffered solution.
  • the 3D-MHF method allows for the production of monodisperse LNPs (polydispersity index (PDI) ⁇ 0.01) at a relatively high throughput
  • the concentration of LNPs in the final solution is low, i.e. the LNPs have a large dilution in the final solution.
  • a large dilution may require additional post-treatment steps to increase the concentration of LNPs in the final solution, in order to render them usable for the envisaged application.
  • Liposomes that encapsulate reagents in a continuous two-phase flow microfluidic network, wherein a solvent-aqueous interface in a microfluidic format is obtained to form the liposomes.
  • the average size of the liposomes can be controlled between 100 nm and 300 nm by manipulation of the liquid flow rates.
  • a disadvantage of the above microfluidic process is that upscaling to industrial level is often limited because of difficulties to maintain the non-turbulent process conditions.
  • Another disadvantage is that the polydispersity index of the obtained nanoparticles can be rather high, in particular larger than 0.1, indicating a rather polydisperse particle size distribution of the LNPs.
  • the present invention aims to solve one or more of the problems of the methods of the state of the art. It is an aim of the invention to provide an improved method for the production of lipid nanoparticles, which allows better control during processing, resulting in lipid nanoparticles having improved properties.
  • the improved method of the invention also allows a high total throughput, rendering the process economically interesting and applicable.
  • the method further allows to produce LNPs having a good to excellent concentration in the final solution comprising the LNPs. In other words, the dilution of the LNPs in the final solution is limited.
  • the invention further aims to provide lipid nanoparticles having improved properties and characteristics compared to lipid nanoparticles of the state of the art.
  • the lipid nanoparticles of the invention have an advantageous average size which is suitable for the envisaged application and use of the LNPs.
  • the LNPs further have an improved size distribution and homogeneity, in particular a particle size distribution which is considered to be rather monodisperse, and thus less polydisperse.
  • the LNPs of the invention also have an increased concentration in the final solution.
  • the method comprises the steps of: a) providing a device comprising: i) a cavity; ii) an output capillary; iii) an outer input capillary; iv) an inner input capillary located inside the outer input capillary; wherein the output capillary is in front of an end of both the outer input capillary and the inner input capillary and all capillaries are coaxially aligned; b) injecting a liquid to the outer input capillary, wherein the liquid comprises a lipid dissolved in a solvent, and wherein the solvent is at least partially miscible in water; c) injecting a first aqueous solution to the inner input capillary, wherein a liquid flow surrounding a first aqueous flow is formed; d) injecting a second aqueous solution to the cavity, wherein a second aqueous flow surrounding the liquid flow surrounding the first aqueous flow is formed, preferably the second aqueous flow being a laminar flow;
  • the liquid comprises a lipid dissolved in a solvent.
  • the solvent is at least partially miscible in water, for example at least 75 % of the solvent is miscible in water, based on the total volume of the solvent.
  • the solvent advantageously comprises an organic solvent.
  • the solvent comprises an alcohol or a mixture of alcohols. Preferred examples are methanol, ethanol, n-propanol, isopropanol, or a mixture of two or more thereof.
  • the lipid is a triglyceride, a phospholipid, a fatty acid, a fatty alcohol, a fatty ester, or a combination of two or more thereof.
  • the liquid comprises between 0.1 % by weight and 10 % by weight of lipids, based on the total weight of the liquid, such as between 0.25 % by weight and 9 % by weight, preferably between 0.5 % by weight and 7.5 % by weight, more preferably between 1 % by weight and 5 % by weight.
  • the liquid can further comprise an agent.
  • the first and/or second aqueous solution can comprise an agent.
  • the agent when present in the liquid, can be a steroid, or a therapeutically active agent, preferably a peptide, a polypeptide or a protein.
  • the agent when present in the first and/or second aqueous solution, is mRNA, siRNA or DNA.
  • the first and the second aqueous solution comprise at least 25 v% of water, such as at least 50 v%, such as at least 60 v%, at least 70 v%, preferably at least 75 v%, for example at least 80 v%, at least 85 v%, more preferably at least 90 v%, or at least 95 v% of water.
  • the first aqueous solution and the second aqueous solution can have the same composition.
  • the first and second aqueous solution can have a different composition.
  • the first and/or the second aqueous solution can be a phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the flow rate ratio is between 0.5 and 50, such as between 0.75 and 40, between 1 and 3, preferably between 2 and 20.
  • the flow rate ratio is herein defined as the sum of the flow rate of the first and second aqueous solutions divided by the flow rate of the liquid.
  • the invention provides lipid nanoparticles
  • lipid nanoparticles are advantageously obtained or obtainable by the method of the invention.
  • the lipid nanoparticles have an average size between 1 nm and 1000 nm, such as between 5 nm and 750 nm, between 10 nm and 500 nm, between 10 nm and 250 nm, between 15 nm and 200 nm, preferably between 20 nm and 200 nm, such as between 20 nm and 100 nm.
  • the average size is measured by means of Dynamic Light Scattering (DLS), and the average size represents the mediane size of the volume distribution of the lipid nanoparticles.
  • DLS Dynamic Light Scattering
  • the lipid nanoparticles have a particle size distribution wherein the polydispersity index (PDI) is equal to or lower than 0.25, such as equal to or lower than 0.2, preferably equal to or lower than 0.15, more preferably equal to or lower than 0.1.
  • PDI polydispersity index
  • the PDI is measured according to ISO 22412:2008(E).
  • the lipid nanoparticles have an average size between 10 nm and 250 nm, preferably between 20 nm and 200 nm, as measured by means of DLS, and a polydispersity index lower than 0.15, preferably lower than 0.1 , as measured according to ISO 22412:2008(E).
  • the lipid nanoparticles comprise between 0.1 % by weight and 50 % by weight of an agent, such as between 0.25 % by weight and 30 % by weight, preferably between 0.5 % by weight and 25 % by weight of an agent.
  • the agent is mRNA, a steroid, a peptide, a polypeptide or a protein.
  • the present invention further discloses the use of lipid nanoparticles of the invention, as set out in the appended claims.
  • the lipid nanoparticles are advantageously used in a drug delivery system.
  • the lipid nanoparticles are used for the encapsulation, the protection and the delivery of mRNA inside cells.
  • LNPs for the encapsulation, protection and delivery of mRNA is for the treatment of diseases such as cancer, genetic disorders and infectious diseases, and for vaccines used to protect against viruses, such as the SARS-CoV-2 coronavirus causing the Covid-19 disease.
  • Fig. 1 discloses a schematic representation of the method of the present invention.
  • Fig. 2 discloses a representation of the phases used in the invention, in particular the interfaces formed between the respective phases.
  • Fig. 3 discloses an example of the average size of LNPs of the invention as a function of the flow rate ratio defined as the total flow rate of the aqueous solutions divided by the flow rate of the liquid.
  • the present invention is related to a method to produce lipid nanoparticles (LNPs).
  • the method comprises the step of providing a device 2 comprising input capillaries 3, a cavity 6 and an output capillary 7.
  • the cavity also comprises an input tubing 8.
  • the input capillaries 3 comprises two coaxial capillaries; a first, outer, coaxial capillary 4 and a second, inner, coaxial capillary 5.
  • the input capillaries 3, and in particular the coaxial capillaries 4, 5, are adapted to receive a fluid.
  • the fluid is a liquid, such as, without being limited thereto, a liquid solution, an emulsion, or a dispersion.
  • the output capillary 7 is coaxially aligned with the input capillary.
  • the output capillary 7 is preferably positioned downstream of the input capillary 3.
  • the input capillary 3 is positioned at a first end of the cavity 6 and the output capillary 7 at the other end of the cavity 6.
  • the tubing 8 is also adapted to receive a fluid in a regulated manner.
  • the method of the invention further comprises the step of providing a liquid 20 to the first, outer, coaxial capillary 4.
  • the liquid 20 comprises a lipid and a solvent.
  • the liquid 20 comprises a lipid dissolved in a solvent.
  • the solvent is at least partially miscible in water.
  • At least partially miscible in water it is meant that, when performing the method, at least 50 %, such as at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, preferably at least 90 %, more preferably at least 95 %, at least 97.5 %, at least 99 % of the solvent is miscible in water, expressed in percentage of the total volume of the solvent.
  • the solvent can comprise one solvent or a mixture of two or more solvents.
  • the solvent can comprise an organic solvent, an inorganic solvent, of a combination of two or more thereof.
  • the solvent is an organic solvent.
  • the solvent is a non-aqueous solvent.
  • a non-aqueous solvent is meant in the light of the present invention a solvent comprising at most 10 % by weight of water, on the total weight of the solvent, preferably at most 5 % by weight of water, even more preferably at most 1 % by weight of water.
  • the solvent can comprise an alcohol or a mixture of two or more alcohols. Alternatively or additionally, the solvent can comprise acetone.
  • the alcohol is advantageously alcohol that is at least partially miscible in water.
  • the alcohol is a C1-4-alcohol, meaning an alcohol comprising between 1 and 4 carbon atoms, such as methanol, ethanol, n-propanol, isopropanol (2-propanol), n-butanol, or 2-methyl-1- propanol (isobutanol).
  • the lipid can be a triglyceride, a phospholipid, a fatty acid, a fatty alcohol, a fatty ester or polyethylene glycol (PEG), or a combination of two or more thereof.
  • the lipid can be ionizable.
  • the lipid is a combination of an ionizable lipid and a PEG lipid.
  • the lipid comprises a phospholipid, for example, the lipid can consist of a phospholipid.
  • the liquid 20 comprises between 0.1 % by weight and 10 % by weight of lipids, based on the total weight of the liquid 20, such as between 0.2 % by weight and 9 % by weight, between 0.25 % by weight and 8 % by weight, between 0.5 % by weight and 7.5 % by weight, between 0.75 % by weight and 7 % by weight, preferably between 0.8 % by weight and 6 % by weight, such as between 0.9 % by weight and 5.5 % by weight, more preferably between 1 % by weight and 5 % by weight.
  • the liquid can further comprise one or more additives.
  • An example of an additive is a surfactant, for example dimethyldioctadecylammonium bromide (DDAB).
  • DDAB dimethyldioctadecylammonium bromide
  • DDAB is advantageously used to stabilize the lipid nanoparticles produced with the method of the invention.
  • the method of the invention further comprises the step of providing a first aqueous solution 21 to the second, inner, coaxial capillary 5.
  • a liquid flow surrounding a first aqueous flow 21-in-20 is formed.
  • the first aqueous solution comprises at least 25 % by volume of water, such as at least 30 %, 40 %, 50 %, 60 %, 70 %, 75 %, 80 %, 90 % or 95 % by volume of water.
  • the first aqueous solution is a phosphate-buffered saline (PBS) solution.
  • PBS solution can by any PBS solution known in the state of the art, in particular PBS solutions suitable for use in biological and biotechnological applications.
  • the PBS solution comprises water and sodium chloride, and has a pH value between 6.5 and 8, for example between 7 and 7.5, such as around 7.2.
  • the method of the invention further comprises the step of providing a second aqueous solution 22 to the cavity 6.
  • a second aqueous solution 22 to the cavity 6
  • the first aqueous solution 21 to the second, inner, coaxial capillary 5 a second aqueous flow surrounding a liquid flow surrounding a first aqueous flow 23 (21-in-20-in-22) is formed.
  • the second aqueous solution comprises at least 25
  • the second aqueous solution is a phosphate-buffered saline (PBS) solution.
  • PBS phosphate-buffered saline
  • the first aqueous solution and the second aqueous solution can have the same composition, such as the same amount of water.
  • the composition of the first and the second aqueous solution is different.
  • the liquid 20 can further comprise an agent.
  • the agent can be an active agent, preferably a therapeutically active agent.
  • a suitable therapeutically active agent examples include, without being limited thereto, a peptide, a polypeptide, a protein, cyclosporine, steroids, cannabidiol (CBD), tetrahydrocannabinol (THC), rapamycin, or antibiotics.
  • the therapeutically active agent is a peptide, a polypeptide or a protein.
  • peptide it is meant in the light of the present invention a molecule comprising a backbone of 2 to 20 amino acid residues.
  • polypeptide it is meant in the light of the present invention a molecule comprising a backbone of more than 20 amino acid residues.
  • suitable peptides or polypeptides are, without being limited thereto, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (LHRH), oxytocin, insulin, corticotropin (ACTH).
  • the first (21) and/or second (22) aqueous solution can comprise an agent.
  • the agent can be, without being limited thereto, mRNA, siRNA or DNA, or an active agent, such as a therapeutically active agent.
  • the solvent comprised in the liquid 20 will advantageously diffuse from the liquid 20 to the first aqueous solution 21 and to the second aqueous solution 22. Upon diffusion of the solvent from the liquid 20, a phase separation of the lipid comprised in the liquid 20 will advantageously take place, wherein lipid nanoparticles 1 (LNPs) are formed.
  • LNPs lipid nanoparticles 1
  • the agent When an agent is present, the agent will be encapsulated in the lipid nanoparticles.
  • the lipid nanoparticles comprising an encapsulated agent can protect the agent against certain environments.
  • the lipid nanoparticles are used in a drug delivery system, they can deliver the agent at the targeted location, such as the target cells, by protecting it against non-favorable environments prior to arriving at the targeted location.
  • the liquid 20 advantageously flows in the cavity at a flow rate between 0.1 mI/min and 1000 mI/min, such as between 0.5 mI/min and 750 mI/min, preferably between 1 mI/min and 500 mI/min, for example between 2.5 mI/min and 400 mI/min, more preferably between 5 mI/min and 300 mI/min, such as between 10 mI/min and 250 mI/min, between 20 mI/min and 200 mI/min, between 25 mI/min and 175 mI/min, or between 50 mI/min and 150 mI/min, for a device having an inner input capillary having a diameter of 30 pm, an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm.
  • mI/min and 1000 mI/min such as between 0.5 mI/min and 750 mI/min, preferably between 1 mI/min and
  • the flow rate of the liquid can be higher.
  • the first aqueous solution 21 advantageously flows in the cavity at a flow rate between 1 mI/min and 1000 mI/min, such as between 5 mI/min and 750 mI/min, preferably between 10 mI/min and 500 mI/min, between 20 mI/min and 400 mI/min, between 30 mI/min and 300 mI/min, between 40 mI/min and 250 mI/min, more preferably between 50 mI/min and 200 mI/min, such as between 75 mI/min and 150 mI/min, for a device having an inner input capillary having a diameter of 30 p , an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm.
  • 1 mI/min and 1000 mI/min such as between 5 mI/min and 750 mI/min, preferably between 10 mI/min and 500 mI/min, between 20 mI/min
  • the flow rate of the first aqueous solution can be higher.
  • the second aqueous solution 22 advantageously flows in the cavity at a flow rate between 5 pl/min and 1000 pl/min, such as between 10 pl/min and 900 pl/min, between 20 pl/min and 800 pl/min, between 30 pl/min and 750 pl/min, preferably between 40 pl/min and 700 pl/min, for example between 50 pl/min and 600 pl/min, between 75 pl/min and 550 pl/min, more preferably between 100 pl/min and 500 pl/min, such as between 125 pl/min and 450 pl/min, between 150 pl/min and 400 pl/min, of between 175 pl/min and 350 pl/min, for a device having an inner input capillary having a diameter of 30 pm, an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm.
  • 5 pl/min and 1000 pl/min such as between 10 pl/min and 900 pl/min, between 20 pl/min and 800 pl/min, between 30 pl/min and 750 pl/min, preferably
  • the flow rate of the second aqueous solution can be higher.
  • the flow rate of the liquid and the flow rate of the first and second aqueous solution together is the so-called total flow rate (TFR).
  • TFR total flow rate
  • the total flow rate at which a process can take place without impacting the products obtained, in this case LNPs, is one of the measures for the scalability of the process.
  • a higher the total flow rate means a higher throughput, and thus a process that is suited for production at industrial scale.
  • the flow rate ratio at which the production of LNPs takes place is an indication for the concentration of LNPs in the final solution.
  • the final solution is the combination of the first aqueous solution, the second aqueous solution, and the solvent diffused into the first and second aqueous solutions.
  • a higher flow rate ratio indicates for a given flow rate of the liquid a larger flow rate of the first and second aqueous solutions, and thus relatively speaking a smaller amount of lipids, and a lower concentration of LNPs in the final solution.
  • a higher flow rate ratio means a higher dilution of the produced LNPs in the final solution.
  • a low dilution, or a high concentration, of LNPs in the final solution is required.
  • a subsequent step may be required to decrease the dilution, i.e. increase the concentration, of the LNPs in the final solution.
  • the flow rate ratio is also a measure for the average size of the
  • a higher FRR typically allows to obtain smaller LNPs.
  • the FRR is also a measure for the polydispersity of the produced LNPs.
  • the polydispersity is related to the particle size distribution (PSD) of the produced LNPs, and is expressed by the polydispersity index (PDI).
  • PSD particle size distribution
  • PDI polydispersity index
  • a monodisperse PSD is preferably used.
  • a higher FRR typically indicates a more polydisperse PSD, thus a higher PDI.
  • One of the problems with the methods of the state of the art is that they have a low total flow rate (at most 100 mI/min) for a flow rate ratio below 100, limiting the scalability to industrial processes, or that they show good total flow rates, but very high flow rate ratios, such as FRRs of more than 100, even more than 200, 500, up to 5000 and more, resulting in the addition of a concentration step to the method, such as a filtration step, rendering the process more complex.
  • the method of the present invention solves these problems, and can be used at a total flow rate of at least 100 mI/min, such as a total flow rate of at least 300 mI/min, in combination with a flow rate ratio between 0.5 and 50, such as between 1 and 25, or between 2 and 20.
  • the inventors have found that by using the method of the present invention, the surface of the interface between the liquid and an aqueous solution - in the present invention the first aqueous solution and the second aqueous solution - is increased, which allows improved control of the process, even at industrial scale.
  • the increase of the surface of the liquid - aqueous solution interface allows more controlled flow, i.e. a more stable flow, and a less turbulent or less chaotic flow, of both the liquid and the aqueous solutions, and a more controlled diffusion of the solvent into the aqueous solutions.
  • the increase in process control leads to a low flow rate ratio, resulting in a good polydispersity (PDI ⁇ 0.1), LNPs having small sizes ( ⁇ 200 nm), and a low dilution of the LNPs in the final solution.
  • the increase in process control further allows for an increase in the total flow rate of the process.
  • the present invention is further related to lipid nanoparticles obtained or obtainable by means of the method of the invention.
  • the lipid nanoparticles have an average size between 1 nm and 1000 nm, such as between 2 nm and 500 nm, between 5 nm and 400 nm, between 10 nm and 300 nm, between 15 nm and 250 nm, between 20 nm and 200 nm, or between 25 nm and 150 nm, for example between 50 nm and 100 nm.
  • the LNPs have an average size between 10 nm and 500 nm, more preferably between 20 nm and 200 nm.
  • the size of the LNPs is measured by Dynamic Light Scattering
  • the lipid nanoparticles of the invention have a polydispersity index (PDI) equal to or lower than 0.25, for example equal to or lower than 0.2, equal to or lower than 0.15, preferably equal to or lower than 0.1 , such as equal to or lower than 0.09, equal to or lower than 0.08, equal to or lower than 0.075, equal to or lower than 0.07, equal to or lower than 0.06, or equal to or lower than 0.05.
  • the polydispersity index is measured according to the test standard ISO 22412:2008(E).
  • the lipid nanoparticles of the invention comprise an agent.
  • the agent can be an agent as described above.
  • the agent is mRNA, a steroid, a peptide or a polypeptide.
  • the lipid nanoparticles comprise between 0.05 % and 50 % by weight of the agent, based on the total weight of the lipid nanoparticle, such as between 0.075 % and 45 % by weight, between 0.1 % and 40 % by weight, between 0.2 % and 35 % by weight, between 0.25 % and 30 % by weight, such as between 0.5 % and 25 % by weight, between 0.75 % and 20 % by weight, between 1 % and 15 % by weight, or between 2 % and 10 % by weight.
  • the invention is further related to the use of the lipid nanoparticles of the invention.
  • the LNPs can be used in a drug delivery system.
  • the lipid nanoparticles comprise an agent, i.e. an encapsulated agent, they can protect the agent against certain environments.
  • the lipid nanoparticles comprising an agent are used in a drug delivery system, they can deliver the agent at a targeted location, such as target cells, by protecting it against non-favorable environments prior to arriving at the targeted location.
  • the lipid nanoparticles can also be used for the encapsulation, protection and delivery of mRNA inside cells.
  • the LNPs can be used in a non-viral gene delivery system, for example in a mRNA delivery system.
  • Examples of the use of LNPs for the encapsulation, protection and delivery of mRNA is for the treatment of diseases such as cancer, genetic disorders and infectious diseases, and for vaccines used to protect against viruses, such as the SARS-CoV-2 coronavirus causing the Covid19 disease.
  • Lipid nanoparticles according to the prior art were produced by using phospholipids dissolved in ethanol as the liquid.
  • the first aqueous solution was a phosphate buffered saline (PBS) comprising water and sodium chloride and having a pH value of 7.2.
  • PBS phosphate buffered saline
  • the PBS solution was provided to the cavity of a single emulsion device, and the liquid was provided to the cavity through an input capillary, wherein an aqueous flow surrounding a liquid flow was obtained.
  • the ethanol diffused into the PBS solution surrounding the liquid via the ethanol-PBS interface, and lipid nanoparticles were obtained.
  • the total flow rate was 100 mI/min.
  • the flow rate ratio (FRR), here defined as the flow rate of the aqueous solution divided by the flow rate of the liquid, was varied between 2 and 49, and the average size of the lipid nanoparticles was measured by means of Dynamic Light Scattering, wherein the measured diameter (the average size) is the mediane size of the volume distribution of the lipid nanoparticles.
  • Lipid nanoparticles according to the invention were produced by using lipids dissolved in ethanol as the liquid.
  • the first aqueous solution and the second aqueous solution had the same composition, and were a phosphate buffered saline (PBS) comprising water and sodium chloride and having a pH value of 7.2.
  • PBS phosphate buffered saline
  • the liquid was provided to the cavity through a first, outer, coaxial capillary.
  • the first PBS solution was provided to the cavity through a second, inner, coaxial capillary.
  • the second PBS solution was provided to the cavity, wherein a second aqueous flow surrounding a liquid flow surrounding a first aqueous flow was obtained.
  • FRR flow rate ratio
  • Fig. 3 represents the average size of the lipid nanoparticles (y-axis) as function of the FRR (x-axis) for the prior art lipid nanoparticles (Example 1 , showed as curve ‘SE’) and for the lipid nanoparticles of the invention (Example 2, showed as curve ⁇ E’). It is clear that for all FRRs, the average size of the LNPs of the invention is smaller than the average size of the prior art LNPs. In other words, a required average size of the LNPs can be obtained at a lower FRR with the method of the invention than with the prior art methods.
  • the method of the invention allows improved control of the process conditions, which, in combination with the increased surface of the liquid-aqueous solution interface, results in a reduction of the FRR for a given average size of the LNPs.
  • Lipid nanoparticles were produced using the method of the invention.
  • the liquid comprised lipids dissolved in ethanol.
  • Phosphate buffered saline (PBS) was used as the first aqueous solution and as the second aqueous solution.
  • the total flow rate, TFR was 300 mI/min, and the flow rate ratio was varied between 2 and 20.
  • the average size of the obtained lipid nanoparticles was measured by Dynamic Light Scattering.
  • the polydispersity index (PDI) was measured according to ISO 22412:2008(E).
  • the obtained LNPs showed excellent properties: their average size varied between 20 nm and 200 nm and the polydispersity index (PDI) of the LNP size distribution was below 0.1, indicating a rather monodisperse particle size distribution for the LNPs.
  • Equivalent experiments carried out with a single emulsion as in comparative example 1 showed a PDI that was about 0.15 higher.

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Abstract

The present invention discloses a method for producing lipid nanoparticles comprising providing a device comprising an input capillary which comprises two coaxial capillaries, a cavity and an output capillary coaxially aligned with the input capillary; providing a liquid to a first, outer, coaxial capillary, the liquid comprising a lipid dissolved in a solvent, and the solvent being at least partially miscible in water; providing a first aqueous solution to a second, inner, coaxial capillary, forming a liquid flow surrounding a first aqueous flow; providing a second aqueous solution to the cavity, forming a second aqueous flow surrounding a liquid flow surrounding a first aqueous flow; diffusion of the solvent from the liquid to the first and second aqueous solution, thereby inducing phase separation of the lipid, wherein lipid nanoparticles are formed. The invention further discloses lipid nanoparticles obtained by the method and the use thereof in drug delivery systems.

Description

METHOD FOR PRODUCING LIPID NANOPARTICLES AND LIPID NANOPARTICLES RESULTING THEREFROM
Technical field
[0001] The present invention is related to a method for producing lipid nanoparticles. The invention is further related to lipid nanoparticles obtained or obtainable with the method of the present invention, and to the use of such lipid nanoparticles, in particular for drug delivery applications and drug administration.
Background art
[0002] Over the last decades, nanotechnology for drug delivery has been developed rapidly because of its suitability and feasibility in delivering both low weight drug molecules as macromolecules. Examples of molecules that can be delivered are proteins, peptides, polypeptides, proteins or genes. Drug delivery by nanotechnology further allows localized or targeted delivery to the cells or the tissue of interest.
[0003] Many of the drug molecules of interest today are small molecules.
In particular, there is a demand for delivery methods of more specialized and complex therapies, such as oligonucleotides, which include RNA, mRNA, siRNA and even DNA- based molecules. Such oligonucleotides can trigger an effect at the genetic level to combat diseases. One example is the delivery of mRNA to a cell to provide the expression of therapeutic proteins. The disadvantage of oligonucleotides is that they are susceptible to degradation in the body.
[0004] The development and use of lipid nanoparticles (LNPs) is becoming more popular because of their ability to act as drug carriers for complex therapeutic agents, such as oligonucleotides (nucleic acid polymers) and (poly)peptides. LNPs have proven their capacity to provide a stable matrix for drug molecules and therapeutic agents, allowing their delivery to the targeted location, e.g. cells or tissue. LNPs can be seen as a new generation of liposomes, which are known for drug delivery since the 1970s. LNPs tend to have a more complex internal lipid architecture with low or minimal internal aqueous presence, rendering the LNPs suited for stable and efficient encapsulation of different complex therapeutic agents and molecules.
[0005] WO 2019/016819 describes the production of LNPs by means of nanoprecipitation. A solution comprising a lipid dissolved in a non-aqueous solvent is contracted with an anti-solvent, such as heptane, wherein the lipid is insoluble in the anti- solvent, and the non-aqueous solvent is miscible with the anti-solvent. The anti-solvent can further comprise water-soluble particles. The method can be used to introduce an agent, such as a therapeutically active agent, for example a peptide, a polypeptide, in the LNPs, for use in drug delivery and drug administration. LNPs having an average size between 200 nm and 600 nm can be obtained, and can be used for nasal drug administration.
[0006] WO 2019/094405 discloses a method to produce a LNP for encapsulation and sustained release of therapeutic agents. A cationic agent, a therapeutic agent and a first water-immiscible solvent is combined with a first aqueous solution. The mixture comprising a complex comprising the cationic agent and the therapeutic agent is then combined with a second water-immiscible solvent, wherein an aqueous phase and an organic phase is obtained. The organic phase, comprising the complex, is separated from the aqueous phase, and combined with a sterol and a first water-miscible solvent. The complex is then dispersed in a second aqueous solution to form the LNPs.
[0007] WO 2013/093 648 discloses a method for preparing a LNP encapsulating a double stranded RNA molecule. A lipid solution, comprising a cationic lipid, a helper lipid, a sterol and a PEG lipid dissolved in a water-miscible organic solvent is injected in a first aqueous solution, comprising the RNA molecule, at continuous stirring of the first aqueous solution. In a next step, the organic solvent is removed from the mixture by diafiltration against a second aqueous buffered solution.
[0008] Disadvantages of method using a water-immiscible solvent is that solvent removal requires the use of filtration and/or centrifugation, which may impact the quality of the LNPs. Filtration and centrifugation typically reduce the yield of the processes, making these processes less suited for large scale production (industrial scale production, i.e. production at high throughput).
[0009] Further, processes requiring mixing are more difficult to reproduce because of the risk for inhomogeneous mixing. Another disadvantage is that these methods often require mechanical forces, such as high shear forces, to obtain the nanoparticles, especially when relatively small nanoparticles are to be obtained. A high shear force may result in the deterioration of an agent incorporated into the nanoparticles.
[0010] ‘A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array’, Renee R. Hood, Don L. DeVoe et al., Lab Chip, 14, pages 2403-2409 (2014) discloses a microscale device for one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The method comprises the radially symmetrical diffusion of an alcohol-solvated lipid into an aqueous flow stream.
[0011] Whereas the 3D-MHF method allows for the production of monodisperse LNPs (polydispersity index (PDI) < 0.01) at a relatively high throughput, the concentration of LNPs in the final solution is low, i.e. the LNPs have a large dilution in the final solution. A large dilution may require additional post-treatment steps to increase the concentration of LNPs in the final solution, in order to render them usable for the envisaged application.
[0012] ‘Controlled Vesicle Self-Assembly in Microfluidic Channels with
Hydrodynamic Focusing’, Jahn et al., Journal of the Americal Chemical Society, 126(9), pages 2674-2675 (2004) discloses the formation of liposomes that encapsulate reagents in a continuous two-phase flow microfluidic network, wherein a solvent-aqueous interface in a microfluidic format is obtained to form the liposomes. The average size of the liposomes can be controlled between 100 nm and 300 nm by manipulation of the liquid flow rates.
[0013] A disadvantage of the above microfluidic process, is that upscaling to industrial level is often limited because of difficulties to maintain the non-turbulent process conditions. Another disadvantage is that the polydispersity index of the obtained nanoparticles can be rather high, in particular larger than 0.1, indicating a rather polydisperse particle size distribution of the LNPs.
Aim of the invention
[0014] The present invention aims to solve one or more of the problems of the methods of the state of the art. It is an aim of the invention to provide an improved method for the production of lipid nanoparticles, which allows better control during processing, resulting in lipid nanoparticles having improved properties. The improved method of the invention also allows a high total throughput, rendering the process economically interesting and applicable. The method further allows to produce LNPs having a good to excellent concentration in the final solution comprising the LNPs. In other words, the dilution of the LNPs in the final solution is limited.
[0015] The invention further aims to provide lipid nanoparticles having improved properties and characteristics compared to lipid nanoparticles of the state of the art. In particular, the lipid nanoparticles of the invention have an advantageous average size which is suitable for the envisaged application and use of the LNPs. The LNPs further have an improved size distribution and homogeneity, in particular a particle size distribution which is considered to be rather monodisperse, and thus less polydisperse. The LNPs of the invention also have an increased concentration in the final solution.
Summary of the invention
[0016] In a first aspect according to the invention, there is provided a method for producing lipid nanoparticles as set out in the appended claims.
[0017] The method comprises the steps of: a) providing a device comprising: i) a cavity; ii) an output capillary; iii) an outer input capillary; iv) an inner input capillary located inside the outer input capillary; wherein the output capillary is in front of an end of both the outer input capillary and the inner input capillary and all capillaries are coaxially aligned; b) injecting a liquid to the outer input capillary, wherein the liquid comprises a lipid dissolved in a solvent, and wherein the solvent is at least partially miscible in water; c) injecting a first aqueous solution to the inner input capillary, wherein a liquid flow surrounding a first aqueous flow is formed; d) injecting a second aqueous solution to the cavity, wherein a second aqueous flow surrounding the liquid flow surrounding the first aqueous flow is formed, preferably the second aqueous flow being a laminar flow; and e) diffusing the solvent from the liquid to the first and the second aqueous solution, thereby inducing phase separation of the lipid, wherein lipid nanoparticles are formed. [0018] A preferred device for implementing the invention is described in
EP3648878 which is hereby incorporated by reference.
[0019] The liquid comprises a lipid dissolved in a solvent. The solvent is at least partially miscible in water, for example at least 75 % of the solvent is miscible in water, based on the total volume of the solvent. The solvent advantageously comprises an organic solvent. Advantageously, the solvent comprises an alcohol or a mixture of alcohols. Preferred examples are methanol, ethanol, n-propanol, isopropanol, or a mixture of two or more thereof.
[0020] Advantageously, the lipid is a triglyceride, a phospholipid, a fatty acid, a fatty alcohol, a fatty ester, or a combination of two or more thereof. Advantageously, the liquid comprises between 0.1 % by weight and 10 % by weight of lipids, based on the total weight of the liquid, such as between 0.25 % by weight and 9 % by weight, preferably between 0.5 % by weight and 7.5 % by weight, more preferably between 1 % by weight and 5 % by weight.
[0021] The liquid can further comprise an agent. Alternatively or additionally, the first and/or second aqueous solution can comprise an agent. Preferably, when present in the liquid, the agent can be a steroid, or a therapeutically active agent, preferably a peptide, a polypeptide or a protein. Preferably, when present in the first and/or second aqueous solution, the agent is mRNA, siRNA or DNA.
[0022] Advantageously, the first and the second aqueous solution comprise at least 25 v% of water, such as at least 50 v%, such as at least 60 v%, at least 70 v%, preferably at least 75 v%, for example at least 80 v%, at least 85 v%, more preferably at least 90 v%, or at least 95 v% of water. The first aqueous solution and the second aqueous solution can have the same composition. Alternatively, the first and second aqueous solution can have a different composition. The first and/or the second aqueous solution can be a phosphate-buffered saline (PBS).
[0023] Advantageously, the flow rate ratio (FRR) is between 0.5 and 50, such as between 0.75 and 40, between 1 and 3, preferably between 2 and 20. The flow rate ratio is herein defined as the sum of the flow rate of the first and second aqueous solutions divided by the flow rate of the liquid.
[0024] In a second aspect, the invention provides lipid nanoparticles
(LNPs) as set out in the appended claims. The lipid nanoparticles are advantageously obtained or obtainable by the method of the invention.
[0025] Advantageously, the lipid nanoparticles have an average size between 1 nm and 1000 nm, such as between 5 nm and 750 nm, between 10 nm and 500 nm, between 10 nm and 250 nm, between 15 nm and 200 nm, preferably between 20 nm and 200 nm, such as between 20 nm and 100 nm. The average size is measured by means of Dynamic Light Scattering (DLS), and the average size represents the mediane size of the volume distribution of the lipid nanoparticles.
[0026] Advantageously, the lipid nanoparticles have a particle size distribution wherein the polydispersity index (PDI) is equal to or lower than 0.25, such as equal to or lower than 0.2, preferably equal to or lower than 0.15, more preferably equal to or lower than 0.1. The PDI is measured according to ISO 22412:2008(E).
[0027] Advantageously, the lipid nanoparticles have an average size between 10 nm and 250 nm, preferably between 20 nm and 200 nm, as measured by means of DLS, and a polydispersity index lower than 0.15, preferably lower than 0.1 , as measured according to ISO 22412:2008(E). [0028] Advantageously, the lipid nanoparticles comprise between 0.1 % by weight and 50 % by weight of an agent, such as between 0.25 % by weight and 30 % by weight, preferably between 0.5 % by weight and 25 % by weight of an agent. Preferably, the agent is mRNA, a steroid, a peptide, a polypeptide or a protein.
[0029] The present invention further discloses the use of lipid nanoparticles of the invention, as set out in the appended claims. The lipid nanoparticles are advantageously used in a drug delivery system. Advantageously, the lipid nanoparticles are used for the encapsulation, the protection and the delivery of mRNA inside cells. Examples of the use of LNPs for the encapsulation, protection and delivery of mRNA is for the treatment of diseases such as cancer, genetic disorders and infectious diseases, and for vaccines used to protect against viruses, such as the SARS-CoV-2 coronavirus causing the Covid-19 disease.
Brief description of the figures
[0030] Fig. 1 discloses a schematic representation of the method of the present invention.
[0031] Fig. 2 discloses a representation of the phases used in the invention, in particular the interfaces formed between the respective phases.
[0032] Fig. 3 discloses an example of the average size of LNPs of the invention as a function of the flow rate ratio defined as the total flow rate of the aqueous solutions divided by the flow rate of the liquid.
Detailed description of embodiments
[0033] In a first aspect, the present invention is related to a method to produce lipid nanoparticles (LNPs).
[0034] Referring to Fig. 1, the method comprises the step of providing a device 2 comprising input capillaries 3, a cavity 6 and an output capillary 7. The cavity also comprises an input tubing 8. The input capillaries 3 comprises two coaxial capillaries; a first, outer, coaxial capillary 4 and a second, inner, coaxial capillary 5. The input capillaries 3, and in particular the coaxial capillaries 4, 5, are adapted to receive a fluid. Preferably, the fluid is a liquid, such as, without being limited thereto, a liquid solution, an emulsion, or a dispersion. Advantageously, the output capillary 7 is coaxially aligned with the input capillary. The output capillary 7 is preferably positioned downstream of the input capillary 3. Advantageously, the input capillary 3 is positioned at a first end of the cavity 6 and the output capillary 7 at the other end of the cavity 6. The tubing 8 is also adapted to receive a fluid in a regulated manner. [0035] Still referring to Fig. 1 , and referring to Fig. 2, the method of the invention further comprises the step of providing a liquid 20 to the first, outer, coaxial capillary 4. The liquid 20 comprises a lipid and a solvent. According to a preferred embodiment, the liquid 20 comprises a lipid dissolved in a solvent. Advantageously, the solvent is at least partially miscible in water. With ‘at least partially miscible in water’ it is meant that, when performing the method, at least 50 %, such as at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, preferably at least 90 %, more preferably at least 95 %, at least 97.5 %, at least 99 % of the solvent is miscible in water, expressed in percentage of the total volume of the solvent.
[0036] The solvent can comprise one solvent or a mixture of two or more solvents. The solvent can comprise an organic solvent, an inorganic solvent, of a combination of two or more thereof. Advantageously, the solvent is an organic solvent. Advantageously, the solvent is a non-aqueous solvent. With a non-aqueous solvent is meant in the light of the present invention a solvent comprising at most 10 % by weight of water, on the total weight of the solvent, preferably at most 5 % by weight of water, even more preferably at most 1 % by weight of water.
[0037] The solvent can comprise an alcohol or a mixture of two or more alcohols. Alternatively or additionally, the solvent can comprise acetone. The alcohol is advantageously alcohol that is at least partially miscible in water. Preferably, the alcohol is a C1-4-alcohol, meaning an alcohol comprising between 1 and 4 carbon atoms, such as methanol, ethanol, n-propanol, isopropanol (2-propanol), n-butanol, or 2-methyl-1- propanol (isobutanol).
[0038] The lipid can be a triglyceride, a phospholipid, a fatty acid, a fatty alcohol, a fatty ester or polyethylene glycol (PEG), or a combination of two or more thereof. The lipid can be ionizable. According to a preferred embodiment, the lipid is a combination of an ionizable lipid and a PEG lipid. According to a preferred embodiment, the lipid comprises a phospholipid, for example, the lipid can consist of a phospholipid. [0039] Advantageously, the liquid 20 comprises between 0.1 % by weight and 10 % by weight of lipids, based on the total weight of the liquid 20, such as between 0.2 % by weight and 9 % by weight, between 0.25 % by weight and 8 % by weight, between 0.5 % by weight and 7.5 % by weight, between 0.75 % by weight and 7 % by weight, preferably between 0.8 % by weight and 6 % by weight, such as between 0.9 % by weight and 5.5 % by weight, more preferably between 1 % by weight and 5 % by weight.
[0040] The liquid can further comprise one or more additives. An example of an additive, without being limited thereto, is a surfactant, for example dimethyldioctadecylammonium bromide (DDAB). DDAB is advantageously used to stabilize the lipid nanoparticles produced with the method of the invention.
[0041] Still referring to Fig. 1 , and referring to Fig. 2, the method of the invention further comprises the step of providing a first aqueous solution 21 to the second, inner, coaxial capillary 5. Advantageously, by providing the liquid 20 to the first, outer, coaxial capillary 4 and the first aqueous solution 21 to the second, inner, coaxial capillary 5, a liquid flow surrounding a first aqueous flow (21-in-20) is formed.
[0042] Advantageously, the first aqueous solution comprises at least 25 % by volume of water, such as at least 30 %, 40 %, 50 %, 60 %, 70 %, 75 %, 80 %, 90 % or 95 % by volume of water. According to a preferred embodiment, the first aqueous solution is a phosphate-buffered saline (PBS) solution. The PBS solution can by any PBS solution known in the state of the art, in particular PBS solutions suitable for use in biological and biotechnological applications. Preferably, the PBS solution comprises water and sodium chloride, and has a pH value between 6.5 and 8, for example between 7 and 7.5, such as around 7.2.
[0043] Still referring to Fig. 1 , and referring to Fig. 2, the method of the invention further comprises the step of providing a second aqueous solution 22 to the cavity 6. Advantageously, by providing the second, aqueous solution 22 to the cavity 6, the liquid 20 to the first, outer, coaxial capillary 4, and the first aqueous solution 21 to the second, inner, coaxial capillary 5, a second aqueous flow surrounding a liquid flow surrounding a first aqueous flow 23 (21-in-20-in-22) is formed.
[0044] Advantageously, the second aqueous solution comprises at least 25
% by volume of water, such as at least 30 %, 40 %, 50 %, 60 %, 70 %, 75 %, 80 %, 90 % or 95 % by volume of water. According to a preferred embodiment, the second aqueous solution is a phosphate-buffered saline (PBS) solution. The PBS solution is as described above.
[0045] The first aqueous solution and the second aqueous solution can have the same composition, such as the same amount of water. Alternatively, the composition of the first and the second aqueous solution is different.
[0046] The liquid 20 can further comprise an agent. The agent can be an active agent, preferably a therapeutically active agent.
[0047] Examples of a suitable therapeutically active agent are, without being limited thereto, a peptide, a polypeptide, a protein, cyclosporine, steroids, cannabidiol (CBD), tetrahydrocannabinol (THC), rapamycin, or antibiotics. Preferably, the therapeutically active agent is a peptide, a polypeptide or a protein. With peptide it is meant in the light of the present invention a molecule comprising a backbone of 2 to 20 amino acid residues. With polypeptide it is meant in the light of the present invention a molecule comprising a backbone of more than 20 amino acid residues. Examples of suitable peptides or polypeptides are, without being limited thereto, thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone (LHRH), oxytocin, insulin, corticotropin (ACTH).
[0048] Alternatively or additionally, the first (21) and/or second (22) aqueous solution can comprise an agent. The agent can be, without being limited thereto, mRNA, siRNA or DNA, or an active agent, such as a therapeutically active agent.
[0049] Still referring to Fig. 1, the solvent comprised in the liquid 20 will advantageously diffuse from the liquid 20 to the first aqueous solution 21 and to the second aqueous solution 22. Upon diffusion of the solvent from the liquid 20, a phase separation of the lipid comprised in the liquid 20 will advantageously take place, wherein lipid nanoparticles 1 (LNPs) are formed. When an agent is present, the agent will be encapsulated in the lipid nanoparticles. The lipid nanoparticles comprising an encapsulated agent, can protect the agent against certain environments. When the lipid nanoparticles are used in a drug delivery system, they can deliver the agent at the targeted location, such as the target cells, by protecting it against non-favorable environments prior to arriving at the targeted location.
[0050] Referring back to Fig. 2, the liquid 20 advantageously flows in the cavity at a flow rate between 0.1 mI/min and 1000 mI/min, such as between 0.5 mI/min and 750 mI/min, preferably between 1 mI/min and 500 mI/min, for example between 2.5 mI/min and 400 mI/min, more preferably between 5 mI/min and 300 mI/min, such as between 10 mI/min and 250 mI/min, between 20 mI/min and 200 mI/min, between 25 mI/min and 175 mI/min, or between 50 mI/min and 150 mI/min, for a device having an inner input capillary having a diameter of 30 pm, an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm. It is to be noted that for devices having input capillaries and an output capillary having larger dimensions, such as an inner input capillary having a diameter of 90 pm, an outer input capillary having a diameter of 160 pm and an output capillary having a diameter of 450 pm, the flow rate of the liquid can be higher.
[0051] The first aqueous solution 21 advantageously flows in the cavity at a flow rate between 1 mI/min and 1000 mI/min, such as between 5 mI/min and 750 mI/min, preferably between 10 mI/min and 500 mI/min, between 20 mI/min and 400 mI/min, between 30 mI/min and 300 mI/min, between 40 mI/min and 250 mI/min, more preferably between 50 mI/min and 200 mI/min, such as between 75 mI/min and 150 mI/min, for a device having an inner input capillary having a diameter of 30 p , an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm. It is to be noted that for devices having input capillaries and an output capillary having larger dimensions, such as an inner input capillary having a diameter of 90 pm, an outer input capillary having a diameter of 160 pm and an output capillary having a diameter of 450 pm, the flow rate of the first aqueous solution can be higher.
[0052] The second aqueous solution 22 advantageously flows in the cavity at a flow rate between 5 pl/min and 1000 pl/min, such as between 10 pl/min and 900 pl/min, between 20 pl/min and 800 pl/min, between 30 pl/min and 750 pl/min, preferably between 40 pl/min and 700 pl/min, for example between 50 pl/min and 600 pl/min, between 75 pl/min and 550 pl/min, more preferably between 100 pl/min and 500 pl/min, such as between 125 pl/min and 450 pl/min, between 150 pl/min and 400 pl/min, of between 175 pl/min and 350 pl/min, for a device having an inner input capillary having a diameter of 30 pm, an outer input capillary having a diameter of 70 pm and an output capillary having a diameter of 160 pm. It is to be noted that for devices having input capillaries and an output capillary having larger dimensions, such as an inner input capillary having a diameter of 90 pm, an outer input capillary having a diameter of 160 pm and an output capillary having a diameter of 450 pm, the flow rate of the second aqueous solution can be higher.
[0053] The flow rate of the liquid and the flow rate of the first and second aqueous solution together is the so-called total flow rate (TFR). The total flow rate at which a process can take place without impacting the products obtained, in this case LNPs, is one of the measures for the scalability of the process. A higher the total flow rate means a higher throughput, and thus a process that is suited for production at industrial scale. With the method of the invention, it is possible to obtain a total flow rate of at least 100 pl/min, such as at least 200 pl/min, at least 250 pl/min, at least 300 pl/min, at least 400 pl/min, at least 500 pl/min, such as even 1 ml/min.
[0054] The ratio of the flow rate of the first and second aqueous solutions
(thus the sum of the flow rate of the first aqueous solution and the flow rate of the second aqueous solution) and the flow rate of the liquid is the so-called flow rate ratio (FRR). The flow rate ratio at which the production of LNPs takes place is an indication for the concentration of LNPs in the final solution. In the light of the present invention, the final solution is the combination of the first aqueous solution, the second aqueous solution, and the solvent diffused into the first and second aqueous solutions. A higher flow rate ratio indicates for a given flow rate of the liquid a larger flow rate of the first and second aqueous solutions, and thus relatively speaking a smaller amount of lipids, and a lower concentration of LNPs in the final solution. In other words, a higher flow rate ratio means a higher dilution of the produced LNPs in the final solution. For almost all applications of the LNPs, a low dilution, or a high concentration, of LNPs in the final solution is required. When the dilution is high, a subsequent step may be required to decrease the dilution, i.e. increase the concentration, of the LNPs in the final solution.
[0055] The flow rate ratio is also a measure for the average size of the
LNPs. A higher FRR typically allows to obtain smaller LNPs. The FRR is also a measure for the polydispersity of the produced LNPs. The polydispersity is related to the particle size distribution (PSD) of the produced LNPs, and is expressed by the polydispersity index (PDI). A PDI below 0.1 , and in particular below 0.05, indicates a rather monodisperse particle size distribution, while a PDI higher than 0.1 , and in particular a PDI equal to or higher than 0.7, indicates a very polydisperse particle size distribution. For most applications of LNPs, a monodisperse PSD is preferably used. A higher FRR typically indicates a more polydisperse PSD, thus a higher PDI.
[0056] For most applications of LNPs, a FRR of at most 100, even at most
50, is required to obtain a sufficiently low polydispersity and sufficiently small LNPs. With the method of the invention, it is possible to obtain a flow rate ratio of at most 50, preferably at most 20, such as between 0.5 and 50, for example between 1 and 25, or between 2 and 20, resulting in a polydispersity index lower than 0.1 and an average particle size of the LNPs between 20 nm and 200 nm.
[0057] One of the problems with the methods of the state of the art is that they have a low total flow rate (at most 100 mI/min) for a flow rate ratio below 100, limiting the scalability to industrial processes, or that they show good total flow rates, but very high flow rate ratios, such as FRRs of more than 100, even more than 200, 500, up to 5000 and more, resulting in the addition of a concentration step to the method, such as a filtration step, rendering the process more complex.
[0058] The method of the present invention solves these problems, and can be used at a total flow rate of at least 100 mI/min, such as a total flow rate of at least 300 mI/min, in combination with a flow rate ratio between 0.5 and 50, such as between 1 and 25, or between 2 and 20.
[0059] The inventors have found that by using the method of the present invention, the surface of the interface between the liquid and an aqueous solution - in the present invention the first aqueous solution and the second aqueous solution - is increased, which allows improved control of the process, even at industrial scale. In particular, the increase of the surface of the liquid - aqueous solution interface allows more controlled flow, i.e. a more stable flow, and a less turbulent or less chaotic flow, of both the liquid and the aqueous solutions, and a more controlled diffusion of the solvent into the aqueous solutions. The increase in process control leads to a low flow rate ratio, resulting in a good polydispersity (PDI < 0.1), LNPs having small sizes (< 200 nm), and a low dilution of the LNPs in the final solution. The increase in process control further allows for an increase in the total flow rate of the process.
[0060] The present invention is further related to lipid nanoparticles obtained or obtainable by means of the method of the invention. Advantageously, the lipid nanoparticles have an average size between 1 nm and 1000 nm, such as between 2 nm and 500 nm, between 5 nm and 400 nm, between 10 nm and 300 nm, between 15 nm and 250 nm, between 20 nm and 200 nm, or between 25 nm and 150 nm, for example between 50 nm and 100 nm. Preferably, the LNPs have an average size between 10 nm and 500 nm, more preferably between 20 nm and 200 nm.
[0061] The size of the LNPs is measured by Dynamic Light Scattering
(DLS). The measured diameter of the lipid nanoparticles, referred to as the average size of the LNPs, is the mediane size of the volume distribution of the lipid nanoparticles. [0062] Advantageously, the lipid nanoparticles of the invention have a polydispersity index (PDI) equal to or lower than 0.25, for example equal to or lower than 0.2, equal to or lower than 0.15, preferably equal to or lower than 0.1 , such as equal to or lower than 0.09, equal to or lower than 0.08, equal to or lower than 0.075, equal to or lower than 0.07, equal to or lower than 0.06, or equal to or lower than 0.05. The polydispersity index is measured according to the test standard ISO 22412:2008(E). [0063] Advantageously, the lipid nanoparticles of the invention comprise an agent. The agent can be an agent as described above. Preferably, the agent is mRNA, a steroid, a peptide or a polypeptide. Advantageously, the lipid nanoparticles comprise between 0.05 % and 50 % by weight of the agent, based on the total weight of the lipid nanoparticle, such as between 0.075 % and 45 % by weight, between 0.1 % and 40 % by weight, between 0.2 % and 35 % by weight, between 0.25 % and 30 % by weight, such as between 0.5 % and 25 % by weight, between 0.75 % and 20 % by weight, between 1 % and 15 % by weight, or between 2 % and 10 % by weight.
[0064] The invention is further related to the use of the lipid nanoparticles of the invention. The LNPs can be used in a drug delivery system. When the lipid nanoparticles comprise an agent, i.e. an encapsulated agent, they can protect the agent against certain environments. When the lipid nanoparticles comprising an agent are used in a drug delivery system, they can deliver the agent at a targeted location, such as target cells, by protecting it against non-favorable environments prior to arriving at the targeted location.
[0065] The lipid nanoparticles can also be used for the encapsulation, protection and delivery of mRNA inside cells. The LNPs can be used in a non-viral gene delivery system, for example in a mRNA delivery system. Examples of the use of LNPs for the encapsulation, protection and delivery of mRNA is for the treatment of diseases such as cancer, genetic disorders and infectious diseases, and for vaccines used to protect against viruses, such as the SARS-CoV-2 coronavirus causing the Covid19 disease.
Examples
Example 1 (comparative)
[0066] Lipid nanoparticles according to the prior art were produced by using phospholipids dissolved in ethanol as the liquid. The first aqueous solution was a phosphate buffered saline (PBS) comprising water and sodium chloride and having a pH value of 7.2. The PBS solution was provided to the cavity of a single emulsion device, and the liquid was provided to the cavity through an input capillary, wherein an aqueous flow surrounding a liquid flow was obtained. The ethanol diffused into the PBS solution surrounding the liquid via the ethanol-PBS interface, and lipid nanoparticles were obtained. The total flow rate was 100 mI/min. The flow rate ratio (FRR), here defined as the flow rate of the aqueous solution divided by the flow rate of the liquid, was varied between 2 and 49, and the average size of the lipid nanoparticles was measured by means of Dynamic Light Scattering, wherein the measured diameter (the average size) is the mediane size of the volume distribution of the lipid nanoparticles.
Example 2
[0067] Lipid nanoparticles according to the invention were produced by using lipids dissolved in ethanol as the liquid. The first aqueous solution and the second aqueous solution had the same composition, and were a phosphate buffered saline (PBS) comprising water and sodium chloride and having a pH value of 7.2. The liquid was provided to the cavity through a first, outer, coaxial capillary. The first PBS solution was provided to the cavity through a second, inner, coaxial capillary. The second PBS solution was provided to the cavity, wherein a second aqueous flow surrounding a liquid flow surrounding a first aqueous flow was obtained. The ethanol diffused into the first and second PBS solutions via the ethanol-PBS interface, and lipid nanoparticles were obtained. The total flow rate was 100 mI/min. The flow rate ratio (FRR), defined as the sum of the flow rate of the first and the second aqueous solution divided by the flow rate of the liquid, was varied between 2 and 32, and the average size of the lipid nanoparticles was measured by means of Dynamic Light Scattering, wherein the measured diameter (the average size) is the mediane size of the volume distribution of the lipid nanoparticles.
[0068] Fig. 3 represents the average size of the lipid nanoparticles (y-axis) as function of the FRR (x-axis) for the prior art lipid nanoparticles (Example 1 , showed as curve ‘SE’) and for the lipid nanoparticles of the invention (Example 2, showed as curve ΌE’). It is clear that for all FRRs, the average size of the LNPs of the invention is smaller than the average size of the prior art LNPs. In other words, a required average size of the LNPs can be obtained at a lower FRR with the method of the invention than with the prior art methods. This results, as explained above, in a lower polydispersity as well (lower PDI), and a lower dilution, or a higher concentration, of the LNPs in the final solution for the method of the invention compared to the prior art method. The method of the invention allows improved control of the process conditions, which, in combination with the increased surface of the liquid-aqueous solution interface, results in a reduction of the FRR for a given average size of the LNPs.
Example 3
[0069] Lipid nanoparticles were produced using the method of the invention. The liquid comprised lipids dissolved in ethanol. Phosphate buffered saline (PBS) was used as the first aqueous solution and as the second aqueous solution. The total flow rate, TFR, was 300 mI/min, and the flow rate ratio was varied between 2 and 20. The average size of the obtained lipid nanoparticles was measured by Dynamic Light Scattering. The polydispersity index (PDI) was measured according to ISO 22412:2008(E).
[0070] The obtained LNPs showed excellent properties: their average size varied between 20 nm and 200 nm and the polydispersity index (PDI) of the LNP size distribution was below 0.1, indicating a rather monodisperse particle size distribution for the LNPs. Equivalent experiments carried out with a single emulsion as in comparative example 1 showed a PDI that was about 0.15 higher.

Claims

1. Method for producing lipid nanoparticles (1) comprising the steps of:
- providing a device (2) comprising input capillaries (3) comprising two coaxial capillaries (4,5), a cavity (6) and an output capillary (7) in front of and coaxially aligned with the input capillaries (3);
- injecting a flow of a liquid (20) to a first, outer, coaxial capillary (4), wherein the liquid (20) comprises a lipid dissolved in a solvent, and wherein the solvent is at least partially miscible in water; - injecting a flow of a first aqueous solution (21) to a second, inner, coaxial capillary (5); wherein the flow of the liquid surrounding the first aqueous flow (21-in-20) is formed;
- injecting a flow of a second aqueous solution (22) to the cavity (6); wherein the second aqueous flow surrounding the flow of the liquid surrounding the first aqueous flow (23; 21-in-20-in-22) is formed in a stable way; and - diffusing the solvent from the liquid (20) to the first aqueous solution (21) and the second aqueous solution (22), thereby inducing phase separation of the lipid, wherein lipid nanoparticles (1) are formed.
2. Method according to claim 1 , wherein the solvent comprises an organic solvent.
3. Method according to any one of the preceding claims, wherein the solvent comprises an alcohol, preferably methanol, ethanol, n-propanol, isopropanol, or a mixture of two or more thereof.
4. Method according to any one of the preceding claims, wherein the lipid is a triglyceride, a phospholipid, a fatty acid, a fatty alcohol, a fatty ester, or a combination of two or more thereof.
5. Method according to any one of the preceding claims, wherein the liquid (20) comprises between 0.1 % by weight and 10 % by weight of lipids, preferably between 1
% by weight and 5 % by weight of lipids, based on the total mass of the liquid (20).
6. Method according to any one of the preceding claims, wherein the liquid (20) further comprises an agent, preferably a steroid, a peptide, a polypeptide or a protein.
7. Method according to any one of the preceding claims, wherein one or both of the first aqueous solution (21) and the second aqueous solution (22) comprises an agent, preferably mRNA.
8. Method according to any one of the preceding claims, wherein the first aqueous solution (21) comprises at least 50 v% of water, preferably at least 75 v% of water, more preferably at least 90 v% of water.
9. Method according to any one of the preceding claims, wherein the second aqueous solution (22) comprises at least 50 v% of water, preferably at least 75 v% of water, more preferably at least 90 v% of water.
10. Method according to any one of the preceding claims, wherein one or both of the first aqueous solution (21) and the second aqueous solution (22) is a phosphate- buffered saline (PBS).
11. Method according to any one of the preceding claims, wherein the ratio of the flow rate of the first and second aqueous solutions (21 , 22) and the flow rate of the liquid (20) (flow rate ratio (FRR)) is between 0.5 and 50, preferably between 2 and 20.
12. Lipid nanoparticles obtained by the method of any one of claims 1 to 11 , wherein the lipid nanoparticles (1) have an average size between 10 nm and 250 nm, preferably between 20 nm and 200 nm, as measured by Dynamic Light Scattering (DLS), and a polydispersity index of less than 0.15, preferably less than 0.1 , as measured according to ISO 22412:2008(E).
13. Lipid nanoparticles according to claim 12, comprising between 0.5 % by weight and 25 % by weight of an agent, preferably mRNA, a steroid, a peptide or a polypeptide, based on the total weight of the nanoparticles.
14. Use of lipid nanoparticles obtained by the method of any one of claims 1 to 11 in a drug delivery system.
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