How to mass-produce large LNPs

Abstract

The present invention relates to the field of lipid nanoparticles (LNPs), more specifically to a method for mass production of large LNPs. The method of the present invention produces LNPs characterized by a minimum average diameter of about 140 nm by a scalable method for rapid production of large quantities of LNPs. The present invention provides a scalable, large-scale, GMP-compliant method for producing large LNPs for delivery of active agents, more specifically for immunogenic delivery of nucleic acid molecules, specifically mRNA.

Description

[Technical field]

[0001] The present invention relates to the field of lipid nanoparticles (LNPs), more specifically to a method for mass production of large LNPs. The method of the present invention produces LNPs characterized by a minimum average diameter of about 140 nm by a scalable method for rapid production of large quantities of LNPs. The present invention provides a scalable, large-scale, GMP-compliant method for producing large LNPs for the delivery of active agents, more specifically for immunogenic delivery of nucleic acid molecules, specifically mRNA. [Background technology]

[0002] One of the major challenges in the field of targeted delivery of bioactive substances is often their instability and low cell penetration ability. This is specifically the case for the delivery of nucleic acid molecules, especially (m)RNA molecules. Therefore, suitable packaging of active agents such as nucleic acid molecules is crucial for proper protection and delivery. Therefore, there is a continuing need for methods and compositions for packaging bioactive substances such as nucleic acids. In this regard, lipid-based nanoparticle compositions such as lipoplexes and liposomes have been used as packaging vehicles for bioactive substances that allow their transport to cells and / or intracellular compartments.

[0003] Another type of packaging composition is lipid nanoparticles (LNPs). LNPs are typically composed of a mixture of four lipids: cationic or ionic lipids, phospholipids, sterols, and PEGylated lipids, and have been developed for non-immunogenic delivery of siRNA and mRNA to the liver after systemic administration. To induce optimal hepatocyte uptake and expression, these LNPs typically exhibit a small size of 70 nm to 100 nm (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3).

[0004] LNPs have also been used for immunogenic delivery of antigen-encoding mRNA to muscle or dermis (Non-Patent Document 4, Non-Patent Document 5). Again, small size (80 nm to 120 nm) LNPs are typically used, as such small size has been shown to be crucial for immunogenicity. Furthermore, small LNPs can efficiently reach draining lymph nodes upon injection, whereas larger LNPs are more frequently retained at the injection site (see Non-Patent Document 6).

[0005] Recently, in contrast to prevailing belief, it has been found that small particles are beneficial for LNP-mediated delivery, but nanoparticles with diameters greater than 140 nm lead to enhanced delivery of active agents (e.g., mRNA) to the spleen, especially after intravenous administration, and thus there is interest in methods to produce large LNPs for this purpose.

[0006] Although the prior art describes methods for producing these large (>100 nm) LNPs, these LNPs have been identified as having poorer phenotypes, such as a lower efficacy in successfully encapsulating nucleic acids as their smaller counterparts (Non-Patent Document 7 and Non-Patent Document 8). Also, mass production of large LNPs in a GMP-compliant manner remains a challenge. The prior art lacks methods for consistently producing large batches of potent large LNPs with high encapsulation rates. These large LNPs are particularly beneficial for the delivery of active agents such as nucleic acids to the spleen, and thus a method for consistently producing LNPs with an average diameter of at least 140 nm on a pharmacologic scale would be beneficial. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Li et al., Nanoletters 2015 [Non-Patent Document 2] Thess et al. Mol ther 2015 [Non-Patent Document 3] Kauffman et al. Biomaterials 2016 [Non-Patent Document 4] Richner et al. Cell 2017 [Non-Patent Document 5] Liang et al., Mol Ther 2017 [Non-Patent Document 6] Reichmuth et al., 2016 [Non-Patent Document 7] MacLachlan, I., 2007 [Non-Patent Document 8] Chen, Sam, et al. 2016 Summary of the Invention

[0008] In a first aspect, the present invention provides a method for mass producing a composition comprising an LNP according to the present invention, comprising the steps of: a) mixing a first composition comprising ionic lipids, phospholipids, sterols and PEG lipids in an organic solvent with a second composition comprising one or more active agents (such as nucleic acids) in an acidic buffer, thereby obtaining a bulk after mixing; b) subjecting the mixed bulk of step a) to tangential flow filtration (TFF); c) subjecting said composition of step b) to a filtration step in an aqueous solution having a pH of <6.5; d) adding a cryobuffer containing a cryoprotectant to the composition of step c); e) subjecting said composition of step d) to a filtration step in an aqueous solution having a pH>6.5; f) sterile filtering the composition of step e), thereby obtaining a composition of lipid nanoparticles having an average diameter of at least 140 nm and comprising the active agent; wherein the TFF in step b) is carried out at a shear rate of less than 4000 / s, and wherein the LNPs comprise less than 1 mol % PEGylated lipid.

[0009] In a particular embodiment of step a), the mixing flow rate of said first composition is lower than the mixing flow rate of said second composition.

[0010] In another specific embodiment of step a), the ratio of the mixing flow rates of the first composition and the second composition is about 1:2.

[0011] In another specific embodiment, said tangential flow filtration of step b) comprises a first ultrafiltration step carried out at a low shear rate compared to the subsequent tangential flow filtration step(s).

[0012] In another specific embodiment, said tangential flow filtration of step b) comprises a first ultrafiltration step carried out at a shear rate of less than 3000 / s, while a subsequent tangential flow filtration step is carried out at a shear rate of less than 4000 / s.

[0013] In another specific embodiment, said organic solvent is chloroform or an alcohol such as methanol or ethanol, in particular ethanol.

[0014] In another particular embodiment, said cryoprotectant is selected from the list comprising maltose, maltodextrin, dextran, mannitol, glucose, trehalose or sucrose, in particular trehalose or sucrose.

[0015] In yet a further embodiment, said active agent is selected from the list comprising small molecules and biomolecules, in particular antibodies, peptides, proteins and / or nucleic acids, more particularly mRNA molecules or DNA molecules.

[0016] In another specific embodiment, the cryobuffer comprises a cryoprotectant and one or more of Tris, citrate or PBS, in particular TRIS and / or PBS.

[0017] In another specific embodiment, said first composition in step a) is at a temperature between 30°C and 45°C.

[0018] In another specific embodiment, said composition of step a) is diluted before step b) and / or said composition of step e) is diluted before step f).

[0019] In another specific embodiment, said filtration in steps c) and / or e) has a filter size / cut-off of 0.20 μm.

[0020] In yet a further embodiment, said aqueous solution of step c) and / or e) is water, in particular water for injection.

[0021] In another specific embodiment, said filtration of steps c) and / or e) is a clarification filtration.

[0022] With specific reference now to the drawings, it is emphasized that the details shown are by way of example only and are for the purpose of illustratively illustrating various embodiments of the invention. These drawings are presented to provide what is believed to be the most useful and facile explanation of the principles and conceptual aspects of the invention. In this regard, no attempt has been made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. This description, taken together with the drawings, will make apparent to those skilled in the art how some forms of the invention may be actually embodied. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 shows size distributions obtained from transmission electron microscopy analysis of samples from Table 1 of Example 1. The average particle size ranges from 150 nm to 200 nm in diameter. [Diagram 2] FIG. 1 shows size distributions obtained from transmission electron microscopy analysis of samples in Table 2 of Example 2. The average particle size ranges from 150 nm to 200 nm in diameter. [Diagram 3]FIG. 13 shows the size distribution after TFF in water (average size: 141 nm diameter, PDI: 0.49). [Figure 4] FIG. 1 shows the size distribution after filtration in water (average size: 92.5 nm, PDI: 0.45). [Diagram 5] FIG. 1 shows the size distribution of LNPs after sterile filtration in cryobuffer (average size: 142 nm, PDI: 0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] As already detailed herein above, the present invention provides a scalable and GMP-compliant method for producing lipid nanoparticles (LNPs) with a larger diameter than is generally used in the art. These LNPs have been found to be highly suitable for the delivery of active agents, more particularly for the immunogenic delivery of nucleic acids, particularly mRNA, leading to enhanced delivery to the spleen after administration (e.g., intravenous administration) to a subject in need thereof. "Immunogenic delivery of nucleic acid molecules" refers to the delivery of nucleic acid molecules to cells, where contact with, internalization and / or expression of said nucleic acid molecules within the cells results in the induction of an immune response. This large-scale production is achieved according to the methods specified herein, in particular the surprisingly low shear rate of tangential flow filtration (TFF) and the surprisingly low proportion of PEGylated lipids used in the methods.

[0025] Thus, in a first aspect, the present invention provides a method for mass producing LNPs comprising an ionic lipid, a phospholipid, a sterol, a PEG lipid and one or more active agents, wherein the LNPs have an average diameter of about 140 nm or greater.

[0026] In particular, the present invention provides a GMP-compliant method for mass production of large LNPs, comprising: a) mixing a first composition comprising an ionic lipid, a phospholipid, a sterol and a PEG lipid in an organic solvent with a second composition comprising one or more active agents in an acidic buffer, thereby obtaining a bulk after mixing; b) subjecting the mixed bulk of step a) to tangential flow filtration (TFF); c) subjecting said composition of step b) to a filtration step in an aqueous solution having a pH of <6.5; d) adding a cryobuffer containing a cryoprotectant to the composition of step c); e) subjecting the composition of step d) to a filtration step in an aqueous solution having a pH of >6.5; f) sterile filtering the composition of step e), thereby obtaining a composition of lipid nanoparticles comprising the active agent and having an average diameter of at least 140 nm; wherein the TFF of step b) is carried out at a shear rate of less than 4000 / s, and wherein the first composition of step a) contains a low proportion (less than 1%) of PEGylated lipid.

[0027] Lipid nanoparticles (LNPs) are commonly known as nano-sized particles composed of a combination of different lipids. Although many different types of lipids can be included in such LNPs, the LNPs produced by the methods of the present invention are typically composed of a combination of ionic lipids, phospholipids, sterols, and PEG lipids.

[0028] As used herein, the term "nanoparticle" refers to any particle having a diameter that makes the particle suitable for systemic administration, particularly intravenous administration, of an active agent, particularly a nucleic acid, and typically has a diameter of less than 1000 nanometers (nm).

[0029] In some embodiments, the LNPs of the present invention have an average diameter of less than 600 nm. In some embodiments, the LNPs of the present invention have an average diameter of less than 400 nm, but in any case, the nanoparticles of the present invention have an average diameter of more than about 140 nm. In specific embodiments, the LNPs of the present invention have an average diameter of about 140 nm or more, about 150 nm or more, about 160 nm or more, about 170 nm or more, about 180 nm or more, about 190 nm or more, or about 200 nm or more. For clarity, when a mixture of LNPs is used, the average diameter referred to refers to the average diameter of the LNPs. In this example, for example, the mixture may contain some LNPs with an average diameter smaller than 140 nm, while the remaining LNPs have an average diameter larger than 140 nm, so long as the combined average diameter of all LNPs is at least 140 nm. As used herein, the term "diameter" always means "average diameter", even if not specifically specified. As used herein, the term "large LNP" always refers to LNPs with a diameter larger than about 140 nm.

[0030] The particle size and polydispersity index of LNPs can be measured using a Malvern Zetasizer dynamic light scattering (DLS) instrument (Malvern Panalytical Ltd., UK). The particle size values ​​presented in this application represent the Z-average diameter of the particle size distribution. Since DLS is a diffusion-based technique that converts the distribution of diffusion coefficients into a particle size distribution, the viscosity of the medium containing the LNP-mRNA plays an important role in obtaining an accurate average diameter.

[0031] The samples are allowed to equilibrate for 120 seconds at 23° C. and measurements based on the LNP-mRNA are performed as follows: before the addition of cryobuffer, the LNP-mRNA sample is in water and the particle size is measured using the viscosity value of water at 23° C. After the addition of cryobuffer, the measured viscosity value taking into account the presence of cryoprotectant at 23° C. is used. Thus, in certain embodiments, the LNPs of the present invention have an average diameter of at least 140 nm, measured using dynamic light scattering (DLS) taking into account the viscosity value of the buffer in which the LNPs are present at 23° C. Alternatively, the size distribution of the LNPs can be determined using other suitable techniques, for example by transmission electron microscopy (TEM) as detailed in Example 2.

[0032] The method of the invention involves several steps to produce LNPs having an average diameter of greater than about 140 nm, and the method is scalable and can be used to produce such LNPs in industrially significant quantities.

[0033] In the context of the present invention, the term "large scale production" refers to production on an industrial scale, in particular the production of large batches of LNPs containing a total payload of at least 250 mg, in particular at least 500 mg, e.g., about 1 g, about 2 g or more of active agent (e.g., mRNA).

[0034] In the context of the present invention, the term "ionic" (or alternatively cationic) in relation to a compound or lipid refers to the presence of ions (usually H + This refers to the presence of any uncharged group in the compound or lipid that can dissociate by gaining an electron (ion) and becoming positively charged itself. Alternatively, any uncharged group in the compound or lipid can gain an electron and become negatively charged. In many cases, the positive charge is the result of the presence of a quaternary nitrogen atom.

[0035] In the context of the present invention, any type of ionizable lipid may be suitably used.

[0036] In the context of the present invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of a phosphate group. These two components are most often linked by a glycerol molecule, so that the phospholipids of the present invention are preferably glycerol-phospholipids. Furthermore, the phosphate group is often modified with simple organic molecules, for example with choline (i.e., to give phosphocholine) or ethanolamine (i.e., to give phosphoethanolamine).

[0037] Suitable phospholipids in the context of the present invention are 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 5-amino-2-phenylpropionyl-sn-glycero-3-phosphocholine ( ... 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

[0038] In the context of the present invention, any suitable sterol may be used, such as a sterol selected from the list comprising cholesterol, ergosterol, campesterol, oxysterol, anthrosterol, desmosterol, nicasterol, sitosterol and stigmasterol, preferably cholesterol.

[0039] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. In a particular embodiment, the PEG lipid is selected from the list comprising PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. More specific examples of such PEG lipids include C14-PEG2000 (1,2-dimyristoyl-rac-glycerol, methoxypolyethyleneglycol-2000 (DMG-PEG2000)) and C18-PEG5000 (1,2-distearoyl-rac-glycerol, methoxypolyethyleneglycol-5000 (DSG-PEG5000)).

[0040] As used herein, the first composition further comprises organic solvent apart from various lipids as defined herein.Such organic solvent is required to keep the lipids contained therein in solution.The organic solvent that can be suitably used in the context of the present invention is, for example, chloroform or alcohol, for example methanol or ethanol, in particular ethanol.

[0041] Moreover, in a further specific embodiment, the first composition is preferably maintained at a slightly elevated temperature, i.e., above room temperature but below the boiling point. Such an elevated temperature can be, for example, between 30° C. and 60° C., for example between 30° C. and 50° C., more specifically between 30° C. and 45° C. This slightly elevated temperature improves the solubility of the lipids dissolved therein, thereby increasing the efficiency of the mixing process, which has been found to be advantageous in the LNP production process.

[0042] In the context of the present invention, the term tangential flow filtration (TFF) refers to a method for separating and purifying biomolecules known in the art. A TFF system typically comprises a filter device, such as a capsule, cassette and holder, or a hollow fiber module, a pump, a tube, a valve or clamp, and a fluid reservoir, and optionally a pressure gauge. Such a TFF system can be used continuously even with high concentrations of active agents (e.g., nucleic acids) without clogging the pores of the filter membrane. The use of a TFF system is advantageous because it allows high-throughput purification of the LNPs to be purified.

[0043] TFF specifically refers to the diffusion of liquid through the filter device, where the feed stream flows parallel to the membrane surface. Pressure is applied to the feed reservoir to force the stream along the filter, and larger particles cannot pass through the filter. To prevent these particles from blocking the filter, they are recirculated back to the feed reservoir. TFF is performed at a specific shear rate, which is the rate at which the feed is applied to the filter. The main applications of TFF in LNP production are concentration and diafiltration (buffer exchange and desalting).

[0044] The filter membrane suitable for use in LNP purification can be any type of material suitable for the above purpose and therefore does not interact with the LNP to be purified. Examples of filter membrane materials include modified or unmodified polyethersulfone (mPES or PES), polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, mixed cellulose esters (ME), ultra-high molecular weight polyethylene (UPE), polyfluorotetraethylene (PTFE), nylon, polysulfone (PS), polyacrylonitrile, polypropylene, polyvinyl chloride, polyvinylidene difluoride (PVDF), and combinations thereof. Filter membranes are characterized by their molecular weight cut-off (MWCO) value, which refers to the minimum molecular weight (in Daltons) of particles that are 90% retained by the filter. Filter membranes preferably have a pore size suitable for retaining LNPs while allowing components smaller in size than the MWCO and therefore the pore size to pass through the filter membrane as filtrate.

[0045] In some embodiments of the invention, a filter membrane is used in TFF having a molecular weight cutoff of 100 kDa to 500 kDa, preferably 100 kDa to 300 kDa, more preferably 100 kDa to 200 kDa, and most preferably about 100 kDa. The filter membrane used in TFF is important because the pore size of the filter membrane determines the size of particles and components, such as LNPs, that can pass through the filter membrane and thus are contained in the filtrate.

[0046] The process using TFF can be further characterized by considering various variables, the transmembrane pressure and flow rate / shear rate being the two most important variables.

[0047] "Transmembrane pressure" (TMP) refers to the driving force that drives components through a filter membrane. In some embodiments, the transmembrane pressure in a TFF process is between 100 mbar and 700 mbar, preferably between 100 mbar and 500 mbar, more preferably between 200 mbar and 400 mbar, or alternatively, the pressure can be expressed as psi, such as in the range of about 2 psi to about 8 psi.

[0048] The term "flow" refers to the volume of solution flowing through the TFF system, particularly in the filter membrane area, and "flow rate" or "shear rate" refers to the volume of solution flowing through the system in a given time. This term refers to the speed required to recirculate the mRNA molecules through the TFF system and to wash the LNPs off the filter surface. Thus, flow rate / shear rate or crossflow rate refers to the speed of solution flow through the filter membrane. In some embodiments, the shear rate in the TFF steps of the present invention is typically less than 4000 / s. In certain embodiments, the first concentration step is performed at a shear rate of about 2800 / s, which corresponds to about 1300 mL / min, while the diafiltration and second ultrafiltration are performed at a shear rate of about 3800 / s, which corresponds to about 1400 ml / min.

[0049] Prior art methods for producing LNPs use higher shear rates than those outlined in the methods of the present invention.

[0050] Shear rate is a measure of the speed at which flow is applied to the TFF filter and is typically greater than 5000 / s in the prior art but less than 4000 / s in the process of the present invention.

[0051] In a particularly preferred embodiment of the present invention, the method for purifying LNPs is carried out using continuous TFF. Continuous TFF refers to a TFF system in which the retentate is used as a feed for another round of TFF. In this specification, the term "feed" refers to a solution or suspension containing LNPs to be purified. Thus, an initial feed containing LNPs is subjected to a first round of filtration using TFF, and the resulting retentate containing LNPs is again used as a feed for another round of TFF by circulating at least LNPs. This is advantageous for improving the purification level by repeating the purification steps in an automated manner, while reducing the risk of losing LNPs due to transfer between systems.

[0052] For this reason, a tangential flow filtration system as used in the present invention may include multiple filtration steps, each of which may be performed at a different flow rate. In particular, a first ultrafiltration step performed at a lower flow rate than the subsequent filtration steps has been found to be advantageous in the context of the present invention. Thus, the present invention provides a method in which the tangential flow filtration in step b) includes a first ultrafiltration step performed at a shear rate of less than 3000 / s, while the other tangential flow filtration steps are preferably performed at a shear rate of less than 4000 / s. In a very specific embodiment, the first ultrafiltration step is performed at a shear rate of about 2800 / s, and the other remaining ultrafiltration steps are performed at a shear rate of about 3800 / s.

[0053] The shear rates used in the context of the present invention are low compared to those commonly used in the prior art. These lower shear rates have been found to be advantageous in processes that yield large particles.

[0054] In the context of the present invention, the term "clarification filtration step" is commonly used in GMP processes. This filtration step aims to recover a liquid composition that is as free as possible from solid impurities. The filters used herein are specifically designed to remove very small particles suspended in the liquid. To achieve this goal, several systems have been developed. For example, the filters can be designed with very closely spaced wires or fibers.

[0055] In particular, the first filtration step applied in the present invention in acidic aqueous conditions is intended to provide a purification in which particles that were not correctly formed during the TFF step, e.g. particles that are too large for further processing, are removed. The second filtration step in cryobuffer, also called the "clarification filtration step", is intended to reduce the so-called bioburden before storage.

[0056] In the context of the present invention, the term "sterile filtration" is also a process that is a filtration method commonly used in GMP processes. This filtration process aims to remove particulates and contaminants from a fluid. Typically, filtration with a pore size of 0.2 micrometers is considered necessary to produce a sterile filtrate.

[0057] In the context of the present invention, the term "cryobuffer" refers to a solution that does not solidify upon cooling and does not substantially impair the integrity and / or stability of the molecules contained therein. In particular, a cryobuffer of the present invention comprises a cryoprotectant and one or more of citrate, Tris and PBS, in particular Tris and / or PBS.

[0058] In the context of the present invention, the term "cryoprotectant" refers to a substance used to protect biological tissue from freezing damage. In a specific embodiment of the present invention, the cryoprotectant is a sugar, such as selected from the list comprising maltose, maltodextrin, dextran, mannitol, trehalose, glucose or sucrose, and may in particular be trehalose or sucrose.

[0059] In the context of the present invention, the term "mixing flow rate" refers to the rate at which a composition is added to a mixing compartment. In an advantageous embodiment of the present invention, the mixing flow rates of the first composition and the second composition are different from each other. More specifically, the mixing flow rate of the first composition is preferably low compared to the mixing flow rate of the second composition. In a very specific embodiment, the ratio of the mixing flow rate of the first composition compared to the mixing flow rate of the second composition is about 1:2, which means that the flow rate of the second composition is about twice the flow rate of the first composition. Alternatively, the ratio of the mixing flow rate of the first composition compared to the second composition is about 1:3, 1:4, 1:5, preferably about 1:2.

[0060] The goal of mixing is to achieve complete and rapid mixing of the multiple samples (i.e., lipid and nucleic acid phases) in the device. Such mixing is typically achieved by enhancing the diffusion effect between the streams of different species. There, several mixing devices can be used, for example, as outlined in Everest al., 2018.

[0061] The inventors have found that the method of the present invention is particularly suitable for mass production of LNPs with a diameter of more than about 140 nm.The method of the present invention is GMP-compliant.The method of the present invention is scalable.The methods described in the prior art can only develop large LNPs at a small laboratory scale and are not scalable.Therefore, the present invention provides a method for producing large LNPs in industrially significant quantities, and the LNPs are GMP-compliant.

[0062] "Active agent" as used in the context of the present invention refers to any component that can be suitably incorporated into an LNP, such as a small molecule or a biomolecule. The present invention is particularly suited to the incorporation of biomolecules such as antibodies, proteins, peptides, nucleic acids, and the like.

[0063] In the context of the present invention, "nucleic acid" refers to deoxyribonucleic acid (DNA) or preferably ribonucleic acid (RNA), more preferably mRNA. Nucleic acid, according to the composition of the method of the present invention, includes genomic DNA, cDNA, mRNA, recombinantly produced molecules and chemically synthesized molecules. The present invention can also be suitably used in the formulation of siRNA molecules, circular RNA / DNA, self-amplifying RNA, etc.

[0064] The nucleic acid, according to the method of the present invention, can be in the form of a molecule that is single-stranded or double-stranded, linear or covalently closed to form a circle. The nucleic acid can be used for introduction into cells, i.e. for transfection of cells, for example in the form of RNA, which can be produced by in vitro transcription from a DNA template. The RNA can further be modified before application by stabilizing sequences, capping, and / or polyadenylation.

[0065] For the avoidance of any doubt, the LNPs produced by the methods of the present invention may comprise a single active agent or may comprise multiple active agents, for example a combination of one or more active agents, for example a nucleic acid molecule encoding an immunomodulatory protein, and / or one or more nucleic acid molecules encoding an antigen-specific protein and / or a disease-specific protein.

[0066] As detailed herein above, the method of the present invention comprises multiple filtration steps that have been found to contribute significantly to the process of obtaining large nanoparticles. These steps can selectively enrich such large particles without compromising their stability and integrity. This is a process that has previously been considered unfeasible for the mass production of LNPs. During such a process, it may be advantageous to apply a dilution step with a suitable buffer / solution before or after one or more of the filtration steps used herein. Thus, in a further embodiment, the composition of step a) may be diluted before step b). In a further embodiment, the composition of step e) may be diluted. The dilution factor applied may depend on the concentration of the solution to be filtered and can be determined by the skilled person.

[0067] For example, for the first dilution of the mixed bulk, it has been found that a 6-fold dilution with water results in a stable mixed bulk in terms of particle size and encapsulation efficiency as a function of time.In particular, the mixed bulk in organic solvents and acidic buffers is slightly unstable and prone to aggregation.Therefore, the dilution step ensures that the bulk is stable for a longer period of time until TFF begins, for better storage / stability between different process steps, such as transport to cleanrooms and / or bulk handling.

[0068] For the second dilution, it may be desirable to dilute the product in the vial to a desired concentration, for example a concentration commonly used in the art, such as 100 μg / ml.

[0069] The larger LNPs produced by the method of the present invention can be used to achieve pharmaceutical compositions or vaccines for immunogenic delivery of one or more active agents (e.g., nucleic acid molecules) by their selective targeting to the spleen.However, the prior art methods for obtaining large LNPs are small-scale laboratory methods.

[0070] A surprising aspect of the method of the present invention that results in the large-scale production of large LNPs is the multiple filtration steps combined with the low shear rate used in the TFF step, in addition to the low percentage of PEGylated lipids in the first composition (less than 1%), as detailed in the Examples section. EXAMPLES

[0071] Example 1 - Method for producing large LNPs In this example, the method of the present invention was carried out to investigate the possibility of developing large LNPs.

[0072] A mixture of Coatsome SS / EC (ionic lipid), DOPE (phospholipid), cholesterol and DSG-PEG 2000 lipid (PEGylated lipid) was mixed in ethanol buffer to a defined molar ratio of 64:8:27:0.5, resulting in a total lipid concentration of 10.13 mg / mL. The mixture was then heated to 41° C. in a water bath.

[0073] An aqueous solution containing mRNA was prepared by diluting the nucleic acid stock with 200 mM sodium acetate to give a solution with an mRNA concentration of 0.126 mg / mL mRNA in 100 mM sodium acetate buffer at pH 4.0.

[0074] The mixture of lipids and mRNA from the above steps was mixed in a mixing device equipped with a pump. The lipid and mRNA compositions were pumped into the mixing device at 66.7 mL / min and 133.3 mL / min, respectively. The entire mixture was then diluted 6-fold with water for injection, and then subjected to buffer exchange and concentration by tangential flow filtration (TFF). This TFF consisted of three sub-steps: first, concentration (70-fold) achieved with a hollow fiber cartridge with an internal diameter of 0.5 mm and a cut-off of 100 kDa at a shear rate of 2800 / s; then, buffer exchange with four wash volumes of water for injection at a shear rate of 3800 / s; and the final step was concentration (2-fold) achieved with a hollow fiber cartridge with an internal diameter of 0.5 mm and a cut-off of 100 kDa at a shear rate of 3800 / s.

[0075] At this stage, the mixture was clarified through a Sartopore 2 (0.45+0.2 μm) sterile filter into water for injection and then diluted with 466 mM trehalose dihydrate, 18 mM Tris(hydroxymethyl)aminomethane (trometamol) buffer containing 137 mM sodium chloride to a pH of 7.4. At this point the formulation was developed.

[0076] The formulation was clarified again through a Sartopore 2 (0.45+0.2 μm) sterile filter in water for injection, then diluted with 466 mM trehalose dihydrate, 18 mM Tris(hydroxymethyl)aminomethane (trometamol) containing 137 mM sodium chloride, pH 7.4, to a concentration of 0.110 mg / mL mRNA. A final sterile filtration was performed through a Sartopore 2 (0.45+0.2 μm) sterile filter.

[0077] The process outlined above was repeated two more times. Particle size, mRNA content and encapsulation, lipid content, pH and osmolality were measured at different stages throughout the process. Analyses for three repeats can be seen in Table 1 (total mRNA batch 250 mg), Table 2 (total mRNA batch 250 mg) and Table 3 (total mRNA batch 100 mg).

[0078] [Table 1]

[0079] [Table 2]

[0080] [Table 3]

[0081] These tables confirm the reproducibility of the method of the invention and, moreover, they show that each of the different steps applied thereto contributes to a regular increase in the average particle size throughout the method, ultimately allowing the achievement of large batches of large nanoparticles.

[0082] Example 2 - LNP batch size validation Dynamic light scattering (DLS) was used to determine the LNP size in Example 1. In this example, a different technique was used to verify these particle sizes: transmission electron microscopy (TEM).

[0083] TEM was performed to visualize the ultrastructure and analyze the size of lipid nanoparticles. The samples evaluated were ENG batch 1 and ENG batch 2 (after freeze-thaw) as defined in Tables 1 and 2 of Example 1. All samples were kept at -80°C until grids were prepared for TEM analysis. All experiments were performed at room temperature (20°C-25°C). After thawing the samples, grids were directly prepared and evaluated in the TEM. Samples were diluted 10-fold in Tris buffer. A small drop of the diluted sample was placed on a 300 mesh carbon-coated copper grid (EM Resolutions, Sheffield, UK) immediately after glow discharge of the grid. A negative stain with 2% uranyl acetate was used to contrast the nanoparticles. After the grid was completely dried (± 30 min), the samples were visualized and evaluated with a FEI Tecnai Spirit G2 BioTwinTEM (ThermoFisher Scientific, Zaventem, Belgium) at 120 kV. At least 15 images were taken randomly across the grid.

[0084] Particle size evaluation was performed using FIJI open source free image analysis software. Particle segmentation was based on an intensity threshold and the area of ​​each selected particle was measured. Based on this area, the average diameter of each particle was calculated. The frequency distribution of particle diameters was plotted in excel. Diameters from 50 nm to 350 nm were plotted on a graph using intervals of 50 nm. The graph shows the percentage of particles belonging to a particular diameter range for each diameter range. Figures 1 and 2 define the size distributions obtained from TEM analysis for each of the LNPs in Tables 1 and 2 of Example 1. As is clear from these figures, the particle sizes determined using DLS (Example 1) are very similar to those determined by TEM, thereby confirming the validity of the DLS size determination method.

[0085] Example 3 - Effect of varying the conditions of the process of the present invention One of the main difficulties in producing large LNPs is their inherent instability, which is further amplified when using small amounts of PEG-lipids as contemplated in the present invention. Thus, standard methods for producing small LNPs are not easily transferable to large LNPs, and various process parameters have had to be fine-tuned, as will be seen by the experimental details below.

[0086] Specifically, the inventors have found that certain shear rates above 4000 / s are inappropriate in the production process as they result in precipitation of the product (see Table 4).

[0087] Table 4: Effect of changing shear rate during the TFF process [Table 4] * Value based on when the TFF process ends.

[0088] Two conclusions can be drawn from the data in Table 4: The shear rates used in other LNP products (4000 / s-6000 / s) are not suitable for the production of large LNPs. Addition of TBS and / or TBS containing cryoprotectants during the TFF process results in precipitation even at low shear rates.

[0089] Thus, it is clear from these data that higher shear rates are not suitable for the production of large LNPs, in contrast to the claimed shear rates below 4000 / s.

[0090] In addition, the inventors found that shear rate alone was not sufficient to successfully produce the desired LNPs. Specifically, further analysis showed that there were two distinct LNP populations in the LNP size distribution after the TFF step, with high PDI values ​​(see FIG. 3). Therefore, the inventors had to take further measures to obtain a population with the desired LNP particle size. Therefore, the next step of the applied method was filtration in water (pH less than 6.5). However, as is evident from Table 5 and FIG. 4, this filtration step itself significantly reduced the average LNP particle size to less than 100 nm. Only after further processing steps, including dilution with cryobuffer and sterile filtration, did the average particle size of the LNPs reach at least 140 nm again, this time with a very low PDI and a single size population (see Table 5 and FIG. 5).

[0091] Table 5: LNP size at different stages during the LNP production process [Table 5]

[0092] In conclusion, both the lower shear rate during TFF and an additional filtration step in water are crucial to obtain the desired quality attributes of large, low PEG-containing LNPs. The combination of TFF in water at low shear rate followed by filtration in water is the only way to obtain a consistent and robust large-scale manufacturing process.

[0093] References Reichmuthet al., 2016 - mRNA vaccine delivery using lipid nanoparticles - TherapeuticDelivery Vol. 7, N°5. Li et al.,2015 - An Orthogonal Array Optimization of Lipid-like Nanoparticles 5 for mRNADelivery in Vivo -Nano Letters 15(12) pg. 8099-8107. Thess etal., 2015 - Sequence-engineered mRNA without Chemical Nucleoside Modifications Enables anEffective Protein Therapy in Large Animals - Molecules Therapy Vol. 23, Issue 9 pg.1456-1464. Kauffmanet al., 2016 - Materials for non-viral intracellular delivery of messenger RNA therapeutics- Journal of Controlled Release, Vol. 240, pg. 227-234. MacLachlan,I. Liposomal formulations for nucleic acid delivery. Antisense Drug Technol.Princ. Strat. Appl. 2007, 2, 237-270. Richner etal., 2017 - Modified mRNA vaccines protect against Zika virus infection - Cell,Vol. 168, Issue6 pg. 1114-1125 Liang etal., 2017 - Efficient Targeting and Activation of Antigen-Presenting Cells InVivo after 15Modified mRNA Vaccine Administration in Rhesus Macaques - Molecular Therapy,vol. 25 Issue 12pg. 2635-2647. Evers etal., 2018; State-of-the-Art Design and Rapid-Mixing Production Techniques ofLipid Nanoparticles for Nucleic Acid Delivery. Small Methods 2018, 2, 1700375 Chen, S., Tam, YYC, Lin, PJ, Sung, MM, Tam, YK and Cullis, PR, 2016. Influenceof particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. Journal of Controlled Release, 235, pp.236-244.

[0094] Drawing translation Figure 1 Percentage of particles in a range Range diameter of particles Figure 2 Percentage of particles in a range Range diameter of particles Figure 3 Intensity(Percent) Intensity(%) Size Figure 4 Intensity(Percent) Intensity(%) Size Figure 5 Intensity(Percent) Intensity(%) Size

Claims

1. 1. A method for mass producing a composition comprising lipid nanoparticles (LNPs) having an average diameter of at least 140 nm, comprising: a) mixing a first composition comprising an ionic lipid, a phospholipid, a sterol and a PEG lipid in an organic solvent with a second composition comprising one or more active agents in an acidic buffer, thereby obtaining a bulk after mixing; b) subjecting the mixed bulk of step a) to tangential flow filtration (TFF); c) subjecting said composition of step b) to a filtration step in an aqueous solution having a pH of <6.5; d) adding a cryobuffer containing a cryoprotectant to the composition of step c); e) subjecting said composition of step d) to a filtration step in an aqueous solution having a pH>6.5; f) sterile filtering the composition of step e), thereby obtaining a composition of lipid nanoparticles having an average diameter of at least 140 nm and containing the active agent; wherein the TFF in step b) is performed at a shear rate of less than 4000 / s, and the LNPs comprise less than 1 mol% PEGylated lipids.

2. The method according to claim 1, wherein in step a) the mixing flow rate of the first composition is lower than the mixing flow rate of the second composition.

3. 3. The method according to claim 1 or 2, wherein in step a), the ratio of the mixing flow rates of the first composition and the second composition is about 1:

2.

4. 4. The method according to claim 1, wherein the tangential flow filtration in step b) comprises a first ultrafiltration step carried out at a low shear rate compared to the other tangential flow filtration steps.

5. 5. The method according to any one of claims 1 to 4, wherein the tangential flow filtration in step b) comprises a first ultrafiltration step carried out at a shear rate of less than 3000 / s while the other tangential flow filtration steps are carried out at a shear rate of less than 4000 / s.

6. The method according to any one of claims 1 to 5, wherein the organic solvent is chloroform or an alcohol such as methanol or ethanol, in particular ethanol.

7. The method according to any one of claims 1 to 6, wherein said active agent is selected from the list comprising small molecules and biomolecules such as nucleic acids, antibodies, proteins, peptides, in particular mRNA molecules.

8. 8. The method according to any one of claims 1 to 7, wherein the cryoprotectant is selected from the list comprising maltose, maltodextrin, dextran, mannitol, trehalose, glucose or sucrose, in particular trehalose or sucrose.

9. 9. The method according to any one of claims 1 to 8, wherein the cryobuffer comprises a cryoprotectant and one or more of citrate, Tris and PBS, in particular one or more of TRIS or PBS.

10. The method according to any one of claims 1 to 9, wherein the first composition of step a) is at a temperature of from 30°C to 45°C.

11. The method according to any one of claims 1 to 10, wherein the mixed bulk obtained in step a) is diluted before step b).

12. The method according to any one of claims 1 to 11, wherein the composition obtained in step e) is diluted before step f).

13. 13. The method according to any one of claims 1 to 12, wherein the filtration in steps c) and / or e) has a filter size of 0.20 μm.

14. The method according to any one of claims 1 to 13, wherein the aqueous solution of step c) and / or e) is water, in particular water for injection.

15. The method according to any one of claims 1 to 14, wherein the filtration of steps c) and / or e) is a clarification filtration.

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