Method for producing lipid nanoparticles

Abstract

The invention relates to a method for producing lipid nanoparticles (LNPs), comprising the steps of (a) performing a reference method for the preparation of LNPs, the reference method comprising the steps of (a1) introducing a first inlet stream of an aqueous solution via a first inlet port of a first mixing chamber, and introducing a second inlet stream of a lipid solution via a second inlet port of the first mixing chamber, thereby mixing the aqueous solution and the lipid solution so as to produce LNPs, and (a2) recovering a first outlet stream comprising the produced LNPs via an outlet port of the first mixing chamber, and (b) producing LNPs according to the reference method in a second mixing chamber, wherein the inner diameters of the first inlet port, second inlet port and outlet port of the second mixing chamber are each by the same factor proportionally bigger by at least 5% than the inner diameters of the first inlet port, second inlet port and outlet port of the first mixing chamber, wherein the linear flow rate of the first outlet stream and the second outlet stream are essentially identical.

Description

METHOD FOR PRODUCING LNPS

[0001] The present application claims the benefit of priority of U.S. provisional patent application no. 63 / 665,551, filed on June 28, 2024, wherein its entire content is incorporated herein in its entirety. Field of the invention

[0002] The invention relates to a method for producing lipid nanoparticles. Background

[0003] Lipid nanoparticles (LNPs) are one of the most powerful nonviral delivery platforms for nucleic acid payload as proved by the recent breakthroughs in COVID-19 mRNA vaccines. LNPs that encapsulate nucleic acids are typically formulated with amino lipids (ionizable or cationic amino lipids), phosphatidylcholine lipids, cholesterol, and polyethylene glycol-lipid conjugate (PEG-lipid) at certain molar ratio. LNPs can be produced either by conventional methods including direct mixing, thin film, ethanol injection etc. or recently developed methods including microfluidic, T-junction mixing, staggered herringbone mixing (SHM) etc.

[0004] T-junction mixing involves two input streams in lipid phase and aqueous phase, respectively, which collide to form particles in the output flow (Figure 1). Given that T-junction mixer provides a controllable mixing environment, leading to reproducible production of LNPs, this approach has been applied to large-scale production of mRNA vaccines and LNP-siRNA. Design parameters including total flow rate (TFR), flow rate ratio (FRR), N to P ratio, and lipid composition etc. greatly impact polydispersity index (PDI),size and cargo encapsulation of LNPs manufactured via T-junction mixing (Evers et al., 2018).

[0005] In pharmaceutical industry, T-junction mixing has been applied to large-scale production of LNP for delivery of RNA therapeutics and vaccines. However, prior to large-scale production by T-junction mixing, LNP formulation parameters must be optimized in small-scale experiments, which usually rely on pipette mixing or microfluidic mixing techniques. It can be challenging to adopt the same parameters obtained by microfluidics in process development stage for subsequent large-scale production by T- junction mixing since the two methods are different in principle. For a defined LNP formulation, design of experiments (DoE) using the same equipment is usually required to determine the process parameters for large-scale production at high flow rates by T-junction mixing, which consumes large quantity of materials and is not cost-effective.

[0006] Due to the complexity in the process parameter optimization, LNP manufacturing scale-up is still a challenge for biopharma industry. LNP manufacturing scale-up process with the use of different equipment tailored to different batch sizes might not be cost effective. For example, in LNP manufacturing with T-junction mixing, low flow rate is preferred in initial process development stage with use of small sizes of T-junction mixers to optimize process parameters for subsequent large-scale production with use of large sizes of T-junction mixers at high flow rate (size of T-junction mixer is referred to inner diameter of output flow of T-junction mixer here and afterwards). Thus, scale-up methods with use of different equipment to achieve robust process controllability and maintain the LNP attributes, especially particle size are highly desired. Objective technical problem to be solved

[0007] The technical problem to be solved is therefore the provision of a method for producing LNPs which allows optimization of all necessaryparameters (e.g., flow rate ratio (FRR), lipid concentration, LNP to payload ratio etc.) on a small-scale and subsequent upscaling without the need for further adjust these parameters. Summary of the invention

[0008] The invention relates to a method for producing lipid nanoparticles (LNPs), comprising the steps of (a) performing a reference method for the preparation of LNPs, the reference method comprising the steps of (a1) introducing a first inlet stream of an aqueous solution via a first inlet port of a first mixing chamber, and introducing a second inlet stream of a lipid solution via a second inlet port of the first mixing chamber, thereby mixing the aqueous solution and the lipid solution so as to produce LNPs, and (a2) recovering a first outlet stream comprising the produced LNPs via an outlet port of the first mixing chamber, (b) producing LNPs according to the reference method in a second mixing chamber, wherein the inner diameters of the first inlet port, second inlet port and outlet port of the second mixing chamber are each by the same factor proportionally bigger by at least 5% than the inner diameters of the first inlet port, second inlet port and outlet port of the first mixing chamber, wherein the linear flow rate of the first outlet stream and the second outlet stream are essentially identical. Brief description of the figures

[0009] Fig.1 shows an illustration of LNP manufacturing via T-junction mixing and two types of T-junction mixers.

[0010] Fig.2 shows an illustration of linear flow rate for T-shape mixers in T-junction mixing.

[0011] Fig.3 shows an illustration of linear flow rate for Y-shape mixers in T-junction mixing.

[0012] Fig. 4 shows size control of DSPC-cholesterol liposomes manufactured with T-shape T-junction mixing with different sizes of mixers at different TFRs by keeping the same linear flow rate (LFR).

[0013] Fig.5 shows size control of polyA LNPs manufactured with T- shape T-junction mixing with different sizes of mixers at different TFRs by keeping the same linear flow rate (LFR).

[0014] Fig.6 shows size control of siRNA LNPs manufactured with T- shape T-junction mixing with different sizes of mixers at different TFRs by keeping the same linear flow rate (LFR).

[0015] Fig.7 shows size control of mRNA LNPs manufactured with T- shape T-junction mixing with different sizes of mixers at different TFRs by keeping the same linear flow rate (LFR).

[0016] Fig. 8 shows size control of DSPC-cholesterol liposomes manufactured with Y-shape T-junction mixing with different sizes of mixers at different TFRs by keeping the same linear flow rate (LFR). Detailed description of the invention

[0017] In one aspect, the invention relates to a method for producing a lipid nanoparticles (LNPs) solution, comprising the steps of (a) performing a reference method for the preparation of LNPs, the reference method comprising the steps of (a1) introducing a first inlet stream of an aqueous solution via a first inlet port of a first mixing chamber at a controlled flow rate, and introducing a second inlet stream of a lipid solution via a second inlet port of the first mixingchamber at a controlled flow rate, thereby mixing the aqueous solution and the lipid solution so as to produce LNPs, and (a2) recovering a first outlet stream of the LNPs via an outlet port of the first mixing chamber, (b) producing LNPs according to the reference method in a second mixing chamber, wherein the inner diameters of the first inlet port, second inlet port and outlet port of the second mixing chamber are each by the same factor proportionally bigger by at least 5% than the inner diameters of the first inlet port, second inlet port and outlet port of the first mixing chamber, wherein the linear flow rate of the first outlet stream and the second outlet stream are essentially identical.

[0018] In one embodiment, the LNPs obtained in step (a2) have a particle size differing by not more than 20%, preferably not more than 15%, from that of the LNPs obtained in step (b).

[0019] In another embodiment, in step (a1), the first inlet stream is introduced at a at a linear flow rate LFRRef1and the second inlet stream is introduced at a at a linear flow rate LFRRef2and in step (b), the first inlet stream is introduced at a at a linear flow rate LFRPro1 and wherein the second inlet stream is introduced at a at a linear flow rate LFRPro2and LFRRef1is essentially identical to LFRPro1and / or LFRRef2is essentially identical to LFRPro2.

[0020] In another embodiment, the ratio between the flow rate of the first inlet stream and the second inlet stream is essentially identical in step (a) and step (b).

[0021] In another embodiment, the angle between the first inlet port and the second inlet port of the first mixing chamber and the second mixing chamber is between about 20 degrees and about 180 degrees.

[0022] In another embodiment, the corresponding angles are essentially identical in the first mixing chamber and the second mixing chamber.

[0023] In another embodiment, the lipid solution and the aqueous solution used in step (a) are essentially identical to those used in step (b).

[0024] In another embodiment, the first mixing chamber and the second mixing chamber are both T-connectors or Y-connectors.

[0025] In another embodiment, the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber are essentially identical.

[0026] In another embodiment, the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber is between 0.25 mm (1 / 100’’) to 9.5 mm (3 / 8’’), preferably between 0.5 mm (1 / 50’’) to 1.6 mm (1 / 16’’).

[0027] In another embodiment, the inner diameter of the first inlet port, the second inlet port and the outlet port of the second mixing chamber is between 0.5 mm (1 / 50’’) to 25.4 mm (1’’), preferably between 2.3 mm (3 / 32’’) to 3.2 mm (1 / 8’’).

[0028] In another embodiment, the flow rate ratio between the first inlet stream and the second inlet stream is in a range from 0.5:1 to 1:10, preferably 1:1 to 1:5, more preferably 1:1 to 1:3, most preferably 1:1 or 1:3.

[0029] In another embodiment, the linear flow rate of the first outlet stream and the second outlet stream is at least 4000 m / h.

[0030] In another embodiment, the total flow rate of the outlet stream of the first mixing chamber is in a range from 10 ml / min to 500 ml / min, 10 ml / min to 400 ml / min or 100 ml / min to 500 ml / min.

[0031] In another embodiment, the total flow rate of the outlet stream of the second mixing chamber is in a range from 300 ml / min to 2000 ml / min, 300 ml / min to 1500 ml / min or 200 ml / min to 2000 ml / min.

[0032] In another embodiment, the aqueous solution comprises a payload, preferably a nucleic acid.

[0033] In another embodiment, the concentration of nucleic acid in the aqueous solution is in a range from about 10 micrograms / ml to about 5000 micrograms / ml, preferably from about 85 micrograms / ml to about 2100 micrograms / ml.

[0034] In another embodiment, the lipid solution is an ethanol solution comprising one or more lipids.

[0035] In another embodiment, the total concentration of the one or more lipids of the ethanol solution is in a range from about 0.1 mg / ml to about 500 mg / ml, preferably from about 5.0 mg / ml to about 125 mg / ml.

[0036] In another embodiment, the one or more lipids comprise at least one cationic lipid.

[0037] In another embodiment, the method as disclosed above, further comprising mixing a dilution buffer with LNPs of step (a2) to produce a diluted LNP solution.

[0038] In another embodiment, the dilution buffer comprises a buffering agent having a pH between 4 to 10, preferably 5.5 to 7.0, and optionally a sodium chloride concentration up to about 200 mM, preferably up to about 100 mM.

[0039] As used herein, the term “lipid nanoparticle (LNP)” refers to a nanoparticle vehicles comprising lipids such as amino lipids (ionizable or cationic amino lipids), phosphatidylcholine lipids, cholesterol, or polyethyleneglycol-lipid conjugates (PEG-lipid) that can be used to deliver a payload e.g. into the cytoplasm. An LNP is usually spherical with an average diameter of 10 to 1000 nanometers. LNPs may carry a payload of nucleic acids, e.g., DNA or RNA, or APIs. In the context of delivery, lipid nanoparticles offer many advantages over other lipid-based delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0040] Referring to Fig. 2 and 3, the term “flow rate” as used herein refers to the flow rate Q indicated in m³ / h and is also called volumetric flow rate. The term “total flow rate (TFR)” refers to the flow rate Q of the outlet stream. The term “linear flow rate (LFR)” is also called flow velocity or linear velocity and refers to the linear flow rate F indicated in m / h. The term “flow rate ratio (FRR)” refers to the ratio between the flow rate Q of the first inlet stream, i.e., the aqueous solution, i.e., and the flow rate Q of the second inlet stream, i.e., the lipid solution.

[0041] The term “essentially identical” or “constant” as used herein means “not deviating by more than 5%”.

[0042] According to the invention, firstly, a reference method is performed, wherein LNPs are prepared in a mixing chamber comprising a first inlet port, a second inlet and an outlet. A first inlet stream of an aqueous solution is introduced via the first inlet port at a linear flow rate LFRRef1 and a second inlet stream of a lipid solution is introduced via second inlet port at a linear flow rate LFRRef2. The first and the second inlet stream and thus the aqueous solution and the lipid solution are mixed in the mixing chamber so that LNPs are formed. The outlet stream having a linear flow rate LFRRefOutand comprising the produced LNPs is then recovered via the outlet port of the mixing chamber. The produced LNPs are in the form of an LNP solution.

[0043] Next, LNPs are produced according to the reference method in a second mixing chamber also comprising a first inlet port, a second inlet port and an outlet port, wherein the inner diameters of the first inlet port, the second inlet port and the outlet port of the second mixing chamber are each by the same factor proportionally bigger by at least 5% than the inner diameters of the first inlet port, second inlet port and outlet port of the first mixing chamber. All other relevant properties of the second mixing chamber such as the angle between the first and the outlet port and the ratios between the inner diameters of the first inlet port, the second inlet port and the outlet port of the mixing chamber are essentially identical to those of the first mixing chamber. Likewise, when producing the LNPs according to the reference method in step (b), all relevant parameters of the reference method are maintained.

[0044] In other words, LNPs are produced by (b1) introducing a first inlet stream of an aqueous solution via the first inlet port of the second mixing chamber at a linear flow rate LFRPro1, and introducing a second inlet stream of a lipid solution via the second inlet port of the second mixing chamber at a linear flow rate LFRPro2, thereby mixing the aqueous solution and the lipid solution so as to produce LNPs, and (b2) recovering a second outlet stream having a linear flow rate LFRProOutcomprising the produced LNPs via the outlet port of the second mixing chamber.

[0045] According to the invention, the LFR of the first outlet stream and the LFR of the second outlet stream are essentially identical, i.e., LFRRefOutis essentially identical to LFRProOut. This is achieved by increasing the TFR of the outlet stream in step (b) in proportion to the increase in size of the inner diameter of the outlet port of the second mixing chamber. For example, if the inner diameter of the outlet port of the second mixing chamber is doubled compared to that of the first mixing chamber, then the TFR of the outlet stream is multiplied by four in order to maintain the same LFR.

[0046] In a preferred embodiment, the LFR of the first inlet stream in the reference method of step (a1) is essentially identical to that of the first inlet stream in the actual method of production of step (b), i.e., LRFRef1is essentially LRFPro1. This is achieved by increasing the flow rate Q of the first inlet stream in step (b) in proportion to the increase in size of the inner diameter of the first inlet port of the second mixing chamber.

[0047] In another preferred embodiment, the LFR of the second inlet stream in the reference method of step (a2) is essentially identical to that of the second inlet stream in the actual method of production of step (b), i.e., LRFRef2is essentially LRFPro2. This is achieved by increasing the flow rate Q of the second inlet stream in step (b) in proportion to the increase in size of the inner diameter of the second inlet port of the second mixing chamber.

[0048] In a particularly preferred embodiments, the LFR of the first inlet stream, the second inlet stream and the outlet stream of the reference method of step (a) are essentially identical to the respective LFR in step (b), i.e., the actual method of producing.

[0049] Other parameters such as the FRR are kept constant between the reference method of step (a) and the step of producing LNPs according to step (b). According to the invention, the flow rate (FRR) is the ratio between the flow rate of the first inlet stream and the flow rate of the second inlet stream.

[0050] The term “essentially identical” or “constant” as used herein means that there is no significant variation between two or more parameters. In one embodiment is means that the parameters do not deviating by more than 5%.

[0051] Prior to the invention, large-scale methods for producing LNPs differed from the small-scale reference method in that different techniques were used (T-junction mixing for large-scale methods, pipette mixing ormicrofluidic mixing techniques for small-scale methods). This meant that the particle size obtained in the reference method is usually different from that obtained with the actual production method and that all parameters have to optimized anew when switching to the large-scale method.

[0052] The present inventors have surprisingly found that it is possible to optimize all parameters necessary for obtaining a solution of LNPs having a defined particle size using a small-scale reference method that can be performed, e.g., in a laboratory. Herein, for the reference method, a mixing chamber is used that is (a) smaller than the one used for the actual method of production, but (b) the corresponding angles are essentially identical in the first mixing chamber and the second mixing chamber and (c) shows the same linear flow rate of the outlet streams.

[0053] That is, both the reference method and the actual method are T- junction mixing methods. But rather than using the same flow rate Q for the reference method and the large-scale method, it is the LFR that is kept constant in both methods. By maintaining a constant LFR between the reference method and the actual large-scale method of producing LNPs, the particle size of the LNPs produced by the actual method of production differs by not more than 20%, preferably not more than 15%, from that of the LNPs prepared by the reference method, without having to use a large amount of material when performing the reference method on a small-scale. The method of the invention is therefore a method of upscaling a method for producing LNPs.

[0054] Thus, for a given type of T-junction mixer, LNP manufacturing scale-up at fixed FRR can be implemented from the initial use of small sizes of T-junction mixers at low TFRs in process development phase directly to the use of large sizes of T-junction mixers at high TFRs in large-scale manufacturing phase.

[0055] Preferably, the particle size of the produced LNPs is between 20 nm and 500 nm, 40 nm to 250 nm, more preferred between 60 and 150 nm, most preferably between 50 and 90 nm.

[0056] In one embodiment, the angle between the first inlet port and the second inlet port of each mixing chamber is between about 20 degrees and about 180 degrees. The skilled person is able to determine a suitable angle between the first inlet port and the second inlet port depending on the desired properties of the produced LNPs. The angle may take any suitable value. In a preferred embodiment, the angle is essentially identical between the first and the second mixing chamber. In a particularly preferred embodiment, all corresponding angles are essentially identical in the first and the second mixing chamber.

[0057] In one embodiment, the first and the second mixing chamber are both T-connectors, i.e., the angle between the first inlet port and the second inlet port of each mixing chamber is 180 degrees. In another embodiment, the first and the second mixing chamber are both Y-connectors, e.g. the angle between the first inlet port and the second inlet port of each mixing chamber is less than 180 degrees, preferably more than 20 degrees.

[0058] In one embodiment, the inner diameters of the first inlet port, the second inlet port and the outlet port of the first mixing chamber are essentially identical. In this embodiment, the inner diameters of the first inlet port, the second inlet port and the outlet port of the second mixing chamber are also essentially identical.

[0059] In some embodiments, the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber is between 0.25 mm (1 / 100’’) to 9.5 mm (3 / 8’’), preferably between 0.5 mm (1 / 50’’) to 1.6 mm (1 / 16’’).

[0060] In some embodiments, the inner diameter of the first inlet port, the second inlet port and the outlet port of the second mixing chamber is between 0.5 mm (1 / 50’’) to 25.4 mm (1’’), preferably between 2.3 mm (3 / 32’’) to 3.2 mm (1 / 8’’).

[0061] In some embodiments, the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber is between 0.25 mm (1 / 100’’) to 9.5 mm (3 / 8’’) and the inner diameter of the first inlet port, the second inlet port and the outlet port of the second mixing chamber is between 0.5 mm (1 / 50’’) to 25.4 mm (1’’).

[0062] Preferably, the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber is between 0.5 mm (1 / 50’’) to 1.6 mm (1 / 16’’ and the inner diameter of the first inlet port, the second inlet port and the outlet port of the second mixing chamber is between 2.3 mm (3 / 32’’) to 3.2 mm (1 / 8’’).

[0063] In one embodiment, the flow rate ratio FRR between the first inlet stream and the second inlet stream in both the first and the second mixing chamber is in a range from 0.5:1 to 1:10, preferably 1:1 to 1:5, more preferably 1:1 to 1:3, most preferably 1:1 or 1:3.The skilled person knows how to determine a suitable FRR based on the characteristics of the lipid and aqueous solution used and the desired properties of the LNPs.

[0064] In a preferred embodiment, the linear flow rate of the first outlet stream and the second outlet stream are at least 4000 m / h. This ensures a homogenous particle size of the produced LNPs. In further preferred embodiments, the linear flow rate of the first outlet stream and the second outlet stream are in a range from 4000 m / h to 13000 m / h, 5000 m / h to 10000 m / h or 6000 m / h to 13000 m / h.

[0065] In some embodiments, the total flow rate of the first outlet stream is in a range from 10 ml / min to 500 ml / min, 10 ml / min to 400 ml / min or100 ml / min to 500 ml / min. Keeping the TFR low when performing the reference method reduces the material needed for performing the reference method, thus lowering the costs for optimizing the reference method on a lab scale or small-scale.

[0066] In some embodiments, the total flow rate of the second outlet stream is in a range from 180 ml / min to 2000 ml / min, 180 ml / min to 1500 ml / min or 180 ml / min to 2000 ml / min. Compared to the reference method, the method for actually producing LNPs on a large scale uses higher TFR, resulting in larger quantities of LNPs having a uniform particle size.

[0067] The aqueous solution of the first inlet stream can be any aqueous solution known to the skilled person and commonly used for producing LNPs. The aqueous solution may be a buffer. In some embodiments, the aqueous solution is a buffer having a pH of 4. In a preferred embodiment, the aqueous solution comprises a payload with which the produced LNPs are to be loaded, e.g., a nucleic acid, a protein, or an API. In a particularly preferred embodiment, the payload is a DNA or an RNA molecule. In preferred embodiments, the concentration of the nucleic acid in the aqueous solution is in a range from about 10 micrograms / ml to about 5000 micrograms / ml, preferably from about 85 micrograms / ml to about 2100 micrograms / ml.

[0068] The lipid solution of the second inlet stream can be any lipid solution known to the skilled person and commonly used for producing LNPs. In one embodiment, the lipid solution is an ethanol solution comprising one or more lipids. In some embodiments, the total concentration of the one or more lipids in the ethanol solution is in a range from about 0.1 mg / ml to about 500 mg / ml, preferably from about 5.0 mg / ml to about 125 mg / ml.

[0069] In some embodiment, the lipids comprise or consists of amino lipids (ionizable or cationic amino lipids), phosphatidylcholine lipids, cholesterol, or polyethylene glycol-lipid conjugates (PEG-lipid).

[0070] In preferred embodiments, the one or more lipids comprise at least one cationic lipid. Cationic lipids are amphiphiles containing a positive hydrophilic head group, two (or more) lipophilic tails, or a steroid portion and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at about physiological pH. In some embodiments, amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g, pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Use of cationic lipid enhances uptake of the LNPs by the target cells. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the negatively charged backbone of nucleic acids and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration.

[0071] In some embodiments, the reference method further comprises a step of mixing a dilution buffer with the LNPs to produce a diluted LNP solution. In this embodiment, the LNPs produced in the second mixing chamber, i.e., during the actual method of producing, are also mixed with the dilution to produce a diluted LNP solution. In some embodiments, the dilution buffer comprises a buffering agent having a pH between 4 to 10, preferably 5.5 to 7.0, and optionally a sodium chloride concentration up to about 200 mM, preferably up to about 100 mM. Examples Example 1

[0072] Sizes of 1 / 50”, 1 / 16”, 3 / 32” and 1 / 8” T-junction mixers were used to produce LNPs. In one experiment for the manufacturing of DSPC-cholesterol liposomes at fixed FRR (=3) via T-shape T-junction mixers, where 10 mM of total lipid concentration with molar ratio of DSPC: Cholesterol =1:1 was used, TFRs were set for different sizes of T-junction mixers with same linear flow rate (LFR) as indicated in following Table 1.

[0073] As demonstrated in Fig. 4, when TFRs were set to keep the same Linear Flow Rate (LFR) for different sizes of T-junction mixers (1 / 50”, 1 / 16”, 3 / 32”, 1 / 8”), size variations of liposomes with PDI < 0.2 formulated via different size of mixers were less than 20% (red dash line boxed). The higher TFR, the smaller the size variation. Example 2

[0074] In another experiment for the manufacturing of polyA LNP at fixed FRR (=3) via T-shape T-junction mixers, where 10 mM of total lipid concentration with molar ratio of Dlin-MC3-DMA : DSPC: Cholesterol : DMG- PEG2000 =50:10:38.5:1.5 and 0.09 mg / ml polyA were used, different TFRs were set for different sizes of T-junction mixers with same linear flow rate (LFR) as indicated in following Table 2:

[0075] As demonstrated in Fig. 5, when TFRs were set to keep the same Linear Flow Rate (LFR) for different sizes of T-junction mixers (1 / 50”, 1 / 16”, 3 / 32”, 1 / 8”), size variations of polyA LNP with PDI < 0.2 formulated via different size of tees were less than 15% (red dash line boxed). Example 3

[0076] In an experiment for the manufacturing of siRNA LNP at fixed FRR (=3) via T-shape T-junction mixers, where 10mM of total lipid concentration with molar ratio of Dlin-MC3-DMA : DSPC: Cholesterol : DMG- PEG2000 =50:10:38.5:1.5 and 0.09mg / ml siRNA were used, different TFRs were set for different sizes of T-junction mixers with same linear flow rate (LFR) as indicated in following Table 3: Table 3. T-junction mixer size, LFR and TFRs for siRNA LNP manufacturing

[0077] As demonstrated in Fig. 6, when linear flow rate (LFR) was maintained the same, sizes and PDIs of siRNA LNP manufactured via 1 / 50” HPLC tee and 1 / 16” PE tee at varying TFRs were not changed, verifying the scale-up method for siRNA LNP for different sizes of tees.Example 4

[0078] In one separate experiment for the manufacturing of mRNA LNP at fixed FRR (=3) via T-shape T-junction mixers, where 10 mM of total lipid concentration with molar ratio of Dlin-MC3-DMA : DSPC: Cholesterol : DMG- PEG2000 =50:10:38.5:1.5 and 0.09mg / ml mRNA were used, different TFRs were set for different sizes of T-junction mixers with same linear flow rate (LFR) as indicated in Table 3. As demonstrated in Fig.7, when linear flow rate (LFR) was maintained the same, size variations of mRNA LNP manufactured via 1 / 50” HPLC tee and 1 / 16” PE tee at different TFRs were < 10%, verifying the scale-up method for mRNA LNP for different sizes of tees. Example 5

[0079] In another experiment, different sizes of Y-shape T-junction mixers (1 / 16”, 3 / 32” and 1 / 8”) were used to manufacture DSPC-cholesterol liposomes, where 10 mM of total lipid concentration with molar ratio of DSPC: Cholesterol =1:1 was used. As demonstrated in Fig.8, when TFRs were set to keep the same Linear Flow Rate (LFR) for different ID sizes of Y-shape T- junction mixers (1 / 16”, 3 / 32”, 1 / 8”), size variations of DSPC / Cholesterol liposomes with PDI < 0.2 were less than 10% (red dash line boxed).

[0080] These data collectively demonstrated a novel scale-up method for LNP and liposomes manufacturing with T-junction mixing, which particle sizes are maintained with the use of different sizes of mixers by setting TFRs to keep linear flow rates constant. Therefore, LNP and liposomes manufacturing scale-up can be implemented from the use of small T-junction mixers with low TFRs for small batches to the use of large T-junction mixers with high TFRs for large-scale manufacturing.

Claims

Claims 1. A method for producing lipid nanoparticles (LNPs), comprising the steps of (a) performing a reference method for the preparation of LNPs, the reference method comprising the steps of (a1) introducing a first inlet stream of an aqueous solution via a first inlet port of a first mixing chamber, and introducing a second inlet stream of a lipid solution via a second inlet port of the first mixing chamber, thereby mixing the aqueous solution and the lipid solution so as to produce LNPs, and (a2) recovering a first outlet stream comprising the produced LNPs via an outlet port of the first mixing chamber, (b) producing LNPs according to the reference method in a second mixing chamber, wherein the inner diameters of the first inlet port, second inlet port and outlet port of the second mixing chamber are each by the same factor proportionally bigger by at least 5% than the inner diameters of the first inlet port, second inlet port and outlet port of the first mixing chamber, wherein the linear flow rate of the first outlet stream and the second outlet stream are essentially identical.

2. The method according to claim 1, wherein the LNPs obtained in step (a2) have a particle size differing by not more than 20% from that of the LNPs obtained in step (b).

3. The method according to claim 1 or 2, wherein in step (a1), the first inlet stream is introduced at a at a linear flow rate LFRRef1 and the second inlet stream is introduced at a at a linear flow rate LFRRef2and in step (b), the first inlet stream is introduced at a at a linear flow rate LFRPro1 and wherein the second inlet stream is introduced at a at alinear flow rate LFRPro2 and LFRRef1 is essentially identical to LFRPro1 and / or LFRRef2 is essentially identical to LFRPro2.

4. The method according to claim 1, wherein the ratio between the flow rate of the first inlet stream and the second inlet stream is essentially identical in step (a) and step (b).

5. The method according to any of claims 1 to 4, wherein the angle between the first inlet port and the second inlet port of the first mixing chamber and the second mixing chamber is between about 20 degrees and about 180 degrees.

6. The method according to any of claims 1 to 5, wherein the corresponding angles are essentially identical in the first mixing chamber and the second mixing chamber.

7. The method according to any of claims 1 to 6, wherein the lipid solution and the aqueous solution used in step (a) are essentially identical to those used in step (b).

8. The method according to any of claims 1 to 7, wherein the first mixing chamber and the second mixing chamber are both T-connectors or Y-connectors.

9. The method according to any of claims 1 to 8, wherein the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber are essentially identical.

10. The method according to any of claims 1 to 9, wherein the inner diameter of the first inlet port, the second inlet port and the outlet port of the first mixing chamber is between 0.25 mm (1 / 100’’) to 9.5 mm (3 / 8’’).

11. The method according to any of claims 1 to 10, wherein the inner diameter of the first inlet port, the second inlet port and the outletport of the second mixing chamber is between 0.5 mm (1 / 50’’) to 25.4 mm (1’’).

12. The method according to any of claims 1 to 11, wherein the flow rate ratio between the first inlet stream and the second inlet stream is in a range from 0.5:1 to 1:

10.

13. The method according to any of claims 1 to 12, wherein the linear flow rate of the first outlet stream and the second outlet stream is at least 4000 m / h.

14. The method according to any of claims 1 to 13, wherein the total flow rate of the outlet stream of the first mixing chamber is in a range from 10 ml / min to 500 ml / min, 10 ml / min to 400 ml / min or 100 ml / min to 500 ml / min.

15. The method according to any of claims 1 to 14, wherein the total flow rate of the outlet stream of the second mixing chamber is in a range from 300 ml / min to 2000 ml / min, 300 ml / min to 1500 ml / min or 200 ml / min to 2000 ml / min.

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