Lipid nanoparticle production system and monitoring and control method thereof

Abstract

The present invention relates to an automated nanoparticle synthesis system and a computer-implemented method of monitoring and controlling the process of manufacturing lipid nanoparticles (LNPs) containing nucleic acid cargo. The invention also relates to a computer program product comprising computer readable instructions that, when loaded and run on a computer system, cause the computer system to perform operations according to the method.

Description

Technical Field

[0001] The present invention relates to an automated nanoparticle synthesis system and a computer-implemented method for monitoring and controlling the process of manufacturing lipid nanoparticles (LNPs) containing nucleic acid cargo. The present invention also relates to a computer program product comprising computer-readable instructions that, when loaded and run on a computer system, cause the computer system to perform operations according to the method. Background Art

[0002] In recent years, LNPs containing nucleic acid cargo have attracted much attention, for example due to the development of messenger RNA (mRNA)-based vaccines. Therefore, mRNA (or any other therapeutic nucleic acid of interest) is encapsulated in LNPs for stable delivery to target cells and efficient transfection of target cells. Typically, LNPs protect their cargo from degradation, deliver the therapeutic agent to the target cell or tissue, and control the release of the cargo at the desired location.

[0003] LNPs are usually composed of amino lipids (ionizable or cationic amino lipids) as the main components, as well as phosphatidylcholine lipids, cholesterol, and polyethylene glycol-lipid conjugates (PEG-lipids). During the manufacturing process, a solution containing a nucleic acid cargo is mixed with a solution containing lipids at an acidic pH, followed by neutralization, buffer exchange, and concentration to obtain LNPs containing nucleic acid cargo.

[0004] However, to date, manufacturing development and screening of LNPs have faced multiple challenges due to the lack of high-throughput methods for producing LNPs that allow for rapid and easy generation and subsequent screening and analysis of a wide range of LNPs. In particular, large-scale screening of LNPs is hampered by current production methods, which allow for the generation of approximately fewer than 20 different LNPs per person per day, while offline analysis of three to four parameters of the LNPs takes two to three days to complete. Enhanced control during the manufacturing process would also improve the scale-up of the process from laboratory scale (< 2 mg) to industrial scale (> 2 g).

[0005] Therefore, there is an urgent need to provide a method for high-throughput preparation and analysis of LNPs containing nucleic acid cargo. In addition, there is an urgent need to provide a method for real-time monitoring and control of the process of manufacturing lipid nanoparticles containing nucleic acid cargo to improve the robustness and reproducibility of the process.

[0006] This need is met by providing the embodiments characterized in the claims. Summary of the invention

[0007] 1. A computer-implemented method for high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, the method comprising the following steps:

[0008] (a) providing one or more solutions containing a nucleic acid cargo;

[0009] (b) providing one or more solutions comprising lipids;

[0010] (c) mixing one or more solutions comprising a nucleic acid cargo with one or more solutions comprising a lipid at an acidic pH;

[0011] (d) neutralizing the pH of the mixed solution obtained in step (c) by diluting to stabilize the LNP obtained in step (c);

[0012] (e) buffer exchanging the neutralized solution obtained in step (d) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (d) and reconstitute them in a cryobuffer to produce one or more LNP formulations;

[0013] in

[0014] During each of steps (c) to (e) and after step (e), one or more of the particle size, polydispersity, and nucleic acid encapsulation efficiency of the one or more LNPs are monitored in real time to generate a data set for each LNP formulation; and the generated data set is associated with the corresponding LNP formulation.

[0015] 2. The computer-implemented method of claim 1, wherein two or more separate LNP formulations are generated in parallel.

[0016] 3. The computer-implemented method of claim 1, wherein one or more solutions comprising a nucleic acid cargo and one or more solutions comprising a lipid are provided in one or more multi-well plates.

[0017] 4. The computer-implemented method of item 1, further comprising stabilizing the LNP obtained in step (c) for a specific period of time before step (d).

[0018] 5. The computer-implemented method of claim 1, wherein one or more LNP formulations are generated in one or more multi-well plates.

[0019] 6. The computer-implemented method of item 1, wherein the nucleic acid is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0020] 7. The computer-implemented method of claim 6, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA) and small interfering RNA (siRNA).

[0021] 8. The computer-implemented method of claim 1, wherein the lipid is an ionizable lipid.

[0022] 9. The computer-implemented method of claim 1, wherein the pH in step (c) is between pH 2 and pH 6.

[0023] 10. The computer-implemented method of claim 4, wherein the time is from 0.1 to 60 minutes.

[0024] 11. The computer-implemented method of claim 1, wherein the pH of the neutralization solution in step (d) is between pH 6 and pH 8.

[0025] 12. The computer-implemented method of item 1, wherein the buffer exchange in step (e) is performed with a buffer comprising phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris) buffered saline.

[0026] 13. The computer-implemented method of claim 1, wherein one or more of the particle size, polydispersity, and nucleic acid encapsulation efficiency of the one or more LNPs can be measured and / or analyzed by at least one of the following: Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS) and multi-angle light scattering (MALS), single particle automated Raman capture analysis (SPARTA), high-speed imaging technology, PATfix ® HPLC platform, nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay.

[0027] 14. The computer-implemented method of claim 1, wherein the method further comprises the step of creating a lipid library based on a data set generated for a corresponding LNP formulation.

[0028] 15. A computer program product comprising computer-readable instructions, which, when loaded and executed on a computer system, causes the computer system to perform operations according to the method of item 1.

[0029] 16. A computer-implemented method for monitoring and controlling a process for manufacturing lipid nanoparticles containing a nucleic acid cargo,

[0030] The process of manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps:

[0031] (i) mixing a solution containing a nucleic acid cargo with a solution containing a lipid at an acidic pH;

[0032] (ii) stabilizing the nanoparticles obtained in step (i) for a specific residence time;

[0033] (iii) neutralizing the pH of the mixed solution obtained in step (i) by diluting to further stabilize the nanoparticles obtained in step (ii);

[0034] (iv) buffer exchanging the neutralized solution obtained in step (iii) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cryobuffer;

[0035] The computer-implemented method for monitoring and controlling the process of manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps:

[0036] (a) monitoring one or more quality parameters of the lipid nanoparticles, such as particle size, polydispersity and nucleic acid encapsulation efficiency, in real time during each of steps (i) to (iv) and after step (iv);

[0037] (b) determining whether the quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is within a predetermined target range;

[0038] (c) if any quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is not within the predetermined target range, feedback control is provided to adjust the operating parameters of the corresponding step in real time so that the corresponding quality parameter enters the predetermined target range.

[0039] 17. The computer-implemented method of claim 16, wherein the nucleic acid is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0040] 18. The computer-implemented method of claim 17, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA) and small interfering RNA (siRNA).

[0041] 19. The computer-implemented method of claim 16, wherein the lipid is an ionizable lipid.

[0042] 20. The computer-implemented method of claim 16, wherein the pH in step (i) is between pH 2 and pH 6.

[0043] 21. The computer-implemented method of claim 16, wherein the residence time is 0.5 to 60 minutes.

[0044] 22. The computer-implemented method of claim 16, wherein the pH of the neutralization solution in step (iii) is between pH 6 and pH 8.

[0045] 23. The computer-implemented method of claim 16, wherein the buffer exchange in step (iv) is performed with a buffer comprising phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris) buffered saline.

[0046] 24. The computer-implemented method of claim 16, wherein the quality parameter comprises one or more of particle size, polydispersity, and nucleic acid encapsulation efficiency of the lipid nanoparticles, and the quality parameter can be measured and / or analyzed by at least one of the following: Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS) and multi-angle light scattering (MALS), single particle automated Raman capture analysis (SPARTA), high-speed imaging technology, PATfix ® HPLC platform, nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay.

[0047] 25. The computer-implemented method of claim 16, wherein the target range of particle size is within a 5% variation of a specific target particle size value, wherein the target particle size value is between 40 and 400 nm.

[0048] 26. The computer-implemented method of claim 16, wherein the target range for polydispersity is 0.04 to 0.1.

[0049] 27. The computer-implemented method of claim 16, wherein the target range for nucleic acid encapsulation efficiency is at least 96% or higher.

[0050] 28. The computer-implemented method of claim 16, wherein the operating parameters adjusted in step (c) are selected from the group consisting of nucleic acid flow rate, lipid flow rate, ratio of nucleic acid flow rate to lipid flow rate, temperature, pressure, nucleation duration, particle growth duration, residence time, maturation period, pH, fluid composition, dilution, dilution rate, mixer type, flow mixing ratio, and agitator / mixer speed.

[0051] 29. A computer program product comprising computer-readable instructions which, when loaded and executed on a computer system, cause the computer system to perform operations according to the method of item 16.

[0052] 30. The computer program product of claim 29, wherein the computer readable instructions include a control algorithm for feedback control of operating parameters, wherein the control algorithm is based on a mechanical hybrid model.

[0053] 31. An automated nanoparticle synthesis system, the system comprising:

[0054] (a) a first dispenser assembly having a first pump integrated with a first valve;

[0055] (b) a second dispenser assembly having a second pump integrated with a second valve;

[0056] (c) a third valve;

[0057] (d) Microfluidic mixer;

[0058] (e) a lipid reservoir for containing a lipid stock solution;

[0059] (f) a nucleic acid storage container for containing a nucleic acid stock solution; and

[0060] (g) waste liquid reservoir, buffer reservoir and ethanol reservoir;

[0061] in

[0062] (i) the lipid reservoir is fluidically connected to the first valve and the nucleic acid reservoir is fluidically connected to the second valve, or vice versa;

[0063] (ii) the first valve is fluidly connected to the first pump and the second valve is fluidly connected to the second pump;

[0064] (iii) the first valve and the second valve are each fluidically connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, wherein ports of the first valve and the second valve that are fluidly connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir are different from ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir;

[0065] (iv) the first valve and the second valve are each fluidly connected to the microfluidic mixer; and

[0066] (v) A third valve is fluidically connected to the microfluidic mixer, a waste reservoir, and one or more collection reservoirs for collecting the produced lipid nanoparticles (LNPs).

[0067] 32. The automatic nanoparticle synthesis system of claim 31, wherein the first pump and / or the second pump is a syringe pump.

[0068] 33. The automatic nanoparticle synthesis system of claim 31, wherein the first valve, the second valve and / or the third valve are 6-way valves.

[0069] 34. The automatic nanoparticle synthesis system of claim 31, wherein the fluid connection is achieved via a pipeline.

[0070] 35. An automated nanoparticle synthesis system according to claim 31, wherein the system further comprises one or more gas plugs suitable for separating a buffer from a solution containing RNA, one or more gas plugs for separating a buffer from a solution containing lipids, and one or more gas plugs for separating a solution containing lipids from ethanol.

[0071] 36. The automatic nanoparticle synthesis system of claim 31, wherein the microfluidic mixer is a microfluidic mixer chip.

[0072] 37. An automated nanoparticle synthesis system, the system comprising:

[0073] (a) a first dispenser assembly having a first pump integrated with a first valve;

[0074] (b) a second dispenser assembly having a second pump integrated with a second valve;

[0075] (c) Microfluidic mixer;

[0076] (d) one or more liquid handlers, wherein the liquid handlers are configured to automatically load reagents and automatically collect samples in one or more well plates; and

[0077] (e) waste liquid reservoir, buffer reservoir and ethanol reservoir;

[0078] in

[0079] (i) the lipid reservoir is fluidically connected to the first valve and the nucleic acid reservoir is fluidically connected to the second valve, or vice versa;

[0080] (ii) the first valve is fluidly connected to the first pump and the second valve is fluidly connected to the second pump;

[0081] (iii) the first valve and the second valve are each fluidically connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, wherein ports of the first valve and the second valve that are fluidly connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir are different from ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir;

[0082] (iv) the first valve and the second valve are each fluidly connected to the microfluidic mixer; and

[0083] (v) The microfluidic mixer is fluidically connected to the liquid handler.

[0084] 38. The automatic nanoparticle synthesis system of claim 37, wherein the first pump and / or the second pump is a syringe pump.

[0085] 39. The automatic nanoparticle synthesis system of claim 37, wherein the first valve and / or the second valve is a 6-way valve.

[0086] 40. The automated nanoparticle synthesis system of claim 37, the system comprising a first liquid processor and a second liquid processor, wherein the first liquid processor is configured to automatically collect synthesized LNPs, and the second liquid processor is configured to automatically load reagents.

[0087] 41. The automatic nanoparticle synthesis system of claim 37, wherein the fluid connection is achieved via a pipeline.

[0088] 42. An automated nanoparticle synthesis system according to claim 37, wherein the system further comprises one or more gas plugs suitable for separating a buffer from a solution containing RNA, one or more gas plugs for separating a buffer from a solution containing lipids, and one or more gas plugs for separating a solution containing lipids from ethanol.

[0089] 43. The automatic nanoparticle synthesis system of claim 37, wherein the microfluidic mixer is a microfluidic mixer chip.

[0090] 44. The automated nanoparticle synthesis system of claim 37, wherein one or more of the liquid processors are configured to automatically remove a lipid-containing solution and an RNA-containing solution from the well plate. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 ：

[0092] Exemplary method for high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo.

[0093] Figure 2 ：

[0094] The process of the present invention for producing lipid nanoparticles containing a nucleic acid cargo, wherein the process comprises the steps of mixing, neutralizing, buffer exchanging and concentrating the solution containing the product, wherein the formation of lipid nanoparticles containing a nucleic acid cargo is depicted.

[0095] Figure 3 ：

[0096] According to some embodiments, embodiments of an automated nanoparticle synthesis system.

[0097] Figure 4 ：

[0098] According to some embodiments, alternative embodiments of the automated nanoparticle synthesis system.

[0099] Figure 5 ：

[0100] According to some embodiments, Figure 3 An embodiment of a dispensing module of an automated nanoparticle synthesis system is depicted in FIG.

[0101] Figure 6 ：

[0102] According to some embodiments, Figure 3 An embodiment of a collection module of an automated nanoparticle synthesis system depicted in FIG.

[0103] Figure 7 ：

[0104] According to some embodiments, Figure 3 A demonstration version of the automated nanoparticle synthesis system depicted in .

[0105] Figure 8 ：

[0106] According to some embodiments, Figure 3 and 4 An embodiment of a user interface of control software for an automated nanoparticle synthesis system is depicted in FIG.

[0107] Fig. 9 ：

[0108] LNP reproducibility. Five different LNP samples generated using the automated nanoparticle synthesis system of the present invention showed reproducible size and polydispersity index (PDI).

[0109] Fig.10 ：

[0110] Effect of the flow rate ratio between lipid and mRNA and the total flow rate in the device on size and PDI. DETAILED DESCRIPTION

[0111] In a first aspect, the present invention relates to a computer-implemented method for high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, the method comprising the following steps:

[0112] (a) providing one or more solutions containing a nucleic acid cargo;

[0113] (b) providing one or more solutions comprising lipids;

[0114] (c) mixing one or more solutions comprising a nucleic acid cargo with one or more solutions comprising a lipid at an acidic pH;

[0115] (d) maturing / stabilizing the LNPs obtained in step (c) for a specific residence time;

[0116] (e) neutralizing the pH of the mixed solution obtained in step (c) by diluting to further stabilize the LNP obtained in step (d);

[0117] (f) buffer exchanging the neutralized solution obtained in step (e) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (e) and reconstitute them in a cryobuffer to produce one or more LNP formulations;

[0118] in

[0119] During each of steps (c) to (f) and after step (f), one or more of the particle size, polydispersity, and nucleic acid encapsulation efficiency of the one or more LNPs are monitored in real time to generate a dataset for each LNP formulation; and the generated datasets are associated with the corresponding LNP formulation and stored for further use.

[0120] As used herein, the term "computer-implemented method" refers to a method that is automatically performed on a computer. Thus, the actual preparation of LNPs and the monitoring and control of the process of manufacturing lipid nanoparticles, data set generation, and data set storage are automatically performed by a computer.

[0121] In a specific embodiment, the nucleic acid cargo is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. In addition, the RNA can be selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

[0122] In a specific embodiment, the lipid used to produce the LNP in the process of manufacturing the LNP containing the nucleic acid cargo is an ionizable lipid. In addition, the lipid-containing solution may contain other agents selected from other ionizable lipids, structural lipids, auxiliary lipids (e.g., DSPC (distearoylphosphatidylcholine)), cholesterol and polyethylene glycol (PEG)-lipid conjugates.

[0123] In step (c) of the method of the present invention, one or more solutions containing nucleic acid cargo are mixed with one or more solutions containing lipids at an acidic pH, wherein the nucleic acid cargo and the lipid are preferably as defined above. In a preferred embodiment, the nucleic acid cargo is contained in the solution containing the nucleic acid cargo at a concentration of 0.1 to 1 mg / mL. The solvent of the solution containing the nucleic acid cargo is preferably a suitable buffer, such as 20 to 100 mM citrate buffer, pH 3.0. In addition, the lipid is preferably contained in the solution containing the lipid at a concentration of 50 to 60 mol%. The solvent of the solution containing the lipid is preferably a suitable organic solvent, such as ethanol. Therefore, the volume ratio between the solution containing the nucleic acid cargo and the solution containing the lipid can be 1:1 to 5:1, preferably about 3:1.

[0124] In a specific embodiment, the acidic pH is pH 2 to pH 6, preferably pH 3 to pH 6, more preferably pH 4 to pH 6, and most preferably about pH 5. In addition, the temperature at which step (c) is performed is not particularly limited, but is preferably room temperature, or a temperature between room temperature and up to 40°C.

[0125] The means for mixing the solution containing the nucleic acid cargo and the solution containing the lipid are not particularly limited and are known in the art. However, the mixing is preferably high-speed laminar mixing, wherein nanoprecipitation is faster than nucleation. Suitable mixers and mixing devices are not particularly limited and are known in the art.

[0126] Step (d) of the method of the present invention is a step of maturing and / or stabilizing the LNP obtained in step (c) by keeping the mixture obtained in step (c) for a certain residence time before the next step. A person skilled in the art can select a suitable residence time, for example, from about 0.1 to about 60 minutes.

[0127] In step (e) of the method of the present invention, the pH of the mixed solution obtained in step (c) is neutralized by dilution (e.g., online dilution) to further stabilize the LNP. In this step, the mixed solution obtained in step (d) is neutralized by mixing with a solution having an alkaline pH, preferably phosphate buffered saline (PBS).

[0128] In a preferred embodiment, the mixed solution obtained in step (d) above is neutralized to a pH of pH 6.0 to pH 8.0, more preferably a pH of pH 7.0 to pH 8.0, more preferably a pH of pH 7.2 to pH 7.6, and most preferably a pH of about pH 7.4.

[0129] In step (f) of the method of the present invention, the neutralized solution obtained in step (e) is buffer exchanged by ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (e) and reconstitute them in a cryobuffer. The corresponding means for performing the buffer exchange are not particularly limited and are known in the art. Suitable cryobuffers include, for example, phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or tris (hydroxymethyl) aminomethane (Tris) buffered saline (TBS), containing a suitable cryoprotectant, such as sucrose, mannitol, trehalose or sorbitol at a concentration of 0.1 to 10% by weight.

[0130] According to the present invention, during each step in steps (c) to (f) and after step (f), one or more of the particle size, polydispersity and nucleic acid encapsulation efficiency of one or more LNPs are monitored in real time, thereby generating a data set for each LNP preparation; and the generated data set is associated with the corresponding LNP preparation and stored for further use. In a specific embodiment, all three parameters of the particle size, polydispersity and nucleic acid encapsulation efficiency of one or more LNPs are monitored. In other embodiments, only one of the parameters is monitored, or any two of the parameters are monitored.

[0131] The term "real time" as used herein means that according to the method of the present invention, the above parameters (eg quality parameters) of the LNPs prepared in the method of the present invention are monitored while the method is being performed.

[0132] The parameter "particle size" determines the size of the LNP containing the nucleic acid cargo. In a non-limiting embodiment, the particle size is determined by Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), PATfix ® HPLC platform, nanoparticle tracking analysis and / or multi-angle light scattering (MALS) determination.

[0133] The parameter "polydispersity" is a measure of the heterogeneity of the LNPs containing nucleic acid cargo. Polydispersity may occur due to size distribution, agglomeration or aggregation of LNPs in solution. In a non-limiting embodiment, polydispersity is measured by Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), PATfix ® The particle size distribution can be determined using an HPLC platform, nanoparticle tracking analysis, multi-angle light scattering (MALS), and / or other suitable tools known in the art for measuring particle size distribution.

[0134] The parameter "nucleic acid encapsulation efficiency" is a measure of the percentage of nucleic acid cargo successfully encapsulated / adsorbed into LNPs. In a specific embodiment, the nucleic acid encapsulation composition is composed of a Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), Förster resonance energy transfer (FRET) assays and / or single particle automated Raman capture analysis (SPARTA).

[0135] According to the present invention, a data set is generated for each LNP formulation (i.e., for each of the one or more LNPs prepared in the method of the present invention). The data set includes data obtained by monitoring one or more of the particle size, polydispersity and nucleic acid encapsulation efficiency of one or more LNPs during each step in steps (c) to (f) and after step (f). In addition, according to the present invention, the corresponding data set is associated with the corresponding LNP formulation, that is, each data set also includes information on which LNP formulation it belongs to. The data set is then stored for further use, for example, to provide feedback adjustment of the preparation step or to enable evaluation and comparison of different LNP formulations.

[0136] In the specific and preferred embodiment of the inventive method, two or more independent LNP preparations are generated in parallel. Except the technical limitations imposed by liquid handling and sample handling equipment, there is no specific upper limit to the number of LNP preparations that can be generated in parallel. In this context, one or more solutions comprising nucleic acid goods and / or one or more solutions comprising lipids can be provided in one or more porous plates. Suitable porous plates are not particularly limited, and are known in the art, including, for example, 6 holes, 12 holes, 24 holes, 48 ​​holes, 96 holes, 384 holes and nanopore microtiter plates. In further specific and preferred embodiments, one or more LNP preparations are also generated in one or more corresponding porous plates.

[0137] Thus, in a preferred embodiment, the method of the present invention provides the possibility of generating two or more LNP preparations in parallel, for example, 96 LNP preparations in a 96-well plate format. In such an embodiment, the individual LNP preparations can differ from each other in any manner that may be of interest. This includes differences in solutions containing nucleic acids (e.g., different nucleic acids, different nucleic acid amounts, different buffer compositions, different pH, different osmotic pressures), differences in solutions containing lipids (e.g., different lipids, different structural lipids, different auxiliary lipids, different PEG-lipid conjugates, different buffer compositions, different lipid compositions, different lipid amounts, different pH, different osmotic pressures), and / or differences in process parameters in steps (c) to (f) (e.g., different mixers, different mixing ratios, different mixing flow rates, different residence times, different buffer compositions, different pH values, different temperatures, different dilution ratios). In this way, the corresponding LNP preparations can be compared with each other and the effects of different parameters can be evaluated. In addition, this allows for convenient screening methods for, for example, nucleic acid libraries, lipid libraries, and the like.

[0138] The technical means for implementing the method of the present invention are not particularly limited and include robotic liquid handling laboratory robot systems known in the art (e.g., Sartorius Ambr ® system).

[0139] In a specific embodiment, the method of the present invention can be used together with a lipid library to screen new LNP preparations. As mentioned above, this lipid library can be provided in the form of a porous plate. In addition, the lipid library can be stored and frozen, for example in this porous plate. In a specific embodiment, lipids can be lyophilized to remove organic solvents before freezing and storage. In this case, the method of the present invention can represent a kind of integrated method for lipid library creation. The lipid library plate created can then be used to produce LNP, analyze and evaluate LNP in cell assays and animal studies. Then use the result to indicate the iterative direction of the next formula run using the method of the present invention to be run. This iterative process can produce new, improved LNP preparations.

[0140] In addition, the methods of the invention can be combined with screening of the formed LNPs in in vitro and / or in vivo cell assays, optionally followed by animal studies. The results of such assays and studies can influence subsequent iterations of LNP formulation design.

[0141] Furthermore, the method of the present invention may further comprise the step of creating a lipid library based on the dataset generated for the corresponding LNP formulation.

[0142] In a specific embodiment, the data set generated by the method of the present invention is continuously analyzed by one or more algorithms (e.g., during different iterations of the method). In this way, new and improved LNP formulation opportunities can be identified by methods such as reinforcement learning.

[0143] In a second aspect, the invention relates to a computer program product comprising computer readable instructions which, when loaded and run on a computer system, cause the computer system to perform operations according to the method of the invention. In this respect, all definitions of the method according to the first aspect of the invention also apply.

[0144] The computer-implemented method of the present invention for monitoring and controlling the process of manufacturing LNPs containing a nucleic acid cargo provides a tool for advantageously monitoring and controlling the process from laboratory to industrial scale in a robust and reproducible manner.

[0145] In a third aspect, the present invention relates to a computer-implemented method for monitoring and controlling a process for manufacturing lipid nanoparticles containing a nucleic acid cargo.

[0146] According to the present invention, the process for manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps:

[0147] (i) mixing a solution containing a nucleic acid cargo with a solution containing a lipid at an acidic pH;

[0148] (ii) allowing the nanoparticles obtained in step (i) to mature / stabilize for a specific residence time;

[0149] (iii) neutralizing the pH of the mixed solution obtained in step (i) by diluting to further stabilize the nanoparticles obtained in step (ii);

[0150] (iv) buffer exchanging the neutralized solution obtained in step (iii) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cryobuffer;

[0151] Furthermore, according to the present invention, the computer-implemented method for monitoring and controlling the process of manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps:

[0152] (a) monitoring one or more quality parameters of the lipid nanoparticles, such as particle size, polydispersity and nucleic acid encapsulation efficiency, in real time during each of steps (i) to (iv) and after step (iv);

[0153] (b) determining whether the quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is within a predetermined target range;

[0154] (c) if any quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is not within the predetermined target range, feedback control is provided to adjust the operating parameters of the corresponding step in real time so that the corresponding quality parameter enters the predetermined target range.

[0155] In a specific embodiment, the nucleic acid cargo is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA. In addition, the RNA can be selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

[0156] In a specific embodiment, the lipid used to produce the lipid nanoparticles in the process of manufacturing the lipid nanoparticles containing the nucleic acid cargo is an ionizable lipid. In addition, the lipid-containing solution may contain other agents selected from other ionizable lipids, structural lipids, auxiliary lipids (e.g., DSPC (distearoylphosphatidylcholine)), cholesterol, and polyethylene glycol (PEG)-lipid conjugates.

[0157] In step (i) of the process for producing lipid nanoparticles containing a nucleic acid cargo, a solution containing a nucleic acid cargo is mixed with a solution containing lipids at an acidic pH, wherein the nucleic acid cargo and the lipids are preferably as defined above. In a preferred embodiment, the nucleic acid cargo is contained in the solution containing the nucleic acid cargo at a concentration of 0.1 to 1 mg / mL. The solvent of the solution containing the nucleic acid cargo is preferably a suitable buffer, such as 20 to 100 mM citrate buffer, pH 3.0.

[0158] Furthermore, the lipid is preferably contained in the lipid-containing solution at a concentration of 50 to 60 mol%. The solvent of the lipid-containing solution is preferably a suitable organic solvent such as ethanol.

[0159] Therefore, the volume ratio between the solution containing the nucleic acid cargo and the solution containing the lipid can be 1:1 to 5:1, preferably about 3:1.

[0160] In a specific embodiment, the acidic pH is pH 2 to pH 6, preferably pH 3 to pH 6, more preferably pH 4 to pH 6, and most preferably about pH 5. In addition, the temperature of step (i) is not particularly limited, but step (i) is preferably carried out at room temperature, or at a temperature between room temperature and up to 40°C.

[0161] The means of mixing the solution containing the nucleic acid cargo and the solution containing the lipid are not particularly limited and are known in the art. However, the mixing is preferably high-speed laminar mixing, where nanoprecipitation is faster than nucleation.

[0162] Step (ii) of the process for producing lipid nanoparticles containing nucleic acid cargo is a step for maturation and / or stabilization of the lipid nanoparticles obtained in step (i) by maintaining the mixture obtained in step (i) for a certain residence time before neutralization in the next step. A person skilled in the art can select a suitable residence time, for example, from about 0.5 to about 60 minutes.

[0163] In step (iii) of the process for manufacturing lipid nanoparticles containing nucleic acid cargo, the pH of the mixed solution obtained in step (i) is neutralized by dilution (e.g., in-line dilution) to further stabilize the lipid nanoparticles. In this step, the mixed solution obtained in step (i) is neutralized by mixing with a solution having an alkaline pH, preferably phosphate buffered saline (PBS).

[0164] In a preferred embodiment, the mixed solution obtained in step (i) above is neutralized to a pH of pH 6.0 to pH 8.0, more preferably a pH of pH 7.0 to pH 8.0, more preferably a pH of pH 7.2 to pH 7.6, and most preferably a pH of about pH 7.4.

[0165] In step (iv) of the process for producing lipid nanoparticles containing nucleic acid cargo, the neutralized solution obtained in step (iii) is buffer exchanged by ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cryobuffer. The corresponding means for performing the buffer exchange are not particularly limited and are known in the art. Suitable cryobuffers include, for example, phosphate buffered saline (PBC) containing a suitable cryoprotectant, such as sucrose, mannitol, trehalose or sorbitol at a concentration of 0.1 to 10% by weight.

[0166] In a specific embodiment, the process for manufacturing lipid nanoparticles containing nucleic acid cargo further comprises step (ii-a), which is performed between the above steps (ii) and (iii). In step (ii-a), the solution obtained in the above step (ii) is diluted, and then the diluted solution is neutralized in step (iii). Thus, the mixed solution obtained in the above step (i) is diluted with a solution containing 20 to 30% of alcohol (preferably ethanol). The mixed solution is diluted 2 to 4 times based on the volume before dilution.

[0167] In step (a) of the method of the present invention, during each of steps (i) to (iv) and after step (iv), the quality parameters of the lipid nanoparticles, such as particle size, polydispersity and nucleic acid encapsulation efficiency, are monitored in real time. In a specific embodiment, all three of the above quality parameters are monitored. In other embodiments, only one of the quality parameters is monitored, or a combination of any two of the quality parameters is monitored.

[0168] In step (b) of the method of the present invention, it is determined whether the quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is within a predetermined target range.

[0169] In addition, in step (c) of the method of the present invention, if any quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) in the process of manufacturing lipid nanoparticles containing nucleic acid cargo is not within the predetermined target range, feedback control is provided to adjust the operating parameters in real time so that the corresponding quality parameter enters the predetermined target range.

[0170] The term "feedback control to adjust operating parameters" as used herein refers to feedback whether a quality parameter meets a target range or whether a measured value is above or below a target range. Based on this, the operating parameters in each of steps (i) to (iv) in the manufacturing process can be adjusted to bring the corresponding quality parameter into the target range.

[0171] In a specific embodiment, the operating parameters adjusted in step (c) of the method of the present invention are selected from the following group: nucleic acid flow rate, lipid flow rate, the ratio of nucleic acid flow rate to lipid flow rate, temperature, pressure, nucleation duration, particle growth duration, residence time, maturation period, pH, fluid composition, dilution, dilution rate, mixer type, flow mixing ratio and agitator / mixer speed.

[0172] The quality parameter "particle size" determines the size of the lipid nanoparticles containing the nucleic acid cargo. The target range of the particle size is within 5% of a specific target particle size value, where the target particle size value can be between 40 and 400 nm, depending on the intended therapeutic application. In a non-limiting embodiment, the particle size is determined by Virus Counter ® platform (SartoriusStedim Biotech GmbH, Germany), dynamic light scattering (DLS), PATfix ® HPLC platform, nanoparticle tracking analysis and / or multi-angle light scattering (MALS) determination.

[0173] If the particle size is above or below the target range, one or more of the above operating parameters are adjusted to reduce or increase the particle size to the predetermined target range.

[0174] The quality parameter "polydispersity" is a measure of the heterogeneity of the lipid nanoparticles containing the nucleic acid cargo. Polydispersity may occur due to size distribution, agglomeration or aggregation of the lipid nanoparticles in solution. The target range of the particle size is preferably 0.04 to 0.1. In a non-limiting embodiment, the polydispersity is measured by Virus Counter ® platform (SartoriusStedim Biotech GmbH, Germany), dynamic light scattering (DLS), PATfix ® The particle size distribution can be determined using an HPLC platform, nanoparticle tracking analysis, multi-angle light scattering (MALS), and / or other suitable tools known in the art for measuring particle size distribution.

[0175] If the polydispersity is above or below the target range, one or more of the above operating parameters are adjusted to reduce or increase the polydispersity to the predetermined target range.

[0176] The quality parameter "nucleic acid encapsulation efficiency" is a measure of the percentage of nucleic acid cargo successfully encapsulated / adsorbed into lipid nanoparticles. The target range for nucleic acid encapsulation compositions is preferably at least 96% or higher. In a specific embodiment, the nucleic acid encapsulation composition is measured by Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), Förster resonance energy transfer (FRET) assays and / or single particle automated Raman capture analysis (SPARTA).

[0177] If the nucleic acid encapsulating composition is above or below the target range, one or more of the above operating parameters are adjusted to increase the nucleic acid encapsulating composition to the predetermined target range.

[0178] In a specific embodiment, the pressure is measured during steps (i) and (iv) of the manufacturing process. A pressure above the target range indicates that fouling may be present.

[0179] In another embodiment, pH is measured during steps (i) and (ii) of the manufacturing process. The target range for pH is as described above.

[0180] In a fourth aspect, the invention relates to a computer program product comprising computer readable instructions which, when loaded and run on a computer system, cause the computer system to perform operations according to the method of the invention. In this respect, all definitions of the method of the first aspect of the invention also apply.

[0181] In a specific embodiment, the computer readable instructions include a control algorithm for feedback controlling the above-mentioned operating parameters, wherein the control algorithm is based on a mechanical hybrid model.

[0182] The computer-implemented method of the present invention for monitoring and controlling the process of manufacturing lipid nanoparticles containing nucleic acid cargo provides a tool for advantageously monitoring and controlling the process from laboratory to industrial scale in a robust and reproducible manner.

[0183] In this context, in a specific embodiment, the present invention includes creating and using algorithms to optimize the control of LNP manufacturing. Specifically, algorithms can be used to control the effects of values ​​associated with different operating parameters (e.g., nucleic acid flow rate, lipid flow rate, ratio of nucleic acid flow rate to lipid flow rate, temperature, pressure, nucleation duration, particle growth duration, residence time, maturation period, pH, fluid composition, dilution, dilution rate, mixer type, flow mixing ratio, and agitator / blender speed) to maximize the performance of LNP. By continuously training the algorithm model using techniques such as reinforcement learning, the robustness of LNP manufacturing will be continuously improved.

[0184] In a fifth aspect, the present invention relates to an automated nanoparticle synthesis system, the system comprising:

[0185] (a) two dispensers, each of the dispensers comprising a syringe pump and a 6-way valve, wherein the syringe pump is fluidically connected to an input port of the 6-way valve;

[0186] (b) 6-way distribution valve;

[0187] (c) Microfluidic mixer chip;

[0188] (d) waste liquid reservoir, buffer reservoir and ethanol reservoir;

[0189] in

[0190] (i) the dispensers are all fluidically connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir through a 6-way valve; and are fluidically connected to the microfluidic mixer chip;

[0191] (ii) a first one of the dispensers is further fluidly connected to a lipid supply via a 6-way valve;

[0192] (iii) a second one of the dispensers is further fluidly connected to an RNA supplier via a 6-way valve;

[0193] (iv) the microfluidic mixer chip is fluidically connected to an input port of the dispensing valve; and

[0194] (v) the dispensing valve is fluidically connected to the waste reservoir; and fluidically connected to a lipid nanoparticle (LNP) collection tube.

[0195] The automated nanoparticle synthesis system of the present invention comprises a first dispenser assembly having a first pump (e.g., a syringe pump) integrated with a first valve (e.g., a 6-way valve), and a second dispenser assembly having a second pump (e.g., a syringe pump) integrated with a second valve (e.g., a 6-way valve). The system further comprises a 6-way dispenser valve. The microfluidic mixer is used to achieve rapid and controllable mixing of lipids and RNA to generate LNPs ( Figure 3 The automated system is designed to increase the throughput of LNP screening campaigns for different microfluidic mixer designs as well as key flow parameters that influence LNP quality, such as FRR (flow rate ratio) and TFR (total flow rate).

[0196] The automatic nanoparticle synthesis system of the present invention can be divided into (i) a dispensing module for loading and dispensing reagents and pushing liquids to a microfluidic mixing component (e.g., a microfluidic mixing chip) located downstream; Figure 5 ), and (ii) a collection module for collecting LNPs synthesized under different screening conditions and storing them ( Figure 6 ). Figure 7 A demonstration version of the automated nanoparticle synthesis system of the present invention is shown.

[0197] like Figure 5 As shown, as a non-limiting example, the distribution module includes a lipid reservoir for containing a lipid stock solution and a nucleic acid reservoir for containing an aqueous nucleic acid stock solution (e.g., an RNA stock solution). These reservoirs can be commercially available reservoirs, such as pipes, holes, or bags. In other examples, the reservoir can contain other nucleic acid stock solutions, such as DNA, miRNA, cDNA, etc.

[0198] The first reservoir is fluidly connected to a first valve (e.g., a 6-way valve) on the first dispenser, and the second reservoir is fluidly connected to a second valve (e.g., a 6-way valve) on the second dispenser. The first valve is fluidly connected to a first pump (e.g., a syringe pump), and the second valve is fluidly connected to a second pump (e.g., a syringe pump). Each of the first valve and the second valve can be a commercially available valve. In other embodiments, in addition to a syringe pump, each of the first pump and the second pump can be any other suitable pump, such as a peristaltic pump, a centrifugal pump, a membrane pump, and / or a piston pump.

[0199] like Figure 5As shown, the first valve and the second valve are fluidly coupled to a waste liquid reservoir, a buffer reservoir and an ethanol reservoir (through separate ports on the first valve and the second valve and through pipelines). The ports at which the fluid on the first valve and the second valve is coupled to the waste liquid reservoir, the buffer reservoir and the ethanol reservoir are different from the ports at which the fluid is coupled to a lipid reservoir and a nucleic acid reservoir. The first valve and the second valve are each configured to automatically switch between a buffer reservoir, an ethanol reservoir, a lipid reservoir and / or a nucleic acid reservoir. These reservoirs can be commercially available reservoirs, such as pipelines, holes or bags.

[0200] like Figure 5 As shown, each of the first and second valves is fluidically coupled to a mixing assembly (e.g., a microfluidic mixer chip) via a pipeline. The mixing assembly can be used for online mixing of a lipid stock solution and an aqueous nucleic acid solution, using precise flow control of the two stock solutions to form a diluted intermediate product.

[0201] like Figure 3 and 6 As shown, as a non-limiting example of a collection module, a dispensing valve (e.g., a 6-way valve) is coupled to a mixing component (e.g., a microfluidic mixer chip) via a pipeline fluid. One port of the dispensing valve is coupled to a waste reservoir via a pipeline fluid, and other separate ports of the dispensing valve are coupled to one or more lipid nanoparticle (LNP) collection tubes via a pipeline fluid, which are used to collect and store synthetic lipid nanoparticles produced by a mixture of two stock solutions. The dispensing valve directs the waste flow to the waste reservoir while controlling the flow of the formulated lipid nanoparticles into the LNP collection tube. Although Figure 3 and Figure 6 Five LNP collection tubes are shown, but in other embodiments, the number of LNP collection tubes may be less than five or greater than five.

[0202] Figure 4 An alternative embodiment of an automated nanoparticle synthesis system is shown. Figure 3 Like the system shown in Figure 4 The system shown in also includes a first dispenser assembly having a first pump (e.g., a syringe pump) integrated with a first valve (e.g., a 6-way valve), and a second dispenser assembly having a second pump (e.g., a syringe pump) integrated with a second valve (e.g., a 6-way valve).

[0203] and Figure 3 As shown in the system, Figure 4The system shown also includes a waste reservoir, a buffer reservoir, and an ethanol reservoir, wherein the first valve and the second valve are each fluidically coupled to the waste reservoir, the buffer reservoir, and the ethanol reservoir through separate ports on the valve. The first valve and the second valve are each also fluidically coupled to a mixing assembly (e.g., a microfluidic mixer chip) to provide rapid and controllable mixing of lipids and RNA for generating LNPs.

[0204] and Figure 3 The system shown is different. Figure 4 The system shown does not include a dispensing valve for sample collection. Instead, Figure 4 The system shown includes a first liquid handler configured to automatically collect the synthesized LNPs, and a second liquid handler configured to automatically load reagents for a plurality of different combinations in a well plate, such as lipid stock solutions and RNA stock solutions. Figure 4 In the example shown, the first liquid handler is configured to automatically transfer the synthesized LNPs to a well plate (e.g., a 96-well plate) for collection and storage. The second liquid handler is configured to remove lipid stock and nucleic acid stock from the well plate (e.g., a 96-well plate). For example, the well plate may have a lipid-RNA library, wherein the lipid stock is present in one or more wells and the RNA stock is present in one or more wells that do not contain the lipid stock. Other embodiments may have only 1 liquid handler, which is configured to automatically collect the synthesized LNPs and load reagents.

[0205] Can be integrated Figure 4 Additional well plates and one or more liquid handlers in the system shown can further enhance Figure 3 The screening throughput and capacity of the system shown. Figure 4 The system shown does not require sample injection, which allows smaller sample volumes to be used.

[0206] Any system and its components described herein, including various pumps and valves described herein, can be controlled using any known control technology and / or any known control system. For example, in some embodiments, such components can be manually, electronically and / or hydraulically controlled.

[0207] exist Figure 3 In the system shown, up to five combinations of TFR and FRR (from 1 to 8) can be used in one automated run. In addition, screening flow rates ranging from approximately 5 µL / min to 30 mL / min can be used for each phase (aqueous / organic). Furthermore, the system described herein allows the use of sample volumes as small as 100 µL.

[0208] The amount of buffer and ethanol used in the automated nanoparticle synthesis system described herein depends on several factors, including: (i) the length and inner diameter of the tubing that defines the fluid path connecting the pump (e.g., syringe pump), microfluidic mixer chip, dispensing valve, and collection reservoir; (ii) the number of screens performed; and (iii) the number of cycles of cleaning (with ethanol) and priming (with buffer) the system. For example, when the total length of the tubing is 4000 mm and its inner diameter is 0.8 mm, the amount of buffer and ethanol required to perform a single screen and a single cleaning and priming cycle is typically less than 5 mL.

[0209] In a sixth aspect, the present invention relates to the use of one or more gas plugs in the system described herein to separate buffer, lipid-containing solution, RNA-containing solution and / or ethanol solution from each other in the tubing lines providing fluid connections between the components.

[0210] The air plug is a certain amount of air introduced into the pipeline line to provide separation between the buffer, the solution containing lipids, the solution containing RNA and / or the ethanol solution. For example, the microfluidic mixer chip can include an air plug that separates the RNA solution from the buffer and separates the buffer from the lipid solution and separates the lipid solution from the ethanol solution. In one embodiment, the volume of the air plug is between 1 μL and 20 μL, preferably about 5 μL.

[0211] Without an air stopper, RNA and lipids can diffuse within the tubing into the surrounding fluids (e.g., buffer and ethanol). This unwanted diffusion can alter RNA concentration and lipid / RNA mixing ratios during particle synthesis by the microfluidic mixer. In preliminary experiments without an air stopper, RNA was found in discarded samples, whereas RNA was found in most samples with an air stopper. Therefore, an air stopper is particularly important for preparing smaller sample volumes while minimizing reagent waste.

[0212] Implementation of a relevant LNP screening workflow according to the present disclosure may involve the following steps (a) to (f):

[0213] (a) Adding a gas plug to the pipeline line of the nanoparticle synthesis system;

[0214] (b) loading lipids and RNA into a sample loop of the system;

[0215] (c) adding another gas lock to the piping line of the system;

[0216] (d) driving the lipids and RNA to meet at the flow focusing junction of the system;

[0217] (e) Coordinating flows to generate LNPs; and

[0218] (f) Switching the valves of the system to collect LNP.

[0219] In this workflow, the nanoparticle synthesis system is preferably the nanoparticle synthesis system of the present disclosure. Figure 3 The automated nanoparticle synthesis system shown in .

[0220] In some embodiments, the LNP screening based on the above method is performed by a slow protocol involving microfluidic chip cleaning and priming steps between screening tasks. In alternative embodiments, the LNP screening based on the above method is performed by a fast protocol without cleaning and priming steps between screening tasks.

[0221] Figure 8 An exemplary user interface of control software that can be used with the automated nanoparticle synthesis system of the present disclosure is shown. The software provides an easy-to-use interface that displays, for example, the flow rate ratio (FFR), total flow rate (TFR), and sample volume selected by the user. Different microfluidic chip designs can be added to the library for future screening. An advanced manual mode enables the user to control each device in the system more flexibly.

[0222] As used herein, the term "comprising" expressly includes the terms "consisting essentially of" and "consisting of, ie, all terms are used interchangeably herein.

[0223] In addition, as used herein, the term "about" preferably indicates a modification of the specified value, i.e., ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1% or ±0.5%. Thus, for example, the term "about 100" may include a range of 90 to 110, 92 to 108, 94 to 106, 95 to 105, 96 to 104, 97 to 103, 98 to 102, 99 to 101 or 99.5 to 100.5.

[0224] The present invention advantageously provides a method for high-throughput preparation of LNPs containing nucleic acid cargo, so that each person can prepare hundreds of different LNP formulations simultaneously every day. This is combined with online and real-time monitoring of important quality parameters (e.g., particle size, polydispersity, nucleic acid encapsulation efficiency). Therefore, the present invention provides a fast and easy-to-use means for generating, screening and analyzing LNPs in a high-throughput format.

[0225] The present invention according to the fifth aspect will be further described in the following examples, but is not limited thereto.

[0226] Example

[0227] 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) blank LNP formulations were prepared using the automated nanoparticle synthesis system disclosed herein. The DOTAP blank LNP formulation included 4 lipids: DOTAP, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG 2000. The molar ratio of these lipids was 40:10:48:2. The lipids were dissolved in ethanol at a concentration of about 7.5 mM and a citric acid buffer at a pH of 4 and a concentration of about 10 mM as the aqueous phase. In this example, the LNP production scale was 200 uL. Five LNP samples were produced continuously. The flow rate ratio (FRR) was 2 and the total flow rate (TFR) was 200 µL / min.

[0228] After each sample production, the microfluidic chip and production system were cleaned with 10 mM citrate buffer and ethanol. Particle size (Z-average) and polydispersity index (PDI) were measured using a Zetasizer Pro (Malvern).

[0229] The effect of the flow rate ratio between lipid and mRNA and the total flow rate in the device on size and polydispersity index was investigated. The same DOTAP formulation was prepared. Mixing was performed using different FRRs (1, 2, 4, 8) and TFRs (100 and 200 µL / min). In this study, the LNP production volume was 100 µL and the final lipid concentration was 2.5 mM. After each sample production, the microfluidic chip and production system were cleaned with 10 mM citrate buffer and ethanol. Particle size (Z-average) and polydispersity index (PDI) were measured using a Zetasizer Pro (Malvern).

Claims

1. A computer-implemented method for high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, the method comprising the following steps: (a) providing one or more solutions containing a nucleic acid cargo; (b) providing one or more solutions comprising lipids; (c) mixing one or more solutions comprising a nucleic acid cargo with one or more solutions comprising a lipid at an acidic pH; (d) neutralizing the pH of the mixed solution obtained in step (c) by diluting to stabilize the LNP obtained in step (c); (e) buffer exchanging the neutralized solution obtained in step (d) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (d) and reconstitute them in a cryobuffer to produce one or more LNP formulations; in During each of steps (c) to (e) and after step (e), one or more of the particle size, polydispersity, and nucleic acid encapsulation efficiency of the one or more LNPs are monitored in real time to generate a data set for each LNP formulation; and the generated data set is associated with the corresponding LNP formulation.

2. The computer-implemented method of claim 1, wherein two or more separate LNP formulations are generated in parallel.

3. The computer-implemented method of claim 1, wherein one or more solutions comprising a nucleic acid cargo and one or more solutions comprising a lipid are provided in one or more multi-well plates.

4. The computer-implemented method of claim 1 , further comprising stabilizing the LNP obtained in step (c) for a specific period of time prior to step (d).

5. The computer-implemented method of claim 1, wherein one or more LNP formulations are generated in one or more multi-well plates.

6. The computer-implemented method of claim 1, wherein the nucleic acid is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

7. The computer-implemented method of claim 6, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

8. The computer-implemented method of claim 1, wherein the lipid is an ionizable lipid.

9. The computer-implemented method of claim 1, wherein the pH in step (c) is between pH 2 and pH 6.

10. The computer-implemented method of claim 4, wherein the time is from 0.1 minutes to 60 minutes. 11 . The computer-implemented method of claim 1 , wherein the pH of the neutralization solution in step (d) is between pH 6 and pH 8.

12. The computer-implemented method of claim 1, wherein the buffer exchange in step (e) is performed with a buffer comprising phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris) buffered saline.

13. The computer-implemented method of claim 1, wherein one or more of the particle size, polydispersity, and nucleic acid encapsulation efficiency of the one or more LNPs is measured and / or analyzed by at least one of the following: Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS) and multi-angle light scattering (MALS), single particle automated Raman capture analysis (SPARTA), high-speed imaging technology, PATfix ® HPLC platform, nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay.

14. The computer-implemented method of claim 1, wherein the method further comprises the step of creating a lipid library based on a dataset generated for a corresponding LNP formulation.

15. A computer program product comprising computer readable instructions which, when loaded and executed on a computer system, cause the computer system to perform operations according to the method of claim 1.

16. A computer-implemented method for monitoring and controlling a process for manufacturing lipid nanoparticles containing a nucleic acid cargo, The process of manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps: (i) mixing a solution containing a nucleic acid cargo with a solution containing a lipid at an acidic pH; (ii) stabilizing the nanoparticles obtained in step (i) for a specific residence time; (iii) neutralizing the pH of the mixed solution obtained in step (i) by diluting to further stabilize the nanoparticles obtained in step (ii); (iv) buffer exchanging the neutralized solution obtained in step (iii) by ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cryobuffer; The computer-implemented method for monitoring and controlling the process of manufacturing lipid nanoparticles containing nucleic acid cargo comprises the following steps: (a) monitoring one or more quality parameters of the lipid nanoparticles, such as particle size, polydispersity and nucleic acid encapsulation efficiency, in real time during each of steps (i) to (iv) and after step (iv); (b) determining whether the quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is within a predetermined target range; (c) if any quality parameter obtained in step (a) during each of steps (i) to (iv) and after step (iv) is not within the predetermined target range, feedback control is provided to adjust the operating parameters of the corresponding step in real time so that the corresponding quality parameter enters the predetermined target range.

17. The computer-implemented method of claim 16, wherein the nucleic acid is selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

18. The computer-implemented method of claim 17, wherein the RNA is selected from the group consisting of messenger RNA (mRNA), self-amplifying RNA (saRNA), guide RNA (gRNA), antisense oligonucleotide (ASO), transfer RNA (tRNA), short hairpin RNA (shRNA), circular RNA (circRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

19. The computer-implemented method of claim 16, wherein the lipid is an ionizable lipid.

20. The computer-implemented method of claim 16, wherein the pH in step (i) is between pH 2 and pH 6.

21. The computer-implemented method of claim 16, wherein the dwell time is between 0.5 minutes and 60 minutes.

22. The computer-implemented method of claim 16, wherein the pH of the neutralization solution in step (iii) is between pH 6 and pH 8.

23. The computer-implemented method of claim 16, wherein the buffer exchange in step (iv) is performed with a buffer comprising phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris) buffered saline.

24. The computer-implemented method of claim 16, wherein the quality parameters include one or more of particle size, polydispersity, and nucleic acid encapsulation efficiency of lipid nanoparticles, and the quality parameters are measured and / or analyzed by at least one of the following: Virus Counter ® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS) and multi-angle light scattering (MALS), single particle automated Raman capture analysis (SPARTA), high-speed imaging technology, PATfix ® HPLC platform, nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay.

25. The computer-implemented method of claim 16, wherein the target range for particle size is within a 5% variation of a specific target particle size value, wherein the target particle size value is between 40 nm and 400 nm.

26. The computer-implemented method of claim 16, wherein the target range for polydispersity is 0.04 to 0.

1.

27. The computer-implemented method of claim 16, wherein the target range for nucleic acid encapsulation efficiency is at least 96% or higher.

28. The computer-implemented method of claim 16, wherein the operating parameter adjusted in step (c) is selected from the group consisting of nucleic acid flow rate, lipid flow rate, ratio of nucleic acid flow rate to lipid flow rate, temperature, pressure, nucleation duration, particle growth duration, residence time, maturation period, pH, fluid composition, dilution, dilution rate, mixer type, flow mixing ratio, and stirrer / mixer speed.

29. A computer program product comprising computer readable instructions which, when loaded and executed on a computer system, cause the computer system to perform operations according to the method of claim 16.

30. The computer program product of claim 29, wherein the computer readable instructions include a control algorithm for feedback control of an operating parameter, wherein the control algorithm is based on a mechanical hybrid model.

31. An automated nanoparticle synthesis system, the system comprising: (a) a first dispenser assembly having a first pump integrated with a first valve; (b) a second dispenser assembly having a second pump integrated with a second valve; (c) a third valve; (d) Microfluidic mixer; (e) a lipid reservoir for containing a lipid stock solution; (f) a nucleic acid storage container for containing a nucleic acid stock solution; as well as (g) waste liquid reservoir, buffer reservoir and ethanol reservoir; in (i) the lipid reservoir is fluidically connected to the first valve and the nucleic acid reservoir is fluidically connected to the second valve, or vice versa; (ii) the first valve is fluidly connected to the first pump and the second valve is fluidly connected to the second pump; (iii) the first valve and the second valve are each fluidically connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, wherein ports of the first valve and the second valve that are fluidly connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir are different from ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) the first valve and the second valve are each fluidly connected to the microfluidic mixer; and (v) A third valve is fluidically connected to the microfluidic mixer, a waste reservoir, and one or more collection reservoirs for collecting the produced lipid nanoparticles (LNPs).

32. The automated nanoparticle synthesis system of claim 31, wherein the first pump and / or the second pump is a syringe pump.

33. The automated nanoparticle synthesis system of claim 31, wherein the first valve, the second valve and / or the third valve are 6-way valves.

34. The automated nanoparticle synthesis system of claim 31, wherein the fluid connection is achieved via tubing.

35. An automated nanoparticle synthesis system according to claim 31, wherein the system further comprises one or more gas plugs suitable for separating a buffer from a solution containing RNA, one or more gas plugs separating a buffer from a solution containing lipids, and one or more gas plugs separating a solution containing lipids from ethanol.

36. The automated nanoparticle synthesis system of claim 31, wherein the microfluidic mixer is a microfluidic mixer chip.

37. An automated nanoparticle synthesis system, the system comprising: (a) a first dispenser assembly having a first pump integrated with a first valve; (b) a second dispenser assembly having a second pump integrated with a second valve; (c) Microfluidic mixer; (d) one or more liquid handlers, wherein the liquid handlers are configured to automatically load reagents and automatically collect samples in one or more well plates; and (e) waste liquid reservoir, buffer reservoir and ethanol reservoir; in (i) the lipid reservoir is fluidically connected to the first valve and the nucleic acid reservoir is fluidically connected to the second valve, or vice versa; (ii) the first valve is fluidly connected to the first pump and the second valve is fluidly connected to the second pump; (iii) the first valve and the second valve are each fluidically connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, wherein ports of the first valve and the second valve that are fluidly connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir are different from ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) the first valve and the second valve are each fluidly connected to the microfluidic mixer; and (v) The microfluidic mixer is fluidically connected to the liquid handler.

38. The automated nanoparticle synthesis system of claim 37, wherein the first pump and / or the second pump is a syringe pump.

39. The automated nanoparticle synthesis system of claim 37, wherein the first valve and / or the second valve is a 6-way valve.

40. The automated nanoparticle synthesis system of claim 37, comprising a first liquid handler and a second liquid handler, wherein the first liquid handler is configured to automatically collect synthesized LNPs and the second liquid handler is configured to automatically load reagents.

41. The automated nanoparticle synthesis system of claim 37, wherein the fluid connection is achieved via tubing.

42. An automated nanoparticle synthesis system according to claim 37, wherein the system further comprises one or more gas plugs suitable for separating a buffer from a solution containing RNA, one or more gas plugs for separating a buffer from a solution containing lipids, and one or more gas plugs for separating a solution containing lipids from ethanol.

43. The automated nanoparticle synthesis system of claim 37, wherein the microfluidic mixer is a microfluidic mixer chip.

44. The automated nanoparticle synthesis system of claim 37, wherein one or more of the liquid handlers are configured to automatically remove the lipid-containing solution and the RNA-containing solution from the well plate.

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