Lipid nanoparticle production system and method for monitoring and controlling the same

Abstract

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

Description

[Technical Field]

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

[0002] Background of the Invention In recent years, LNPs containing nucleic acid cargo have attracted considerable attention, for example, due to the development of messenger ribonucleic acid (mRNA)-based vaccines. This allows mRNA (or any other therapeutic nucleic acid of interest) to be encapsulated in LNPs for stable delivery and efficient transfection into target cells. In general, LNPs protect the cargo from degradation, deliver the therapeutic agent to target cells or tissues, and control the release of the cargo at the desired location. LNPs are typically composed of amino lipids (ionic or cationic amino lipids) as the main components, as well as phosphatidylcholine lipids, cholesterol, and polyethylene glycol lipid conjugates (PEG lipids). In the manufacturing process, a solution containing a nucleic acid cargo is mixed with a solution containing a lipid at an acidic pH, followed by neutralization, buffer exchange, and concentration to obtain LNPs containing the nucleic acid cargo.

[0003] However, there are some challenges in the manufacturing development and screening of LNPs, because there is currently no method for the large-scale production of LNPs that can rapidly and easily produce a variety of LNPs, followed by screening and analysis.In particular, large-scale screening of LNPs is hindered by current manufacturing methods that can produce less than approximately 20 LNPs per person per day, using offline assays for analyzing 3-4 or fewer parameters of the LNPs, which takes 2-3 days to complete.Increasing control during the manufacturing process will also improve the scale-up of the process from laboratory scale (less than 2 mg) to industrial scale (2 g or more). Therefore, there is a strong need to provide methods for the preparation and analysis of large-scale LNPs containing nucleic acid cargos, and to provide methods for monitoring and controlling the process of producing lipid nanoparticles containing nucleic acid cargos in real time, thereby increasing the robustness and reproducibility of the process. This need is met by providing the embodiments characterized in the claims. Summary of the Invention

[0004] Summary of the Invention The present invention relates to the following items: 1. A computer-implemented method for the high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, comprising: (a) providing one or more solution(s) containing nucleic acid cargo; (b) providing one or more solutions comprising lipids; (c) mixing one or more solutions containing nucleic acid cargo with one or more solutions containing lipids at an acidic pH; (d) neutralizing the pH of the mixed solution obtained in step (c) through dilution to stabilize the LNPs obtained in step (c); (e) performing buffer exchange on the neutralized solution obtained in step (d) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (d) and reformulate them into a cold buffer, thereby producing one or more LNP preparation(s); The method includes the steps of: where: During each of steps (c) through (e), and after step (e), one or more of particle size, polydispersity, and nucleic acid encapsulation of the one or more LNPs are monitored in real time, thereby generating a dataset for each LNP preparation; and the generated dataset is associated with each LNP preparation. The implementation method.

[0005] 2. The computer-implemented method of paragraph 1, wherein two or more individual LNP preparations are generated in parallel. 3. The computer-implemented method of paragraph 1, wherein one or more solutions containing nucleic acid cargo and one or more solutions containing lipids are provided in one or more multiwell 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 paragraph 1, wherein the one or more LNP preparation(s) are generated in one or more multi-well plates. 6. The computer-implemented method of paragraph 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.

[0006] 7. The computer-implemented method of paragraph 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), microRNA (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 in the range of pH 2 to pH 6. 10. The computer-implemented method of claim 4, wherein the time period ranges from 0.1 to 60 minutes. 11. The computer-implemented method of claim 1, wherein the pH of the neutralization solution in step (d) ranges from pH 6 to pH 8. 12. The computer-implemented method of paragraph 1, wherein the buffer exchange in step (e) is performed with a buffer solution, and the buffer solution comprises phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris)-buffered saline.

[0007] 13. One or more of the particle size, polydispersity, and nucleic acid encapsulation of one or more LNPs are selected from 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 and analysis (SPARTA), High-speed imaging technology, PATfix® HPLC platform, Nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay, 2. The computer-implemented method of claim 1, wherein the measurement and / or analysis can be performed by at least one of:

[0008] 14. The computer-implemented method of claim 1, further comprising creating a lipid library based on the dataset generated for each LNP preparation. 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 in accordance with the method set forth in paragraph 1.

[0009] 16. A computer-implemented method for monitoring and controlling a process for producing lipid nanoparticles containing nucleic acid cargo, comprising: wherein the process for producing lipid nanoparticles containing nucleic acid cargo comprises: (i) mixing a solution containing nucleic acid cargo with a lipid-containing solution 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) via dilution in order to further stabilize the nanoparticles obtained in step (ii); (iv) performing buffer exchange on the neutralized solution obtained in step (iii) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cold buffer; The method includes the steps of: The computer-implemented method for monitoring and controlling the process for producing lipid nanoparticles containing nucleic acid cargo comprises: (a) monitoring one or more quality parameters, such as particle size, polydispersity, and nucleic acid encapsulation of the lipid nanoparticles in real time during each of steps (i) to (iv) and after step (iv); (b) determining whether the quality parameters obtained in step (a) during each of steps (i)-(iv) and after step (iv) are within predetermined target ranges; (c) providing a feedback control that adjusts the operating parameters of each step in real time during each of steps (i) to (iv) and after step (iv), if any of the quality parameters obtained in step (a) are not within the predetermined target range, to bring each quality parameter within the predetermined target range; The steps include: The implementation method.

[0010] 17. The computer-implemented method of paragraph 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 paragraph 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), microRNA (miRNA), and small interfering RNA (siRNA). 19. The computer-implemented method of paragraph 16, wherein the lipid is an ionizable lipid. 20. The computer-implemented method of claim 16, wherein the pH in step (i) is in the range of pH 2 to pH 6. 21. The computer-implemented method of claim 16, wherein the residence time is in the range of 0.5 to 60 minutes. 22. The computer-implemented method of claim 16, wherein the pH of the neutralization solution in step (iii) ranges from pH 6 to pH 8. 23. The computer-implemented method of paragraph 16, wherein the buffer exchange in step (iv) is performed with a buffer solution, the buffer solution comprising phosphate-buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (Tris)-buffered saline.

[0011] 24. The quality parameters include one or more of particle size, polydispersity, and nucleic acid encapsulation of the lipid nanoparticles, and the quality parameters are selected from 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 and analysis (SPARTA), High-speed imaging technology, PATfix® HPLC platform, Nanoparticle tracking analysis, and Förster resonance energy transfer (FRET) assay, 17. The computer-implemented method of claim 16, wherein the measurement and / or analysis can be performed by at least one of: 25. The computer-implemented method of paragraph 16, wherein the target range of particle size is within a 5% variation of a specific target particle size value, where the target particle size is between 40 and 400 nm. 26. The computer-implemented method of paragraph 16, wherein the target range of polydispersity is from 0.04 to 0.1. 27. The computer-implemented method of paragraph 16, wherein the nucleic acid encapsulation has a target coverage of at least 96% or greater. 28. The computer-implemented method of paragraph 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 period, particle growth period, residence time, maturation period, pH, fluid composition, dilution degree, dilution ratio, mixer type, stream mixing ratio, and stirring / agitator speed.

[0012] 29. A computer program product comprising computer readable instructions which, when loaded and executed on a computer system, causes the computer system to perform operations in accordance with the method set out in clause 16. 30. The computer program product of paragraph 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 mechanistic hybrid model.

[0013] 31. An automated nanoparticle synthesis 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) third valve; (d) microfluidic mixer; (e) a lipid reservoir for holding lipid storage solution; (f) a nucleic acid reservoir for holding a nucleic acid storage solution; and (g) a waste reservoir, a buffer reservoir, and an ethanol reservoir; Including, where: (i) the lipid reservoir is in fluid communication with a first valve and the nucleic acid reservoir is in fluid communication with a second valve, or vice versa; (ii) the first valve is in fluid communication with the first pump and the second valve is in fluid communication with the second pump; (iii) each of the first valve and the second valve is fluidly connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, and wherein the 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 the ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) each of the first valve and the second valve is fluidly connected to a microfluidic mixer; and (v) a third valve fluidly connected to the microfluidic mixer, to a waste reservoir, and to one or more collection reservoirs for collecting the produced lipid nanoparticles (LNPs); The system.

[0014] 32. The automated nanoparticle synthesis system according to item 31, wherein the first pump and / or the second pump are syringe pumps. 33. The automated nanoparticle synthesis system according to item 31, wherein the first valve, the second valve, and / or the third valve are six-port valves. 34. An automated nanoparticle synthesis system as described in paragraph 31, wherein fluid communication is achieved via tubing. 35. The automated nanoparticle synthesis system described in paragraph 31, wherein the system further comprises one or more air valves adapted to separate the buffer solution from the RNA-containing solution, one or more air valves that separate the buffer solution from the lipid-containing solution, and one or more air valves that separate the lipid-containing solution from the ethanol. 36. The automated nanoparticle synthesis system described in item 31, wherein the microfluidic mixer is a microfluidic mixer chip.

[0015] 37. An automated nanoparticle synthesis 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 configured to automate the loading of reagents and to automate sample collection in one or more well plates; and (e) a waste reservoir, a buffer reservoir, and an ethanol reservoir; Including, where: (i) the lipid reservoir is in fluid communication with a first valve and the nucleic acid reservoir is in fluid communication with a second valve, or vice versa; (ii) the first valve is in fluid communication with the first pump and the second valve is in fluid communication with the second pump; (iii) each of the first valve and the second valve is fluidly connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, and wherein the 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 the ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) each of the first valve and the second valve is fluidly connected to a microfluidic mixer; and (v) the microfluidic mixer is in fluid communication with the liquid handler; The system.

[0016] 38. The automated nanoparticle synthesis system according to item 37, wherein the first pump and / or the second pump are syringe pumps. 39. The automated nanoparticle synthesis system according to item 37, wherein the first valve and / or the second valve is a six-port valve. 40. The automated nanoparticle synthesis system of paragraph 37, comprising a first liquid handler and a second liquid handler, wherein the first liquid handler is configured to automate collection of synthesized LNPs and the second liquid handler is configured to automate loading of reagents. 41. An automated nanoparticle synthesis system as described in paragraph 37, wherein fluid communication is achieved via tubing.

[0017] 42. The automated nanoparticle synthesis system described in paragraph 37, wherein the system further comprises one or more air valves adapted to separate the buffer solution from the RNA-containing solution, one or more air valves that separate the buffer solution from the lipid-containing solution, and one or more air valves that separate the lipid-containing solution from the ethanol. 43. The automated nanoparticle synthesis system described in item 37, wherein the microfluidic mixer is a microfluidic mixer chip. 44. The automated nanoparticle synthesis system described in paragraph 37, wherein one or more of the liquid handlers are configured to automatically remove lipid-containing solutions and RNA-containing solutions from the well plate. [Brief explanation of the drawings]

[0018] Brief description of the diagram [Figure 1] FIG. 1 shows an exemplary method for the large-scale preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo. [Figure 2] FIG. 2 illustrates a process for producing lipid nanoparticles containing nucleic acid cargo of the present invention, wherein the process includes steps of mixing, neutralizing, buffer exchange, and concentrating a solution containing the product, and illustrates the formation of lipid nanoparticles containing nucleic acid cargo. [Figure 3] FIG. 3 illustrates an embodiment of an automated nanoparticle synthesis system, according to some embodiments. [Figure 4] FIG. 4 illustrates an alternative embodiment of an automated nanoparticle synthesis system, according to some embodiments. [Figure 5] FIG. 5 illustrates an embodiment of a dispensing module of the automated nanoparticle synthesis system illustrated in FIG. 3, according to some embodiments.

[0019] [Figure 6] FIG. 6 illustrates an embodiment of a collection module of the automated nanoparticle synthesis system illustrated in FIG. 3, according to some embodiments. [Figure 7] FIG. 7 shows a demonstration of the automated nanoparticle synthesis system illustrated in FIG. 3, according to some embodiments. [Figure 8] FIG. 8 shows an embodiment of a user interface of the control software for the automated nanoparticle synthesis system illustrated in FIGS. 3 and 4, according to some embodiments. [Figure 9] Figure 9 shows the reproducibility of LNPs. Five different LNP samples produced using the automated nanoparticle synthesis system of the present invention show reproducible sizes and polydispersity index (PDI). [Figure 10] FIG. 10 shows the effect of lipid to mRNA flow ratio and total flow rate within the device on size and PDI. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description of the Invention In a first aspect, the present invention relates to a computer-implemented method for the high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, comprising: (a) providing one or more solution(s) containing nucleic acid cargo; (b) providing one or more solutions comprising lipids; (c) mixing one or more solutions containing nucleic acid cargo with one or more solutions containing lipids at an acidic pH; (d) maturation / stabilization of the LNPs obtained in step (c) for a specific residence time; (e) neutralizing the pH of the mixed solution obtained in step (c) by dilution to further stabilize the LNPs obtained in step (d); (f) performing buffer exchange on the neutralized solution obtained in step (e) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (e) and reformulate them into a cold buffer, thereby producing one or more LNP preparation(s); The method includes the steps of: where: During each of steps (c) through (f), and after step (f), one or more of particle size, polydispersity, and nucleic acid encapsulation of the one or more LNPs are monitored in real time, thereby generating a dataset for each LNP preparation; the generated dataset is associated with the respective LNP preparation and stored for further use.

[0021] As used herein, the term "computer-implemented method" refers to a method that is automatically performed on a computer. Thus, the actual preparation of the LNPs, as well as the monitoring and control of the lipid nanoparticle manufacturing process, the generation of the data set, and the storage of the data set, are performed automatically by a computer.

[0022] In certain embodiments, 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), microRNA (miRNA), and small interfering RNA (siRNA). In certain embodiments, the lipids used in the production of LNPs in the process of manufacturing LNPs containing nucleic acid cargo are ionizable lipids. Additionally, the lipid-containing solution may contain an additional agent selected from the group consisting of additional ionizable lipids, structural lipids, helper lipids (e.g., DSPC (distearoylphosphatidylcholine)), cholesterol, and polyethylene glycol (PEG) lipid conjugates.

[0023] In step (c) of the method of the present invention, one or more solutions containing nucleic acid cargos are mixed with one or more solutions containing lipids at an acidic pH, wherein the nucleic acid cargos and lipids are preferably as defined above. In a preferred embodiment, the nucleic acid cargos are contained in the solution containing nucleic acid cargos at a concentration of 0.1 to 1 mg / mL. The solvent for the solution containing nucleic acid cargos is preferably a suitable buffer (e.g., 20 to 100 mM citrate buffer, pH 3.0). Furthermore, the lipids are preferably contained in the solution containing lipids at a concentration of 50 to 60 mol %. The solvent for the solution containing lipids is preferably a suitable organic solvent (e.g., ethanol). Thus, the volume ratio of the solution containing nucleic acid cargos to the solution containing lipids can be 1:1 to 5:1, preferably about 3:1. In certain embodiments, the acidic pH is from pH 2 to pH 6, preferably from pH 3 to pH 6, more preferably from pH 4 to pH 6, and most preferably about pH 5. Moreover, the temperature at which step (c) is carried out is not particularly limited, but is preferably room temperature or a temperature between room temperature and 40° C. The means for mixing the solution(s) containing nucleic acid cargo with the solution(s) containing lipid is not particularly limited and is known in the art.However, the mixing is preferably high-speed laminar flow mixing, in which nanoprecipitation is faster than nucleation.Suitable mixers and mixing devices are not particularly limited and are known in the art.

[0024] Step (d) of the method of the present invention is a step of aging and / or stabilizing the LNPs obtained in step (c) by maintaining the mixture obtained in step (c) for a certain residence time prior to neutralization in the next step. A suitable residence time can be selected by one skilled in the art, for example, from about 0.1 to about 60 minutes. In step (e) of the method of the present invention, the pH of the mixed solution obtained in step (c) is neutralized via dilution (e.g., in-line dilution) to further stabilize the LNPs. In this step, neutralization is carried out by mixing the mixed solution obtained in step (d) with a solution having a basic pH, preferably phosphate buffered saline (PBS).

[0025] In a preferred embodiment, the mixed solution obtained in step (d) above is neutralized to a pH ranging from pH 6.0 to pH 8.0, more preferably to a pH ranging from pH 7.0 to pH 8.0, more preferably to a pH ranging from pH 7.2 to pH 7.6, and most preferably to a pH of about pH 7.4. In step (f) of the method of the present invention, the neutralized solution obtained in step (e) is subjected to buffer exchange via ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (e) and reconstitute them into a low-temperature buffer. The means for performing the buffer exchange are not particularly limited and are known in the art. Suitable low-temperature buffers 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.

[0026] According to the present invention, during each of steps (c) to (f) and after step (f), one or more of particle size, polydispersity, and nucleic acid encapsulation of one or more LNPs are monitored in real time, thereby generating a data set for each LNP preparation; the generated data set is associated with each LNP preparation and stored for further use. In certain embodiments, all three of particle size, polydispersity, and nucleic acid encapsulation 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.

[0027] As used herein, the term "in real time" refers to the fact that, according to the methods of the present invention, the above parameters (e.g., quality parameters) of the LNPs prepared in the methods of the present invention are monitored while the methods are in progress. The parameter "particle size" determines the size of the LNP containing the nucleic acid cargo. In non-limiting embodiments, the particle size is determined by a Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), a PATfix® HPLC platform, nanoparticle tracking analysis, and / or multi-angle light scattering (MALS).

[0028] The parameter "polydispersity" is a measure of the heterogeneity of LNPs containing nucleic acid cargo. Polydispersity can occur due to size distribution, aggregation, or aggregation of LNPs in solution. In a non-limiting embodiment, polydispersity is determined by a Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), a PATfix® HPLC platform, nanoparticle tracking analysis, multi-angle light scattering (MALS), and / or other suitable tools for measuring particle size distribution, as known in the art. The parameter "nucleic acid encapsulation" is a measure of the percentage of nucleic acid cargo successfully encapsulated / adsorbed into the LNP. In certain embodiments, the nucleic acid encapsulation composition is determined by the Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), Förster resonance energy transfer (FRET) assay, and / or single particle automated Raman capture analysis (SPARTA).

[0029] According to the present invention, a dataset is generated for each LNP preparation, i.e., for each of one or more LNPs prepared in the method of the present invention. This dataset includes data obtained by monitoring one or more of the particle size, polydispersity, and nucleic acid encapsulation of one or more LNPs during each of steps (c) through (f) and after step (f). Furthermore, according to the present invention, each dataset is associated with a respective LNP preparation, i.e., each dataset further includes information about which LNP preparation it belongs to. The dataset is then stored for further use, for example, to provide feedback regulation of the preparation steps or to enable evaluation and comparison of different LNP preparations.

[0030] In a specific and preferred embodiment of the method of the present invention, two or more individual LNP preparations are produced in parallel.Other than the technical limitations imposed by the liquid handling and sample handling equipment, there is no specific upper limit to the number of LNP preparations that can be produced in parallel.In this context, one or more solution(s) containing nucleic acid cargo and / or one or more solution(s) containing lipids can be provided in one or more multi-well plate(s).Suitable multi-well plate is not particularly limited, and examples include 6-well, 12-well, 24-well, 48-well, 96-well, 384-well and nanowell microtiter plate known in the art.In a more specific and preferred embodiment, one or more LNP preparation(s) are also produced in one or more respective multi-well plate(s). Thus, in a preferred embodiment, the method of the present invention provides the possibility of generating two or more LNP preparations in parallel (e.g., 96 LNP preparations in a 96-well plate format). In such embodiments, the individual LNP preparations can differ from each other in any manner of interest. This includes differences in the nucleic acid-containing solution (e.g., different nucleic acids, different nucleic acid amounts, different buffer compositions, different pH, different osmolality), differences in the lipid-containing solution (e.g., different lipids, different structural lipids, different helper lipids, different PEG-lipid conjugates, different buffer compositions, different lipid compositions, different lipid amounts, different pH, different osmolality), and / or differences in the process parameters in steps (c)-(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, individual LNP preparations can be compared with each other, and the effects of different parameters can be evaluated. This further enables convenient screening methods for, for example, nucleic acid libraries, lipid libraries, and the like.

[0031] The technical means for carrying out the method of the present invention are not particularly limited and include robotic liquid handling and laboratory robotic systems known in the art (for example, the Sartorius Ambr® system). In certain embodiments, the methods of the present invention can be used with lipid libraries to screen new LNP formulations. Such lipid libraries can be provided in the form of multi-well plates, as indicated above. Furthermore, lipid libraries can be stored and frozen (for example, within such multi-well plates). In certain embodiments, lipids can be lyophilized to remove organic solvents prior to freezing and storage. In this context, the methods of the present invention can represent an integrated approach to lipid library creation. The created lipid library plates can then be used to produce LNPs, which are analyzed and evaluated in cell assays and animal studies. The results are then used to define iterative directions for subsequent formulations performed using the methods of the present invention. This iterative process results in new and improved LNP formulations.

[0032] Furthermore, the methods of the present invention can be combined with screening of the formed LNPs in in vitro and / or in vivo cell assays, optionally followed by animal testing. The results of such assays and studies can influence subsequent iterations of the design of LNP formulations. Additionally, the methods of the present invention can further comprise creating a lipid library based on the dataset generated for each LNP preparation. In certain embodiments, the datasets generated from the methods of the present invention are continuously analyzed by one or more algorithms, for example during different iterations of the method. In this manner, opportunities for new and improved LNP formulations can be identified through approaches such as reinforcement learning.

[0033] In a second aspect, the present invention relates to a computer program product comprising computer readable instructions which, when loaded and executed on a computer system, cause the computer system to perform operations in accordance with the method of the present invention. In this aspect, all definitions of the method according to the first aspect of the invention apply equally. The computer-implemented methods for monitoring and controlling the process of manufacturing LNPs containing nucleic acid cargo of the present invention provide tools to advantageously monitor and control the process in a robust and reproducible manner from laboratory to industrial scale.

[0034] In a third aspect, the invention relates to a computer-implemented method for monitoring and controlling a process for producing lipid nanoparticles containing nucleic acid cargo. According to the present invention, the process for producing lipid nanoparticles containing nucleic acid cargo comprises: (i) mixing a solution containing nucleic acid cargo with a lipid-containing solution at an acidic pH; (ii) maturation / stabilization of the nanoparticles obtained in step (i) for a specific residence time; (iii) neutralizing the pH of the mixed solution obtained in step (i) via dilution in order to further stabilize the nanoparticles obtained in step (ii); (iv) performing buffer exchange on the neutralized solution obtained in step (iii) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cold buffer; The method includes the steps of: Further, in accordance with the present invention, the computer-implemented method for monitoring and controlling the process for producing lipid nanoparticles containing nucleic acid cargo comprises: (a) monitoring one or more quality parameters, such as particle size, polydispersity, and nucleic acid encapsulation of the lipid nanoparticles in real time during each of steps (i) to (iv) and after step (iv); (b) determining whether the quality parameters obtained in step (a) during each of steps (i)-(iv) and after step (iv) are within predetermined target ranges; (c) providing a feedback control that adjusts the operating parameters of each step in real time during each of steps (i) to (iv) and after step (iv) if any of the quality parameters obtained in step (a) are not within the predetermined target range, in order to bring each quality parameter within the predetermined target range; The steps include:

[0035] In certain embodiments, 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), microRNA (miRNA), and small interfering RNA (siRNA). In certain embodiments, the lipids used in the production of lipid nanoparticles in the process of producing lipid nanoparticles containing nucleic acid cargo are ionizable lipids.In addition, the lipid-containing solution may contain additional agents selected from the group consisting of additional ionizable lipids, structural lipids, helper lipids (e.g., DSPC (distearoylphosphatidylcholine)), cholesterol, and polyethylene glycol (PEG) lipid conjugates.

[0036] In step (i) of the process for producing lipid nanoparticles containing nucleic acid cargo, a solution containing nucleic acid cargo is mixed with a solution containing lipid at an acidic pH, where the nucleic acid cargo and lipid are preferably as defined above. In a preferred embodiment, the nucleic acid cargo is contained in the solution containing nucleic acid cargo at a concentration of 0.1 to 1 mg / mL. The solvent for the solution containing nucleic acid cargo is preferably a suitable buffer (e.g., 20 to 100 mM citrate buffer, pH 3.0). Furthermore, the lipid is preferably contained in the lipid-containing solution at a concentration of 50 to 60 mol %. The solvent for the lipid-containing solution is preferably a suitable organic solvent (for example, ethanol). Thereby, the volume ratio of the solution containing the nucleic acid cargo to the solution containing the lipids can be 1:1 to 5:1, preferably about 3:1.

[0037] In certain embodiments, the acidic pH is from pH 2 to pH 6, preferably from pH 3 to pH 6, more preferably from pH 4 to pH 6, and most preferably about pH 5. Moreover, 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 40°C. The means for mixing the solution containing the nucleic acid cargo with the solution containing the lipids is not particularly limited and is known in the art, however, the mixing is preferably high-speed laminar mixing where nanoprecipitation is faster than nucleation. Step (ii) of the process for producing lipid nanoparticles containing nucleic acid cargo is a step of aging and / or stabilizing the lipid nanoparticles obtained in step (i) by maintaining the mixture obtained in step (i) for a certain residence time prior to neutralization in the next step. A suitable residence time can be selected by one skilled in the art, for example, from about 0.5 to about 60 minutes. In step (iii) of the process for producing lipid nanoparticles containing nucleic acid cargo, the pH of the mixed solution obtained in step (i) is neutralized via dilution (e.g., in-line dilution) to further stabilize the lipid nanoparticles. In this step, neutralization is carried out by mixing the mixed solution obtained in step (i) with a solution having a basic pH, preferably phosphate-buffered saline (PBS).

[0038] In a preferred embodiment, the mixed solution obtained in step (i) above is neutralized to a pH ranging from pH 6.0 to pH 8.0, more preferably to a pH ranging from pH 7.0 to pH 8.0, more preferably to a pH ranging from pH 7.2 to pH 7.6, and most preferably to a pH of about pH 7.4. In step (iv) of the process for producing lipid nanoparticles containing nucleic acid cargo, the neutralized solution obtained in step (iii) is subjected to buffer exchange via ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them into a low-temperature buffer. The means for performing the buffer exchange are not particularly limited and are known in the art. Suitable low-temperature buffers include, for example, phosphate-buffered saline (PBS) containing a suitable cryoprotectant, such as sucrose, mannitol, trehalose, or sorbitol, at a concentration of 0.1 to 10% by weight. In a specific embodiment, the process for producing lipid nanoparticles containing nucleic acid cargo further comprises step (ii-a) performed between steps (ii) and (iii). In step (ii-a), the solution obtained in step (ii) is diluted, and then the diluted solution is neutralized in step (iii). Accordingly, the mixed solution obtained in step (i) is diluted with a solution containing 20-30% alcohol, preferably ethanol. The mixed solution is diluted 2-4 times based on the volume before dilution.

[0039] In step (a) of the method of the present invention, during each of steps (i) to (iv) and after step (iv), quality parameters such as particle size, polydispersity, and nucleic acid encapsulation of lipid nanoparticles are monitored in real time.In certain embodiments, all three of the quality parameters are monitored.In other embodiments, only one of the quality parameters is monitored, or any combination of two of the quality parameters is monitored. In step (b) of the method of the present invention, during each of steps (i) to (iv), as well as after step (iv), it is determined whether the quality parameters obtained in step (a) are within predetermined target ranges. Furthermore, in step (c) of the method of the present invention, during each of steps (i) to (iv) of the process for producing lipid nanoparticles containing nucleic acid cargo, and if any of the quality parameters obtained in step (a) after step (iv) are not within the predetermined target range, feedback control is provided to adjust the operating parameters in real time to bring each quality parameter within the predetermined target range.

[0040] As used herein, the term "feedback control to adjust operating parameters" refers to feedback regarding whether a target range for a quality parameter is being met or whether a measured value is above or below the target range, based on which the operating parameters in each of steps (i)-(iv) in the manufacturing process can be adjusted to bring the respective quality parameter within the target range. In certain embodiments, the operating parameters adjusted in step (c) of the methods of the present invention 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 period, particle growth period, residence time, maturation period, pH, fluid composition, dilution degree, dilution ratio, mixer type, stream mixing ratio, and stirring / agitator speed.

[0041] The quality parameter "particle size" determines the size of lipid nanoparticles containing nucleic acid cargo. The target particle size range is within 5% variation of a specific target particle size value, where the target particle size can be between 40 and 400 nm depending on the intended therapeutic application. In non-limiting embodiments, particle size is determined by a Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), a PATfix® HPLC platform, nanoparticle tracking analysis, and / or multi-angle light scattering (MALS). If the particle size is above or below the target range, one or more of the above operating parameters are adjusted to decrease or increase the particle size to the predetermined target range.

[0042] The quality parameter "polydispersity" is a measure of the heterogeneity of lipid nanoparticles containing nucleic acid cargo. Polydispersity can occur due to size distribution, aggregation, or aggregation of lipid nanoparticles in solution. The target range of particle size is preferably 0.04 to 0.1. In a non-limiting embodiment, polydispersity is determined by a Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), dynamic light scattering (DLS), a PATfix® HPLC platform, nanoparticle tracking analysis, multi-angle light scattering (MALS), and / or other suitable tools for measuring particle size distribution, as known in the art. If the polydispersity is above or below the target range, one or more of the above operating parameters are adjusted to decrease or increase the polydispersity to the predetermined target range. The quality parameter "nucleic acid encapsulation" is a measure of the percentage of nucleic acid cargo successfully encapsulated / adsorbed into lipid nanoparticles. The target range of nucleic acid encapsulation composition is preferably at least 96% or more. In certain embodiments, nucleic acid encapsulation composition is determined by the Virus Counter® platform (Sartorius Stedim Biotech GmbH, Germany), Förster resonance energy transfer (FRET) assay, and / or single particle automated Raman capture analysis (SPARTA).

[0043] If the nucleic acid encapsulation composition is above or below the target range, one or more of the above operating parameters are adjusted to increase the nucleic acid encapsulation composition into the predetermined target range. In certain embodiments, pressure is measured during steps (i) and (iv) of the manufacturing process. Pressures above the target range are indicative of potential fouling. In another particular embodiment, the pH is measured in steps (i) and (iv) of the manufacturing process. The target range of pH is as described above.

[0044] In a fourth aspect, the present invention relates to a computer program product comprising computer readable instructions which, when loaded and executed on a computer system, cause the computer system to perform operations in accordance with the method of the present invention. In this aspect, all definitions of the method according to the first aspect of the invention apply equally. In certain embodiments, the computer readable instructions include a control algorithm for feedback control of the operating parameter, wherein the control algorithm is based on a mechanistic hybrid model. The computer-implemented methods for monitoring and controlling the process of producing lipid nanoparticles containing nucleic acid cargo of the present invention provide a tool to advantageously monitor and control the process in a robust and reproducible manner from laboratory to industrial scale. In this context, in certain embodiments, the present invention encompasses the creation and use of algorithms to optimize the control of LNP production. Specifically, algorithms may be used to control the influence of values ​​related to 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 period, particle growth period, residence time, maturation period, pH, fluid composition, dilution degree, dilution rate, mixer type, flow mixing ratio, and stirring / agitator speed) to maximize LNP performance. Continuous training of algorithmic models through techniques such as reinforcement learning leads to continuous improvement in the robustness of LNP production.

[0045] In a fifth aspect, the present invention relates to an automated nanoparticle synthesis system, comprising: (a) two dispensers, each comprising a syringe pump and a six-port valve, wherein the syringe pump is in fluid communication with an input port of the six-port valve; (b) Six-port distributor valve; (c) microfluidic mixer chip; (d) a waste reservoir, a buffer reservoir, and an ethanol reservoir; Including, where: (i) both dispensers are fluidly connected to the waste reservoir, the buffer reservoir, and the ethanol reservoir; and to the microfluidic mixer chip via a six-port valve; (ii) a first of said dispensers is further in fluid communication with a lipid source via a six-port valve; (iii) a second of said dispensers is further in fluid communication with an RNA source via a six-port valve; (iv) the microfluidic mixer chip is in fluid communication with the input port of the dispensing valve; and (v) The dispensing valve is fluidly connected to the waste reservoir; and to a lipid nanoparticle (LNP) collection tube.

[0046] The automated nanoparticle synthesis system of the present invention includes a first dispenser assembly having a first pump (e.g., a syringe pump) integrated with a first valve (e.g., a six-port valve) and a second dispenser assembly having a second pump (e.g., a syringe pump) integrated with a second valve (e.g., a six-port valve). The system also includes a six-port distribution valve. A microfluidic mixer is utilized to achieve fast and controllable mixing of lipids and RNA for LNP production (Figure 3). The automated system aims to increase throughput in LNP screening activities by examining various microfluidic mixer designs and key flow parameters that affect LNP quality, such as FRR (flow rate ratio) and TFR (total flow rate). The automated nanoparticle synthesis system of the present invention can be divided into (i) a dispensing module (FIG. 5) for loading and dispensing reagents and pushing the liquids into a microfluidic mixing assembly (e.g., a microfluidic mixing chip) chip positioned downstream, and (ii) a collection module (FIG. 6) for collecting LNPs synthesized under different screening conditions and storing them. Figure 7 shows a demonstration of the automated nanoparticle synthesis system of the present invention.

[0047] 5, and by way of non-limiting example, the dispensing module includes a lipid reservoir for holding a lipid storage solution and a nucleic acid reservoir for holding an aqueous nucleic acid storage solution, such as an RNA storage solution. These reservoirs may be commercially available reservoirs, such as tubes, wells, or bags. In other examples, the reservoirs may hold other nucleic acid storage solutions, such as DNA, miRNA, cDNA, etc. The first reservoir is fluidly connected to a first valve (e.g., a six-port valve) on the first dispenser, and the second reservoir is fluidly connected to a second valve (e.g., a six-port valve) on the second dispenser. The first valve is fluidly coupled to a first pump (e.g., a syringe pump), and the second valve is fluidly coupled to a second pump (e.g., a syringe pump). Each of the first valve and the second valve may be a commercially available valve. In addition to a syringe pump, each of the first pump and the second pump may be any other suitable pump, such as a peristaltic pump, a centrifugal pump, a membrane pump, and / or a piston pump, in other embodiments.

[0048] As shown in Figure 5, each of the first and second valves is fluidly coupled to a waste reservoir, a buffer reservoir, and an ethanol reservoir (through separate ports on the first and second valves and through tubing). The ports on the first and second valves that are fluidly coupled to the waste reservoir, the buffer reservoir, and the ethanol reservoir are different from the ports that are fluidly coupled to the lipid reservoir and the nucleic acid reservoir. Each of the first and second valves is configured to automatically switch between the buffer reservoir, the ethanol reservoir, the lipid reservoir, and / or the nucleic acid reservoir. These reservoirs may be commercially available reservoirs such as tubes, wells, or bags. As seen in Figure 5, each of the first and second valves is fluidly coupled via tubing to a mixing assembly (e.g., a microfluidic mixer chip). The mixing assembly may be used for in-line mixing of a lipid reservoir solution with an aqueous nucleic acid solution to form a diluted intermediate product using precise flow control of the two reservoir solutions.

[0049] As seen in Figures 3 and 6, and as a non-limiting example of a collection module, a distribution valve (e.g., a six-port valve) is fluidly coupled to a mixing assembly (e.g., a microfluidic mixer chip) via tubing. One port of the distribution valve is fluidly coupled to a waste reservoir via tubing, and each of the other ports of the distribution valve is fluidly coupled to one or more lipid nanoparticle (LNP) collection tubes via tubing for collecting and storing synthetic lipid nanoparticles produced from the mixture of two reservoir solutions. The distribution valve directs the waste stream to the waste reservoir while controlling the flow of formulated lipid nanoparticles to the LNP collection tube. Although Figures 3 and 6 show five LNP collection tubes, in other embodiments, there may be fewer or more than five LNP collection tubes.

[0050] Figure 4 shows an alternative embodiment of an automated nanoparticle synthesis system. Similar to the system shown in Figure 3, the system shown in Figure 4 also includes a first dispenser assembly having a first pump (e.g., a syringe pump) integrated with a first valve (e.g., a six-port valve), and a second dispenser assembly having a second pump (e.g., a syringe pump) integrated with a second valve (e.g., a six-port valve). 3, the system shown in FIG. 4 also includes a waste reservoir, a buffer reservoir, and an ethanol reservoir, where a first valve and a second valve are each fluidly coupled to the waste reservoir, the buffer reservoir, and the ethanol reservoir via separate ports on the valve. Each of the first valve and the second valve is also fluidly coupled to a mixing assembly (e.g., a microfluidic mixer chip) to provide fast and controllable mixing of lipids and RNA to generate LNPs.

[0051] Unlike the system shown in FIG. 3 , the system shown in FIG. 4 does not include a distribution valve for sample collection. Instead, the system shown in FIG. 4 includes a first liquid handler configured to automate the collection of synthesized LNPs and a second liquid handler configured to automate the loading of reagents for many different combinations into a well plate, such as lipid and RNA storage solutions. In the example shown in FIG. 4 , the first liquid handler is configured to automatically transfer, collect, and store synthesized LNPs into a well plate (e.g., a 96-well plate). The second liquid handler is configured to remove lipid and nucleic acid storage solutions from the well plate (e.g., a 96-well plate). For example, a well plate may contain a lipid-RNA library, with the lipid storage solution present in one or more wells and the RNA storage solution present in one or more wells that do not contain the lipid storage solution. In other embodiments, there may be only one liquid handler configured to automate both the collection of synthesized LNPs and the loading of reagents.

[0052] The screening process and capacity of the system shown in Figure 3 can be further improved by integrating special well plates and one or more liquid handlers in the system shown in Figure 4. As a result, the system shown in Figure 4 does not require sample injection, allowing for the use of smaller sample volumes. Any of the systems and components thereof described herein, including the various pumps and valves described herein, can be controlled using any known control technique and / or known control system. For example, in some embodiments, such components can be controlled manually, electronically, and / or hydraulically.

[0053] In the system shown in Figure 3, up to five TFR and FRR (1-8) combinations can be used in a single automated run. Furthermore, screening flow rates ranging from approximately 5 μL / min to 30 mL / min for each phase (aqueous / organic) are available. Furthermore, the system described herein allows for the use of sample volumes as small as 100 μL. The amounts of buffer and ethanol used in the automated nanoparticle synthesis system described herein are: (i) The length and inner diameter of the tubing that defines the fluid pathway connecting the pump (e.g., a syringe pump), the microfluidic mixer chip, the distribution valve, and the collection reservoir; (ii) the number of screenings performed; and (iii) the number of cycles for washing (with ethanol) and priming (with buffer) the system; For example, if the total length of the tubing is 4000 mm and the internal diameter is 0.8 mm, the amount of buffer and ethanol required to perform one individual screening and one cycle of washing and priming is typically less than 5 mL.

[0054] In a sixth aspect, the present invention relates to the use of one or more air valves in the systems described herein to separate buffer solutions, lipid-containing solutions, RNA-containing solutions, and / or ethanol solutions from each other in tubing lines that provide fluid connections between the components. Air plug is the amount of air introduced into the tube line to provide separation between buffer solution, lipid-containing solution, RNA-containing solution, and / or ethanol solution.For example, microfluidic mixer chip may include air plug that separates RNA solution from buffer solution, and air plug that separates buffer solution from lipid solution and lipid solution from ethanol solution.In one embodiment, the volume of air plug is between 1 μL and 20 μL, preferably about 5 μL. Without the air stopcock, RNA and lipids can diffuse into the surrounding liquid (e.g., buffer and ethanol) within the tube. Such unwanted diffusion can alter the RNA concentration and lipid / RNA mixture ratio during particle synthesis through the microfluidic mixer. In preliminary experiments without the air stopcock, RNA was found in waste samples, whereas with the air stopcock, RNA was found in most samples. As a result, the air stopcock is particularly important for preparing small sample volumes while minimizing reagent waste.

[0055] Aspects of a relevant LNP screening workflow according to the present disclosure include: (a) Adding air plugs to the tubing lines of the nanoparticle synthesis system; (b) loading lipids and RNA into the sample loop of the system; (c) adding another air valve to the tubing line of the system; (d) extruding lipids and RNA to meet at the junctions where the flow of the system converges; (e) regulating the flow to generate LNP; and (f) switching the valve of the system to collect LNP; This may involve steps (a) to (f). In this workflow, the nanoparticle synthesis system is preferably the automated nanoparticle synthesis system shown in FIG. 3 of the present disclosure. In some embodiments, the LNP screening based on the above-mentioned method is carried out through a time-consuming protocol, which involves washing and priming the microfluidic chip between screening tasks.In alternative embodiments, the LNP screening based on the above-mentioned method is carried out through a short protocol, which does not involve washing and priming between screening tasks. Figure 8 shows an exemplary user interface for control software that can be used with the automated nanoparticle synthesis system of the present disclosure. The software provides an easy-to-use interface that displays, for example, the user-selected flow rate ratio (FFR), total flow rate (TFR), and sample volume. Different microfluidic chip designs can be added to the library for future screening. An advanced manual mode allows for more flexible use and control of every device in the system.

[0056] As used herein, the term "comprising" / "comprises" expressly includes the terms "consisting essentially of" / "consists essentially of" and "consists of" / "consists of", i.e., all terms herein are interchangeable. Furthermore, as used herein, the term "about" preferably refers to a modifier of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5% of the specified value. Thus, as an example, the term "about 100" can include ranges of 90-110, 92-108, 94-106, 95-105, 96-104, 97-103, 98-102, 99-101, or 99.5-100.5.

[0057] The present invention advantageously provides a method for the high-throughput preparation of LNPs containing nucleic acid cargo, resulting in the parallel preparation of hundreds of different LNPs per person per day. This is combined with online, real-time monitoring of key quality parameters (e.g., particle size, polydispersity, nucleic acid encapsulation). Thus, the present invention provides a rapid, easy-to-use means for generating, screening, and analyzing LNPs in a high-throughput format. The invention according to the fifth aspect is further illustrated in the following non-limiting examples.

[0058] example A 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) blank LNP formulation was prepared using the automated nanoparticle synthesis system disclosed herein. The DOTAP blank LNP formulation contained four lipids: DOTAP, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000. The molar ratio of these lipids was 40:10:48:2. The lipids were dissolved in ethanol at a concentration of approximately 7.5 mM and citrate buffer at a pH of 4 and a concentration of approximately 10 mM as the aqueous phase. In this example, the LNP production scale was 200 μL. Five LNP samples were produced consecutively. The flow rate ratio (FRR) was 2, and the total flow rate (TFR) was 200 μL / min. The microfluidic chip and production system were washed with 10 mM citrate buffer and ethanol after each sample production. Particle size (Z-average) and polydispersity index (PDI) were measured using a Zetasizer Pro (Malvern).

[0059] The effects of the flow rate ratio between lipid and mRNA and the total flow rate within the device on size and polydispersity index were investigated. The same DOTAP formulation was prepared. Different FRRs (1, 2, 4, and 8) and TFRs (100 and 200 μL / min) were used for mixing. In this study, the LNP production volume was 100 μL, and the final lipid concentration was 2.5 mM. The microfluidic chip and production system were washed with 10 mM citrate buffer and ethanol after each sample production. Particle size (Z-average) and polydispersity index (PDI) were measured using a Zetasizer Pro (Malvern).

Claims

1. 1. A computer-implemented method for the high-throughput preparation of lipid nanoparticles (LNPs) containing nucleic acid cargo, comprising: (a) providing one or more solution(s) comprising nucleic acid cargo; (b) providing one or more solution(s) comprising lipids; (c) mixing one or more solutions containing nucleic acid cargo with one or more solutions containing lipids at an acidic pH; (d) neutralizing the pH of the mixed solution obtained in step (c) through dilution to stabilize the LNPs obtained in step (c); (e) performing buffer exchange on the neutralized solution obtained in step (d) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the LNPs obtained in step (d) and reconstitute them into a cold buffer, thereby producing one or more LNP preparation(s); The method includes the steps of: where: During each of steps (c) through (e), and after step (e), one or more of particle size, polydispersity, and nucleic acid encapsulation of the one or more LNPs are monitored in real time, thereby generating a dataset for each LNP preparation; and the generated dataset is associated with each LNP preparation. The implementation method.

2. The computer-implemented method of claim 1 , wherein two or more individual LNP preparations are produced in parallel.

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

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

5. 10. The computer-implemented method of claim 1, wherein the one or more LNP preparation(s) are produced in one or more multiwell plate(s).

6. 10. 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. 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), microRNA (miRNA), and small interfering RNA (siRNA).

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

9. 2. The computer-implemented method of claim 1, wherein the pH in step (c) ranges from pH 2 to pH 6.

10. 5. The computer-implemented method of claim 4, wherein the time period ranges from 0.1 to 60 minutes.

11. 2. The computer-implemented method of claim 1, wherein the pH of the neutralization solution in step (d) ranges from pH 6 to pH 8.

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

13. 10. The computer-implemented method of claim 1, wherein one or more of the particle size, polydispersity, and nucleic acid encapsulation of the one or more LNPs can be measured and / or analyzed by at least one of the following: a 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 techniques, a PATfix® HPLC platform, nanoparticle tracking analysis, and a Förster resonance energy transfer (FRET) assay.

14. The computer-implemented method of claim 1 , further comprising creating a lipid library based on the dataset generated for each LNP preparation.

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 claim 1.

16. 1. A computer-implemented method for monitoring and controlling a process for producing lipid nanoparticles containing nucleic acid cargo, comprising: wherein the process for producing lipid nanoparticles containing said nucleic acid cargo comprises: (i) mixing a solution containing a nucleic acid cargo with a lipid-containing solution 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) via dilution to further stabilize the nanoparticles obtained in step (ii); (iv) performing buffer exchange on the neutralized solution obtained in step (iii) via ultrafiltration and / or diafiltration (UF / DF) to concentrate the nanoparticles obtained in step (iii) and reconstitute them in a cold buffer; The method includes the steps of: The computer-implemented method for monitoring and controlling a process for producing lipid nanoparticles containing said nucleic acid cargo comprises: (a) monitoring one or more quality parameters of the lipid nanoparticles, such as particle size, polydispersity, and nucleic acid encapsulation, in real time during each of steps (i)-(iv) and after step (iv); (b) determining whether the quality parameters obtained in step (a) during each of steps (i) to (iv), and after step (iv), are within predetermined target ranges; (c) providing a feedback control during each of steps (i) to (iv), and after step (iv), if any of the quality parameters obtained in step (a) are not within said predetermined target ranges, to adjust the operating parameters of each step in real time to bring each quality parameter within the predetermined target range; The steps include: The implementation method.

17. 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. 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), microRNA (miRNA), and small interfering RNA (siRNA).

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

20. 17. The computer-implemented method of claim 16, wherein the pH in step (i) ranges from pH 2 to pH 6.

21. 17. The computer-implemented method of claim 16, wherein the residence time ranges from 0.5 to 60 minutes.

22. 17. The computer-implemented method of claim 16, wherein the pH of the neutralization solution in step (iii) ranges from pH 6 to pH 8.

23. 17. 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 quality parameters include one or more of particle size, polydispersity, and nucleic acid encapsulation of the lipid nanoparticles, and the quality parameters are measured using 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; The computer-implemented method of claim 16 , wherein the measurement and / or analysis can be performed by at least one of:

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

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

1.

27. 17. The computer-implemented method of claim 16, wherein the nucleic acid encapsulation has a target coverage of at least 96% or greater.

28. 17. 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 period, particle growth period, residence time, maturation period, pH, fluid composition, dilution degree, dilution ratio, mixer type, stream mixing ratio, and stirring / agitator speed.

29. 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 claim 16.

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

31. 1. An automated nanoparticle synthesis 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 holding a lipid storage solution; (f) a nucleic acid reservoir for holding a nucleic acid stock solution; and (g) a waste reservoir, a buffer reservoir, and an ethanol reservoir; Including, where: (i) the lipid reservoir is in fluid communication with a first valve and the nucleic acid reservoir is in fluid communication with a second valve, or vice versa; (ii) the first valve is in fluid communication with the first pump and the second valve is in fluid communication with the second pump; (iii) each of the first valve and the second valve is fluidly connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, and wherein the 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 the ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) each of the first valve and the second valve is fluidly connected to a microfluidic mixer; and (v) a third valve fluidly connected to the microfluidic mixer, to a waste reservoir, and to one or more collection reservoirs for collecting the produced lipid nanoparticles (LNPs); The system.

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

33. 32. The automated nanoparticle synthesis system of claim 31, wherein the first valve, the second valve, and / or the third valve is a six-port valve.

34. 32. The automated nanoparticle synthesis system of claim 31, wherein the fluid communication is achieved through tubing.

35. The automated nanoparticle synthesis system of claim 31, wherein the system further comprises one or more air valves adapted to separate the buffer solution from the RNA-containing solution, one or more air valves that separate the buffer solution from the lipid-containing solution, and one or more air valves that separate the lipid-containing solution from the ethanol.

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

37. 1. An automated nanoparticle synthesis 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 configured to automate the loading of reagents and to automate sample collection in one or more well plates; and (e) a waste reservoir, a buffer reservoir, and an ethanol reservoir; Including, where: (i) the lipid reservoir is in fluid communication with a first valve and the nucleic acid reservoir is in fluid communication with a second valve, or vice versa; (ii) the first valve is in fluid communication with the first pump and the second valve is in fluid communication with the second pump; (iii) each of the first valve and the second valve is fluidly connected to a waste reservoir, a buffer reservoir, and an ethanol reservoir, and wherein the 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 the ports that are fluidly connected to the lipid reservoir and the nucleic acid reservoir; (iv) each of the first valve and the second valve is fluidly connected to a microfluidic mixer; and (v) the microfluidic mixer is in fluid communication with the liquid handler; The system.

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

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

40. 38. 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 automate collection of synthesized LNPs and the second liquid handler is configured to automate loading of reagents.

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

42. The automated nanoparticle synthesis system of claim 37, wherein the system further comprises one or more air valves adapted to separate the buffer solution from the RNA-containing solution, one or more air valves that separate the buffer solution from the lipid-containing solution, and one or more air valves that separate the lipid-containing solution from the ethanol.

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

44. 38. 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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