System for continuous production of a liposome suspension

Abstract

The invention relates to a system (1) for producing a liposome suspension (2), comprising a first line (12) that extends between a first container (10) containing an initial solution (4) of at least one active agent (3) and phospholipids (5) and a millifluidic device (13). The system (1) also comprises a second line (19) that extends between a second container (20) containing carbon dioxide (6) and the millifluidic device (13). The millifluidic device (13) comprises at least one mixer (30), a plurality of tubes (30a, 30b, 30c, 30d), a plurality of reducers (50a, 50b) and a micrometric valve (35). The system (1) does not include an autoclave.

Description

Continuous production system for a liposome suspension

[0001] The invention relates to a system for the continuous production of a liposome suspension, the liposomes being of medium size, in particular having a diameter on the order of one hundred nanometers and an encapsulation rate greater than 80%. It relates to such a system. It also relates to a method for producing a liposome suspension using such a system.

[0002] Document WO2024 / 026569 describes a process for the continuous production of liposomes, comprising the following steps: (a) continuously feeding an autoclave with an aqueous phase comprising a phospholipid, an active agent and carbon dioxide; (b) creating and maintaining temperature and pressure conditions above the critical point of carbon dioxide in the autoclave; and (c) continuously withdrawing and depressurizing a stream of solution outlet from the autoclave containing liposomes.

[0003] The aqueous phase and carbon dioxide are mixed together before entering the autoclave.

[0004] The temperature condition is a temperature below approximately 60 °C and the pressure condition is a pressure below approximately 300 bar.

[0005] Such a process is implemented by a system comprising said autoclave which is equipped with an agitation device, such as a paddle or propeller agitator, and / or which is filled with any material to improve the mixing with the aqueous phase, and in particular the contact between the phospholipid, the active agent and the supercritical carbon dioxide.

[0006] The autoclave includes an outlet pipe with an outlet valve and / or nozzle to promote a reduction in the size of the liposomes obtained during depressurization.

[0007] The resulting liposomes have a size of less than about 200 nm and a narrow size distribution including a polydispersity index of the order of 0.30.

[0008] A primary problem with such a system is that it includes an autoclave, which is an expensive, bulky component requiring regular maintenance and significant monitoring of implementation parameters.

[0009] A second problem inherent in such a system is that the use of a paddle or propeller stirring device in the autoclave is complex to implement in a pressurized system.

[0010] A third problem lies in the fact that the use of a packing, i.e. any material to improve the mixing with the aqueous phase and the contact between the phospholipid, the active agent and the supercritical carbon dioxide requires maintenance.

[0011] A fourth problem lies in the fact that the outlet valve does not have heating elements to compensate for the temperature drop due to depressurization, so the outlet pipe can become blocked due to the freezing of the aqueous solution and carbon dioxide, which can lead to overpressures in the system.

[0012] One aim of the present invention is to propose a continuous production system for a liposome suspension which addresses the aforementioned problems and is simple, easy to implement, inexpensive and compact.

[0013] Another objective of the present invention is to propose a method for implementing such a system which is easy to carry out, quick to implement, and involves a minimum number of steps.

[0014] To this end, the present invention proposes a system for producing a liposome suspension comprising a first line extending between a first container holding an initial solution of at least one active agent and phospholipids and a millifluidic device. The system comprises a second line extending between a second container holding liquid carbon dioxide and the millifluidic device.

[0015] According to the present invention, the millifluidic device comprises at least one mixer, a plurality of tubes, a plurality of reducing fittings and a micrometric valve.

[0016] The system advantageously includes at least one of the following technical features, taken alone or in combination:

[0017] - the system is autoclave-free,

[0018] - the mixer comprises at least a first branch connected to the first line, a second branch connected to the second line, and a third branch connected to a first tube,

[0019] - The millifluidic device comprises successively the first tube, a first reducing fitting, a second tube, the micrometric valve, a third tube, a second reducing fitting and a fourth tube,

[0020] - the second tube and the third tube are identical,

[0021] - the first line includes a first pump capable of circulating the initial solution at a first flow rate of approximately 1 mL / min + / - 1%, and a first pressure, which is between 80 bar and 300 bar, + / - 5%,

[0022] - the second line includes a second pump capable of circulating carbon dioxide at a second flow rate of approximately 9 g / min + / - 1%, and at a second pressure, which is between 80 bar and 300 bar, + / - 5%,

[0023] - the first line includes a first heat exchanger which is designed to bring the initial solution to a first temperature which is between 35 °C and 60 °C,

[0024] - the first line includes a sintered metal,

[0025] - the second line includes a second heat exchanger which is designed to bring the carbon dioxide to a second temperature which is between 35 °C and 60 °C, the second heat exchanger being capable of bringing the carbon dioxide into the supercritical phase at the outlet of the second heat exchanger before its entry into the millifluidic device.

[0026] The present invention also relates to a method for the continuous production of a liposome suspension by means of such a system, the system comprising a first line extending between a first container containing an initial solution of at least one active agent and phospholipids and a millifluidic device, the system comprising a second line comprising a second pump and extending between a second container containing liquid carbon dioxide (which will be brought to a supercritical phase at the outlet of the second heat exchanger before entering the millifluidic device) and the millifluidic device, the millifluidic device comprising at least one mixer, a plurality of tubes, a plurality of reducing fittings and a micrometric valve, the method comprising a step of maintaining the carbon dioxide, between the second pump and the plurality of tubes, at a second pressure which is on the order of 100 bar,to within + / - 5% and at a second temperature between 35°C and 60°C, and a step of obtaining the liposome suspension at the outlet of the tubes in a collector.

[0027] The invention and its advantages will be better understood upon reading the following description and non-limiting embodiments, with reference to the attached figures in which:

[0028] represents a schematic view of a continuous production system for a liposome suspension.

[0029] represents a schematic view of a millifluidic device constituting the system illustrated on the.

[0030] illustrates a distribution curve and photos of liposomes obtained from the system shown on the.

[0031] Figure 1 schematically illustrates a system 1 of the present invention, which is intended to continuously produce a suspension of liposomes 2. Each liposomes produced can be described as lipid vesicles containing an active agent 3, such as a drug or similar substance. The produced liposomes transport the active agent 3 until its release through rupture / opening of the lipid vesicles during the internalization of the liposomes into the cells. Such production is achieved from, on the one hand, an initial solution 4 comprising at least water, the active agent 3, and phospholipids 5, and on the other hand, liquid carbon dioxide 6.The initial solution 4 includes, for example, an aqueous mixture of phospholipids 5, such as phosphatidylcholine, ethanol, the active agent 3, such as lutein or messenger RNA, a stabilizer, such as cholesterol, and / or an agent enabling the formation of a stealth liposome, such as polyethylene glycol.

[0032] To do this, system 1 includes a first container 10 containing the initial solution 4.

[0033] System 1 also includes a first pump 11 for circulating the initial solution 4 within a first line 12 extending from the first container 10 to a millifluidic device 13. The millifluidic device 13, which is the element of system 1 within which the liposome suspension 2 is produced, is described in more detail below. The first pump 11 is, for example, a high-pressure liquid pump for circulating the initial solution 4 at a pressure between 80 and 300 bar.

[0034] System 1 also includes a first flow controller 14 to measure a first flow rate D1 of the initial solution 4 inside the first line 12, preferably at the outlet of the first container 10, and a first pressure controller 15 to measure a first pressure P1 inside the first line 12, preferably at the outlet of the first container 10. As an example, the first flow rate D1 is on the order of 1 mL / min to within + / - 1%, and the first pressure P1 is on the order of 100 bar, to within + / - 5%.

[0035] System 1 also includes a first heat exchanger 16, such as a high-pressure heat exchanger in the form of a 316L stainless steel coil containing the fluid to be heated and equipped with a heating element to raise the temperature of the solution to a first temperature T1 between 35 and 60 °C, preferably around 40 °C to within + / -0.1%.

[0036] System 1 also includes a first safety valve 17a to prevent accidental overpressures and a first valve 17b to allow or prohibit circulation of the initial solution 4 within the first line 12.

[0037] System 1 also includes a metal sinter 18 which is intended to homogenize the initial solution 4. This metal sinter 18 has a porosity of the order of two microns, to within + / -10%.

[0038] Thus, the first line 12 includes successively, from the first container 10 to the millifluidic device 13, the first pump 11, the first flow controller 14, the first pressure controller 15, the first heat exchanger 16, the first safety valve 17a, the first valve 17b and the sintered metal 18.

[0039] System 1 also includes a second line 19 which extends between a second container 20 containing carbon dioxide 6 and the milli-fluidic device 13. The second container 20 is, for example, a carbon dioxide 6 storage cylinder at 60 bar.

[0040] System 1 also includes a second valve 21 to allow or prohibit the flow of carbon dioxide 6 within the second line 19.

[0041] System 1 also includes a temperature regulator 22, such as a cold bath or similar device, to bring the carbon dioxide 6 to a temperature of approximately 0 °C, within + / -0.1%. The temperature regulator 22 cools the liquid carbon dioxide 6 to 0 °C, ensuring that it remains liquid at 60 bar and can be pumped.

[0042] System 1 also includes a second pump 23 for circulating carbon dioxide 6 within the second line 19, which extends from the second container 20 to the millifluidic device 13. The second pump 23 is, for example, a high-pressure, double-piston liquid pump or similar. The second pump 23 is capable of raising the carbon dioxide 6 to a second pressure P2, which is greater than 73.8 bar, in particular between 80 bar and 300 bar. Ideally, the second pump 23 delivers a flow rate of 9 g / min.

[0043] System 1 also includes a second flow controller 24 to measure a second flow rate D2 of carbon dioxide 6 inside the second line 19, preferably at the outlet of the second container 20, and a second pressure controller 25 to measure the second pressure P2 inside the second line 19, particularly at the outlet of the second pump 23. As an example, the second flow rate D2 is on the order of 9 g / min, to within + / - 1%, and the second pressure P2 is on the order of 100 bar, to within + / - 5%.

[0044] System 1 also includes a second heat exchanger 26, such as a high-pressure heat exchanger in the form of a 316L stainless steel coil and equipped with a heating element for raising the temperature of carbon dioxide 6 to a second temperature T2 between 35 °C and 60 °C. At the outlet of the coil, the CO2 is in the supercritical phase.

[0045] The second heat exchanger 26 is of the same type as the first heat exchanger 16. The second heat exchanger 26 prevents pressure surges of the carbon dioxide 6 and maintains a constant mixing flow rate D of carbon dioxide 6 and a constant mixing temperature T of carbon dioxide 6. For example, the mixing flow rate D is approximately 9 g / min, ±1%, and the mixing temperature T of carbon dioxide 6 is approximately 40 °C, ±0.10%.

[0046] System 1 also includes a first temperature sensor 27 to measure a third temperature T3 at the outlet of the second heat exchanger 26 and to verify that it is equivalent to the second temperature T2, which is the setpoint temperature of the second heat exchanger 26.

[0047] System 1 also includes a pressure sensor 28 to measure a third pressure P3 at the outlet of the second heat exchanger 26.

[0048] System 1 also includes a second safety valve 29a to prevent accidental overpressures and a third valve 29b to allow or prohibit the circulation of carbon dioxide 6 within the second line 19.

[0049] Thus, the second line 12 includes successively, from the second container 20 to the millifluidic device 13, the second valve 21, the temperature regulator 22, the second pump 23, the second flow controller 24, the second pressure controller 25, the second heat exchanger 26, the first temperature sensor 27, the pressure sensor 28, the second safety valve 29a and the third valve 29b.

[0050] The mixing of carbon dioxide 6 and the initial solution 4 takes place inside the millifluidic device 13, which replaces the autoclave of the prior art, the mixing of the flows due to hydrodynamics replacing the use of an agitator and packing of the prior art.

[0051] It is noted that at the outlet of the first heat exchanger 16 and the second heat exchanger 26, the first line 12 and the second line 19 are at the same temperature and pressure. Furthermore, the coiled heat exchangers 16 and 26 are made of high-pressure 316L stainless steel and have an internal volume that prevents fluid surges, whether of the initial solution 4 or carbon dioxide 6, in the process.

[0052] The millifluidic device 13 advantageously comprises a mixer 30, such as a tee shown in Figure 1, or such as a cross shown in Figure 2, a plurality of tubes 30a, 30b, 30c, 30d, a plurality of fittings 50a, 50b, and a micrometer or fine-tuning valve 35. The tubes 30a, 30b, 30c, 30d are made of 316L stainless steel.

[0053] The initial solution 4 and carbon dioxide 6 are brought into contact inside the mixer 30. For this purpose, the mixer 30 comprises a first branch 31, which is connected to the first line 12, through which the initial solution 4 flows, and a second branch 32, through which the carbon dioxide 6 flows. The initial solution 4 and the carbon dioxide 6 are at the same pressure and temperature. The mixer 30 also comprises a third branch 33, through which the initial solution 4 and the carbon dioxide 6 flow. It should be noted that the first branch 31 and the second branch 32 are orthogonal to each other, and that the third branch 33 is on the same axis as the first branch 31. The third branch 33 is connected to a first tube 30a, through which the initial solution 4 and the carbon dioxide 6 flow.

[0054] According to the variant illustrated in Figure 1, the mixer 30, arranged in a cross shape, includes a fourth arm 34, parallel to the second arm 32, to which a second thermocouple-type temperature sensor 60 is connected. The second temperature sensor 60 can either monitor the temperature of the flows entering the first tube 30a, or serve as a temperature control probe if the first tube 30a is equipped with a heating element to ensure the heating of the flows via the first heat exchanger 16 and / or the second heat exchanger 26. The heating element in the first tube 30a is optional, as the coil-shaped heat exchangers 16 and 26, which precede the millifluidic device 13, provide the heating.

[0055] The dimensions of tubes 30a, 30b, 30c, 30d are described below and are schematically illustrated on the diagram, while also being valid for the variant illustrated on the diagram, the proportions not being respected for better readability and clarity of the figures.

[0056] The first tube 30a has a first external diameter X1 of one quarter inch, or 6.35 mm, a first internal diameter Y1 of 3.87 mm, and a first length L1 measured between its ends of 10 cm. One end of the first tube 30a is in fluidic contact with the third branch 33, while the other end is in fluidic contact with a first reducing fitting 50a.

[0057] The first 50a reducing fitting is a quarter-inch to eighth-inch reducing fitting. The first 50a reducing fitting is inserted between the first 30a tube and a second 30b tube.

[0058] The second tube 30b has a second external diameter X2 of one-eighth of an inch, or 3.175 mm, a second internal diameter Y2 of 1.75 mm, and a second length L2 of 5 cm between its ends. One end of the second tube 30b is in fluidic contact with the first reducing fitting 50a, while the other end is in fluidic contact with the micrometer valve 35. The micrometer valve 35 precisely controls the depressurization at a constant flow rate. The micrometer valve 35 is equipped with a heating element 36, a type of heating cable connected to a temperature controller, which heats the body of the micrometer valve 35 to prevent the carbon dioxide 6 and the initial solution 4 from freezing within the valve body 5.

[0059] The micrometric valve 35 is interposed between the second tube 30b and a third tube 30c, preferably identical to the second tube 30b.

[0060] Thus, the third tube 30c has a third external diameter X3 of one-eighth of an inch, or 3.175 mm, a second internal diameter Y3 of 1.75 mm, and a second length L3 of 5 cm between its ends. One end of the third tube 30c is in fluidic contact with the micrometer valve 35, while the other end is in fluidic contact with a second reducing fitting 50b.

[0061] The second 50b reducing fitting is a one-eighth-inch to one-sixteenth-inch reducing fitting. The second 50b reducing fitting is inserted between the third 30c tube and a fourth 30d tube.

[0062] The fourth tube 30d has a fourth external diameter X4 of one sixteenth of an inch, or 1.5875 mm, a fourth internal diameter Y4 of 0.5 mm, and a fourth length L4, measured between its ends, of 13 cm. One end of the fourth tube 30d is in fluidic contact with the second reducing fitting 50b, while the other end is in fluidic contact with a manifold 38.

[0063] We note that the first external diameter X1 is greater than the second external diameter X2, the second external diameter X2 being equal to the third external diameter X3, and the third external diameter X3 being greater than the fourth external diameter X4. We also note that the first external diameter X1 is twice the second external diameter X2 and the third external diameter X3, and that the second external diameter X2 and the third external diameter X3 are twice the fourth external diameter X4.

[0064] We also note that the first internal diameter Y1 is greater than the second internal diameter Y2, the second internal diameter Y2 being equal to the third internal diameter Y3, the third internal diameter Y3 being greater than the fourth internal diameter Y4.

[0065] The purpose of the millifluidic device 13 thus constituted is to ensure the contact and mixing between carbon dioxide 6 and the initial solution 4 for the formation of the suspension of nanometer-sized liposomes 2 at the outlet of the fourth tube 30d. The carbon dioxide 6 and initial solution 4 phases begin to come into contact in the mixer 30 and mixing continues throughout the entire millifluidic device 13.

[0066] Furthermore, these arrangements aim to prevent controlled depressurization. More specifically, at the outlet of the fourth tube 30d, the collector 38 allows for the depressurization and collection of the liposome suspension 2 and the venting of carbon dioxide 6 in a gaseous state through a vent 40. In other words, the liposome suspension 2 is formed at the outlet of the fourth tube 30d in the collector 38, allowing for depressurization, the withdrawal of the liposome suspension 2, and the venting of the carbon dioxide 6, now back in a gaseous state at ambient pressure and temperature, towards the vent 40. The fourth tube 30d ensures a nanometric size for the liposomes.

[0067] The collector 38 is, for example, a container or other component that allows for the withdrawal of the liposome suspension 2 and the discharge of carbon dioxide 6 to the vent 40. The collector 38 is, for example, associated with a flow meter 39, such as a float flow meter, which is designed to control an outlet flow rate D' of carbon dioxide 6. The outlet flow rate D' is approximately 9 g / min, within ±1%. In other words, the outlet flow rate D' is advantageously equivalent to the second flow rate D2. The mass flow meter 39 allows for the control of the outlet flow rate D' of gaseous carbon dioxide 6, which must be the same as that delivered by the second pump 23, i.e., 9 g / min.

[0068] Several examples of the implementation of such a process from such a system 1 are listed below. According to the following ten examples, the initial solution 4 comprises a solution containing water and ethanol in respective mass proportions of 79 and 21%, as well as solutes in a mass proportion of 0.1% relative to the ethanol solution, the solutes being Phosphatidylcholine, Cholesterol and Polyethylene glycol in respective mass proportions of 65%, 30% and 5%, as well as an active agent concentration of 33 mg / L.

[0069] According to a first embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 87 ± 5 nm with an encapsulation rate of 98.1 ± 1.6%. The experimental conditions are such that the second pressure P2 is equal to 150 bar and the second temperature T2 is equal to 35 °C.

[0070] According to a second embodiment, the liposomes obtained have an average diameter of 88 nm with an encapsulation rate of 96%. The experimental conditions are such that the second pressure P2 is equal to 170 bar and the second temperature T2 is equal to 35 °C. Figure 1 illustrates, on the one hand, a distribution curve of liposome occurrence as a function of the diameter of the liposomes obtained, which shows a unimodal distribution, and on the other hand, transmission electron micrographs of liposomes.

[0071] According to a third embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 81 nm with an encapsulation rate of 95.8%. The experimental conditions are such that the second pressure P2 is equal to 200 bar and the second temperature T2 is equal to 35 °C.

[0072] According to a fourth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 83 nm with an encapsulation rate of 96.8%. The experimental conditions are such that the second pressure P2 is equal to 250 bar and the second temperature T2 is equal to 35 °C.

[0073] According to a fifth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 78 nm with an encapsulation rate of 96.9%. The experimental conditions are such that the second pressure P2 is equal to 150 bar and the second temperature T2 is equal to 40 °C.

[0074] According to a sixth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 73.2 nm with an encapsulation rate of 95%. The experimental conditions are such that the second pressure P2 is equal to 200 bar and the second temperature T2 is equal to 40 °C.

[0075] According to a seventh embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 79.8 nm with an encapsulation rate of 97.7%. The experimental conditions are such that the second pressure P2 is equal to 250 bar and the second temperature T2 is equal to 40 °C.

[0076] According to an eighth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 78 nm with an encapsulation rate of 96.3%. The experimental conditions are such that the second pressure P2 is equal to 150 bar and the second temperature T2 is equal to 45 °C.

[0077] According to a ninth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 67 nm with an encapsulation rate of 96.9%. The experimental conditions are such that the second pressure P2 is equal to 200 bar and the second temperature T2 is equal to 45 °C.

[0078] According to a tenth embodiment, in liposome suspension 2, the liposomes obtained have an average diameter of 79.3 nm with an encapsulation rate of 97.8%. The experimental conditions are such that the second pressure P2 is equal to 250 bar and the second temperature T2 is equal to 45 °C.

Claims

System (1) for producing a suspension of liposomes (2) comprising a first line (12) extending between a first container (10) containing an initial solution (4) of at least one active agent (3) and phospholipids (5) and a millifluidic device (13), and a second line (19) extending between a second container (20) containing carbon dioxide (6) in liquid form and the millifluidic device (13), characterized in that the millifluidic device (13) comprises at least one mixer (30), a plurality of tubes (30a, 30b, 30c, 30d), a plurality of reducing fittings (50a, 50b) and a micrometer valve (35). System (1) according to claim 1, characterized in that the mixer (30) comprises at least a first branch (31) in connection with the first line (12), a second branch (32) in connection with the second line (19) and a third branch (33) in connection with a first tube (30a). System (1) according to claim 2, characterized in that the millifluidic device (13) successively comprises the first tube (30a), a first reducing fitting (50a), a second tube (30b), the micrometric valve (35), a third tube (30c), a second reducing fitting (50b) and a fourth tube (30d). System (1) according to claim 3, characterized in that the second tube (30b) and the third tube (30c) are identical. System (1) according to any one of the preceding claims, characterized in that the first line (12) comprises a first pump (11) capable of circulating the initial solution (4) at a first flow rate (D1) which is on the order of 1 mL / min at + / - 1%, and a first pressure (P1), which is between 80 bar and 300 bar, at + / - 5%. System (1) according to any one of the preceding claims, characterized in that the second line (19) comprises a second pump (23) capable of circulating carbon dioxide (6) at a second flow rate (D2) which is on the order of 9 g / min to within + / - 1%, and at a second pressure (P2), which is between 80 bar and 300 bar, to within + / - 5%. System (1) according to any one of the preceding claims, characterized in that the first line (12) comprises a first heat exchanger (16) which is designed to bring the initial solution (4) to a first temperature (T1) which is between 35 °C and 60 °C. System (1) according to any one of the preceding claims, characterized in that the first line (12) comprises a metal sinter (18). System (1) according to any one of the preceding claims, characterized in that the second line (19) comprises a second heat exchanger (26) designed to bring the carbon dioxide (6) to a second temperature (T2) which is between 35 °C and 60 °C. A method for the continuous production of a liposome suspension (2) via a system (1) according to claim 1, the system (1) comprising a first line (12) extending between a first container (10) containing an initial solution (4) of at least one active agent (3) and phospholipids (5) and a millifluidic device (13), the system (1) comprising a second line (19) comprising a second pump (23) and extending between a second container (20) containing carbon dioxide (6) and the millifluidic device (13), the millifluidic device (13) comprising at least one mixer (30), a plurality of tubes (30a, 30b, 30c, 30d), a plurality of reducing fittings (50a, 50b), and a micrometer valve (35), characterized in that the method comprises a carbon dioxide holding step (6) between the second pump (23) and the plurality of tubes (30a, 30b, 30c, 30d), at a second pressure (P2) which is on the order of 100 bar,to within + / - 5% and at a second temperature (T2) between 35 °C and 60 °C, and a step of obtaining the liposome suspension (2) at the outlet of the tubes (30a, 30b, 30c, 30d) in a collector (38).

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