Free-flow electrophoresis microcell device and its use

Abstract

A free-flow electrophoresis microcell device comprising, in sequence, an electrophoresis plate X, a waterproof plate Y, and a cooling plate Z including a heat transfer system, wherein the plates are stacked on a floor including an electrophoresis chamber, and each electrophoresis chamber includes a recess of height h corresponding to the thickness of the electrophoresis plate X of 25 to 200 mm, 4 to 9 consecutive inlets aligned so that the inlets and outlets face each other, 4 to 12 consecutive outlets, and supply channels and recovery channels connected to the inlets and outlets, respectively.

Description

Technical Field

[0001] The present invention relates to a free-flow electrophoresis microcell device and its use.

Background Art

[0002] High-performance liquid chromatography (HPLC) and electrophoresis in a region using a carrier are techniques for the analytical separation and / or preparative separation of molecules, including biomolecules, present in a mixture. However, since chromatography used in industrial processes for separation and / or purification requires a stationary phase, it is costly. Furthermore, since zone electrophoresis requires a carrier, there are limitations to its use on an industrial scale as an adjustment method from the perspectives of cost and quantity.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, in order to enable the purification and / or separation of products expected in terms of volume and quantity on an industrial scale, it is necessary to develop an adjustable and robust device that requires limited maintenance, operates in a continuous flow, and is easy to operate and modularly designed for implementation.

[0005] One object of the present invention is to provide a device for purifying and / or separating molecules by free-flow electrophoresis. Another object of the present invention is a method for purifying and / or separating molecules, particularly biomolecules, that is adjustable on an industrial scale. Another object of the present invention is a method for installing such a device on an industrial scale. [Means for solving the problem]

[0006] The first object of the present invention is a free-flow electrophoresis microcell device comprising vertically continuous plates X, Y, and Z, wherein the surface of the plates is YZY (XYZY) p Stacked according to sequence, X represents an electrophoresis plate (1) made of an inert substance, Y represents a waterproof plate (2) made of sapphire or alumina Al2O3 containing 99% α-Al2O3, which is made of an inert material, is electrically insulating and thermally conductive, and is made of α-Al2O3. Z represents the cooling plate (3) including the heat transfer system (4), p is an integer between 1 and 100, representing the number of stages in the device and the number of plates X. Each row (5) is: - Defined by the plate YZYXYZY in the following sequence, -Plate X is located between the two plates Y, -The two plates Z are each adjacent to plate Y, - Each of the two plates Y located at the ends of the YZYXYZY sequence covers plate Z, so that each plate Z is located between the two plates Y. The device also includes means for fastening all plates that enable the waterproofing of the device, The aforementioned device has each plate X, It comprises i electrophoresis chambers (Fi), where i is an integer from 1 to 100, particularly 50, preferably 10. Each electrophoresis chamber (6) is ○ Including the recessed part, It has the shape of a rectangular prism with four sides (a, b, c, d) and two top and bottom faces (e, f). The recess has a length Loe and a width Lae. The height h of the electrophoresis plate X, depending on its thickness, is 25 μm to 20 mm, particularly 50 to 200 μm, or 1.0 mm to 5.0 mm. The sides (a,b) are parallel to each other, face (a) is enclosed by two sides (A1,A2) of dimension Lae, and face (b) is enclosed by two sides (B1,B2) of dimension Lae. The sides (c,d) are parallel to each other, face (c) is enclosed by two sides (C1,C2) of dimension Loe, and face (d) is enclosed by two sides (D1,D2) of dimension Loe. ○ Includes n consecutive entrances E(1), E(2) to E(n-1), En, where n is an integer from 4 to 9, preferably 5 or 6, distributed on the plane (a) between A1 and A2, and aligned in directions parallel to A1 and A2. ○ m consecutive exits S(1), S(2) include S(m-1), S(m), where m is an integer from 4 to 12, preferably 5 or 7, distributed on the plane (b) between B1 and B2, aligned in directions parallel to B1 and B2, so that in directions parallel to C1 and D1, S(1) faces E(1) and S(m) faces E(n). The plate Y ensures the watertightness of the electrophoresis chamber, The chambers (Fi) are arranged such that the sides C1 of each recess are parallel to each other. The device described above, - A supply channel is configured to connect the inlet E(1) of each electrophoresis chamber to the micro / millimeter flow supply circuit of the liquid cathode, - A supply channel is configured to connect the inlet E(n) of each electrophoresis chamber to a micro / millimeter flow supply circuit that supplies liquid cathodes, - A supply channel is configured to connect at least one of the inlets E(2) to E(n-1) of each electrophoresis chamber to a micro / millimeter flow supply circuit for the initial solution containing the product to be purified and / or separated. - The remaining inlets of each electrophoresis chamber are configured to be connected to a supply channel that connects to a micro / millimeter flow supply circuit of at least one buffer, -Includes a recovery channel configured to connect each outlet S(1) to S(m) of each electrophoresis chamber to a micro / millimeter flow recovery circuit, The device is parallel to A1 and perpendicular to C1, and is operating in the presence of an electric field generated between the liquid cathode and the liquid anode. - The liquid cathode is circulated from the inlet E(1) to the outlet S(1). -The liquid anode is circulated from the inlet E(n) to the outlet S(m), -In the electrophoresis chamber (Fi), a solution containing the separated and / or purified products and at least one buffer is circulated between the liquid cathode and the liquid anode from inlet E(2) to E(n-1) to outlet S(2) to S(m-1). - At one of the outlets S(2) to S(m-1) of each electrophoresis chamber in the recovery circuit, the purified product and / or separated product contained in the initial solution are recovered.

[0007] "Free-flow electrophoresis" refers to electrophoresis that does not use a stationary phase, that is, electrophoresis that does not use a solid phase to support the movement of species during electrophoresis.

[0008] A "plate" is typically a rigid element in the shape of a rectangular parallelepiped, with at least two faces parallel to each other, the latter primarily representing the total surface area of ​​the element; that is, the area of ​​the two parallel faces accounts for more than half of the total surface area. These two faces are referred to as the "front" or "bottom or top" of the plate. The thickness of the plate is determined by the distance between these two surfaces of the plate.

[0009] "Continuous plates in the vertical direction" means a stack of plates, where the surfaces of the individual plates are in contact.

[0010] An "electrophoresis plate" refers to a plate named X, which contains i electrophoresis chambers (Fi). Plate X is made from a material that is inert to electrophoresis.

[0011] "Electrophoresis chamber" refers to a part of electrophoresis plate X. -A recess provided in the electrophoresis plate X, partitioned by the side wall, - An inlet and an outlet located on the side wall of the recess, and the like. The electrophoresis chamber is part of Plate X, and free-flow electrophoresis is performed.

[0012] The "electrophoresis cell" is a structural unit of the device. The electrophoresis cell includes an electrophoresis chamber, the wall of Plate Y that closes the electrophoresis chamber, and supply channels and recovery channels connected to the inlet and outlet of the electrophoresis chamber. The electrophoresis cell includes a recess closed by the side walls of Plate X and Plate Y, an inlet and an outlet of the recess, and supply channels and recovery channels.

[0013] A device composed of electrophoresis cells is called an electrophoresis chip. The "cooling plate" is named Z and means a plate including a heat transfer system. The function of the heat transfer system is to carry a heat transfer fluid to enable temperature control. The heat transfer system consists of, for example, a network of channels formed by the recesses of Plate Z.

[0014] The "waterproof plate" named Y means a plate aimed at ensuring the fluid water tightness of the device. In fact, each Plate X is located between two Plates Y, which ensure the waterproofness of the electrophoresis chamber of Plate X. Similarly, each Plate Z is arranged between two Plates Y, which ensure the waterproofness of the heat transfer system of Plate Z. Plate Y is made of an inert substance, has electrical insulation and thermal conductivity, thereby ensuring the chemical inertness and electrical inertness of the device required for the free-flow electrophoresis method, and ensuring the thermal conductivity between the heat transfer system and the electrophoresis chamber to enable temperature control of the device. Plate Y is made of 99% α-Al2O3 sapphire or alumina Al2O3.

[0015] In a preferred embodiment of the present invention, Plate Y is made of sapphire. In another aspect of the present invention, plate Y is made of alumina Al2O3 with 99% α-Al2O3. "Sapphire" is a material made from corundum, that is, alumina Al2O3 containing 99% by weight of the α-Al2O3 phase.

[0016] Advantageously, plate Y has a Mohs hardness of 9 (Coridon) and a thermal conductivity of 30-50 W / m / K. Plate Y also possesses high mechanical strength. As a non-exclusive example, sapphire plate Y is supplied by Saint-Gobain (LuxiumSolutions). Sapphire plates have the advantage of being transparent.

[0017] This device consists of stacked plates fastened together by a clamping mechanism, with the surfaces of adjacent plates in direct contact. The adjacent plates are continuous plates. Each plate of the device has up to two adjacent plates, i.e., two adjacent plates, positioned on both sides of the plate.

[0018] Each stage of this device consists of a series of plates stacked according to the following sequence: plates YZYXYZY. The number of plates X defines the number of stages in the device. Each stage consists of a plate X surrounded by two cooling plates Z, with each of plates X and Z adjacent to two plates Y, thereby providing airtightness of the heat transfer system of the i electrophoresis chambers of plates Z and X, and thermal conductivity between plate X and the cooling plates. Plates X and Z are separated by plate Y, thereby separating the fluid in the heat transfer system of plate Z from the fluid circulating within the electrophoresis chamber of plate X, so that they are not in fluidic communication and the exchange that takes place is only thermal.

[0019] In a multi-stage device, the YZY sequence plate located between two plates X is common to two consecutive stages. In a two-stage device with a YZYXYZYXYZY sequence, the central YZY sequence is common to both stages. In a three-stage device with the sequence YZYXYZYXYZYXYZY, the two central YZY sequences are common to two consecutive stages: the first and second stages, and the second and third stages, respectively.

[0020] The device is advantageous to include 1 to 100 electrophoresis chambers for each electrophoresis plate distributed on the floor. It is advantageous for the chambers to be arranged in rows for each plate X on each floor. The range "1-100" includes integers in the ranges of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, and 90-100, in particular the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

[0021] The electrophoresis chamber in which free-flow electrophoresis is performed includes a recess in plate X, which is closed by the side wall within plate X, and the recess has a general rectangular parallelepiped shape.

[0022] The term "rectangular parallelepiped" generally refers to a rectangular parallelepiped portion with length Loe, width Lae, and height h, corresponding to the thickness of plate X (Figure 2). When defining the general shape of the recess, means present within the electrophoresis chamber, such as channeling means located in the inlet and outlet, as well as in the protrusions, are not considered.

[0023] A rectangular prism consists of four sides (a, b, c, d) and two faces (e, f), each of which has a top and bottom face (Figure 2). The sides (a,b) are parallel to each other and have the same dimensions. Face (a) is enclosed by two edges (A1, A2) of dimension Lae, and is spaced at intervals of h. Face (b) is enclosed by two edges (B1, B2) of dimension Lae, and is spaced at intervals of h. The sides (c,d) are parallel to each other and have the same dimensions. Face (c) is enclosed by two edges (C1, C2) of dimension Loe, and is spaced at intervals of h. Face (d) is enclosed by two edges (D1, D2) of dimension Loe, and is spaced at intervals of h. The upper surface (e) and the lower surface (f) are parallel to each other and have the same dimensions. They form the surfaces that border the recesses carved into plate X. Face (e) is enclosed by sides (A1, B1, C1, D1) that form a rectangle with width Lae and length Loe. Face (f) is enclosed by sides (A2, B2, C2, D2) that form a rectangle with width Lae and length Loe.

[0024] Height h corresponds to the thickness of plate X, the height of the electrophoresis chamber, and the recess of the electrophoresis chamber. The height h is between 100 μm and 20 mm. The range from 25 μm to 20 mm includes 25-50 μm, 50-75 μm, 75-100 μm, 100-200 μm, 200-300 μm, 300-400 μm, 400-500 μm, 500-600 μm, 600-700 μm, 700-800 μm, 800-900 μm, 900 μm to 1.0 mm, 1.0-2.0 mm, 2.0-3.0 mm, 3.0-4.0 mm, 4.0-5.0 mm, and 5.0 This range includes ~6.0mm, 6.0~7.0mm, 7.0~8.0mm, 8.0~9.0mm, 9.0~10.0mm, 10.0~11.0mm, 11.0~12.0mm, 12.0~13.0mm, 13.0~14.0mm, 14.0~15.0mm, 15.0~16.0mm, 16.0~17.0mm, 17.0~18.0mm, 18.0~19.0mm, and 19.0~20.0mm. Advantageously, the height h is 25-200 μm. Ideally, the height h should be between 1.0 and 5.0 mm.

[0025] "Inlet" means a passage that allows liquid fluid flow from the outside to the inside of a system that includes a closed recess configured to contain the fluid, such as an electrophoresis chamber or a heat transfer system.

[0026] "Outlet" means a passage that allows liquid fluid flow from the inside to the outside of a system that includes a closed recess configured to contain the fluid, such as an electrophoresis chamber or a heat transfer system.

[0027] (a) On this side, n inlets in the recesses of the electrophoresis chamber are continuously distributed and referred to as E(1) to E(n), i.e., E(1), E(2) to E(n-1), and E(n). The number of inlets n is 4 to 9, i.e., 4, 5, 6, 7, 8, 9, preferably 5 or 6. Entrances E(1) to E(n) are located between edges A1 and A2 and are aligned at approximately equal distances from each other in directions parallel to A1 and A2.

[0028] "Alignment" means that the inlet or outlet is close to the same line. Therefore, the inlets can be positioned slightly forward or backward of plane (a) and / or slightly above or below each other. The inlet positions are configured to introduce various flows into the electrophoresis chamber and flow through the chamber parallel to side C1.

[0029] (b) On this side, m outlets are continuously distributed and referred to as S(1) to S(m), respectively, S(1), S(2) to S(m-1), and S(m). The number of outlets is 4 to 12, i.e., 4, 5, 6, 7, 8, 12, preferably 5 or 7. Exits S(1) to S(m) are located between sides B1 and B2 and are aligned at approximately equal distances from each other in a direction parallel to B1 and B2.

[0030] Entrances E(1) and E(n) are located near the ends of edges A1 and A2, that is, as close as possible to face (c) or (d). Exits S(1) and S(m) are located near the ends of edges B1 and B2, that is, as close as possible to face (c) or (d).

[0031] The inlet E(1) is configured for introducing the liquid cathode. The outlet S(1) is configured to discharge the liquid cathode after circulation within the electrophoresis chamber. The inlet E(n) is configured for introducing the liquid anode. The outlet S(m) is configured to discharge the liquid anode after circulation within the electrophoresis chamber.

[0032] The chamber inlet and outlet are configured such that E(1) faces E(n) and S(1) faces S(m), thereby enabling electrophoresis by inducing an electric field within the electrophoresis chamber between the liquid anode and liquid cathode during fluid circulation. The liquid anode and liquid cathode are electrolytes, i.e., solutions containing ions.

[0033] A "liquid cathode" refers to an electrolyte solution configured to act as a cathode during electrophoresis.

[0034] A "liquid anode" refers to an electrolyte solution configured to act as an anode during electrophoresis.

[0035] The presence of a liquid cathode and liquid anode generates an electric field within the electrophoresis chamber when the electrolyte becomes charged. The liquid cathode and liquid anode are electrolytes capable of generating an electric field, which can be increased or decreased by the action of a generator that charges the liquid anode and liquid cathode, and / or by an increase in the concentration of ions.

[0036] One of the inlets E(2) to E(n-1) is configured to introduce the solution to be purified and / or separated into the electrophoresis chamber. One of the outlets S(2) to S(m-1) is configured for recovering the purified and / or separated solution after electrophoresis performed in the electrophoresis chamber. At least one of the inlets E(2) to E(n-1), separate from the inlet for the solution to be purified and / or separated, is configured to introduce the buffer into the electrophoresis chamber. The inlet and outlet of the electrophoresis chamber are connected by channels to a supply or recovery circuit configured for the circulation of a microstream or millistream of fluid.

[0037] A "microcurrent circuit" refers to a set of channels with cross-sections on the order of micrometers.

[0038] A "millimeter-flow circuit" refers to a set of channels with cross-sections on the order of millimeters.

[0039] Inlets E(1) to E(n) are connected to the supply circuit by supply channels. The outlets S(1) to S(m) are connected to the recovery circuit by the recovery channel.

[0040] The device of the present invention is - The inlet E(1) of each electrophoresis chamber (Fi) is connected to a micro / millimeter flow supply circuit that supplies liquid cathodes. - The inlet E(n) of each electrophoresis chamber (Fi) is connected to a micro / millimeter flow supply circuit that supplies liquid anodes. - Connect at least one of the inlets E(2) to E(n-1) of each electrophoresis chamber (Fi) to a micro / millimeter flow feed circuit that supplies the initial solution containing the product to be purified and / or separated. - From each electrophoresis chamber, the remaining inlets are fed into a micro / millimeter flow circuit that supplies at least one buffer solution. - The outlet S(1) of each electrophoresis chamber (Fi) is connected to a micro / millimeter flow circuit for recovering the liquid cathode after circulation within the electrophoresis chamber. - The outlet S(m) of each electrophoresis chamber (Fi) is routed to a micro / millimeter flow circuit for collecting the liquid anode after circulation within the electrophoresis chamber. - At least one of the outlets S(2) to S(m-1) of each electrophoresis chamber (Fi) is connected to a micro / millimeter flow circuit for recovering the purified and / or separated products contained in the initial solution to be purified and / or separated. - Includes a channel for connecting other outlets to at least one micro / millimeter flow recovery circuit.

[0041] Therefore, the device of the present invention operates with respect to each electrophoresis chamber (Fi) in the presence of an electric field applied parallel to A1 and perpendicular to C1. -The liquid cathode is circulated along the surface (c) from the inlet E(1) to the outlet S(1). -The liquid anode is circulated along the surface (d) from the inlet E(n) to the outlet S(m). -In the electrophoresis chamber (Fi), a solution containing the separated and / or purified products and at least one buffer is circulated between the liquid cathode and the liquid anode from inlet E(2) to E(n-1) to outlet S(2) to S(m-1). - At one of the outlets S(2) to S(m-1) of each electrophoresis chamber in the recovery circuit, the purified product and / or separated product contained in the initial solution are recovered.

[0042] Surprisingly, the inventors have introduced a cooling circuit using a sapphire or alumina plate Al2O3 containing 99% α-Al2O3 as a means of separation between the heat transfer system and the electrophoresis chamber. -The thickness h of the electrophoresis chamber, which can be varied from micrometers to millimeters, and as a result, can increase the processing capacity of the solution to be purified or separated, constitutes a device. -Purification and / or separation of temperature-sensitive molecules, , - We found that excellent temperature control and homogeneity can be induced by introducing a temperature gradient into the electrophoresis chamber in a manner that makes this possible.

[0043] In this invention, the material properties of plate Y, which is made from sapphire or alumina Al2O3 containing 99% α-Al2O3, are essential features of the device of this invention. In fact, temperature control in free-flow electrophoresis is an important parameter, not only in terms of protein denaturation, but especially in maintaining a laminar flow system.

[0044] In the prior art, the use of a cooling circuit with a separation plate made of glass, quartz, ceramic, or thermoplastic material in the device described in Patent Document 1 is not efficient enough to achieve an electrophoresis chamber thickness on the order of millimeters, particularly due to the low thermal conductivity of the plate. For example, the thermal conductivity of a glass plate is on the order of 1 W / (mK), which is 1 / 40th of the thermal conductivity of sapphire used in the present invention. In Patent Document 1, the plate bordering the microfluidic circuit has the sole function of protecting the film etched by the microfluidic circuit.

[0045] Furthermore, the use of sapphire plate Y provides a robust and waterproof device that can be disassembled and reassembled, and whose device components are easy to clean and maintain. The modularity of the device allows for the reuse of plates Y and Z. Advantageously, sapphire's mechanical strength allows for assembly systems and enables high-pressure flow within the device while ensuring excellent sealing between each stage. This is in contrast to glass, which cracks very easily even at low pressure and is brittle; in the presence of water, cracks in glass can spread throughout the entire cell, potentially causing serious waterproofing problems.

[0046] Thus, by using a sapphire plate Y as an adjacent plate to close the flow circuit, the thermal conductivity of sapphire enables robustness and temperature control of the device. As a result, the present invention provides a free-flow electrophoresis device that can process larger volumes than the device described in Patent Document 1, because the height of the electrophoresis chamber is on the order of millimeters, for industrial applications of continuous purification and / or separation of samples by free-flow electrophoresis.

[0047] According to a particular embodiment, the number of outlets m is greater than or equal to the number of inlets n in order to induce fine separation of the product to be separated or purified. Advantageously, the number of exits (m) is greater than the number of inlets (n). By increasing the number of discharges, the recovery rate can be increased. According to a particular embodiment, n is equal to 5 and m is equal to 5. According to a particular embodiment, n is equal to 5 and m is equal to 7.

[0048] According to a particular embodiment, the present invention relates to a device as defined above, - The outlet S(1) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for collecting the liquid cathode. - The outlet S(m) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for collecting the liquid anode. - One of the outlets S(2) to S(m-1) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for recovering the purified and / or separated products contained in the initial solution. - Each electrophoresis chamber includes a recovery channel configured to connect the remaining outlets to at least one micro / millimeter flow recovery circuit of at least one buffer solution.

[0049] The device's recovery channels are configured to recover the purified and / or separated products contained in the liquid cathode, liquid anode, buffer(s), and / or the initial solution after circulation within the electrophoresis chamber. Since these recoveries are performed in four separate recovery circuits, the liquid anode and liquid cathode maintain an electric field without contact before or during the recovery of the purified and / or separated products at the outlet within the device's electrophoresis chamber. These recovery channels are also configured to recover the purified and / or separated products separately from at least one buffer used.

[0050] "Separation" means the separation of at least two products present in the initial solution that can be recovered separately, and each of the two products can be recovered at a different outlet of the device of the present invention.

[0051] "Purification" refers to the process of separating the product from other species present in the initial solution.

[0052] In certain embodiments, the present invention relates to a device as defined above, comprising a single electrophoresis plate containing a single electrophoresis chamber in which p is equal to 1, i is equal to 1, and in particular the height h is 25 to 200 μm or 1.0 to 5.0 mm. The present invention, in which p is equal to 1 and i is equal to 1, consists of a plate sequence YZYXYZY, includes a single electrophoresis chamber, and is advantageous in that the height h is 25-200 μm or 1.0-5.0 mm.

[0053] This constitutes the experimental tool configuration. Therefore, it is advantageous to identify the effects of various parameters such as the properties of the buffer, electric field, temperature, and the number and supply rates of inlets and outlets in the electrophoresis chamber in order to optimize the purification and separation conditions of the solution to be purified or separated.

[0054] In a particular embodiment, the present invention relates to a device as defined above, wherein p is equal to 1, i is 2 to 10, and comprises a single electrophoresis plate X having 2 to 10, preferably 10, electrophoresis chambers. The present invention, in which p is equal to 1 and i is 2 to 10, consists of a plate YZYXYZY in the following sequence, i.e., a single stage consisting of at least two electrophoresis chambers, preferably 10 electrophoresis chambers.

[0055] In a particular embodiment, the present invention relates to a device as defined above, wherein p is 2 to 10, i is 2 to 10, and comprises 2 to 10 electrophoresis plates X, each electrophoresis plate X comprising 2 to 10 electrophoresis chambers, in particular where p is equal to 10 and i is equal to 10. Advantageously, the height of the electrophoresis chamber is 1.0 to 20 mm, particularly 1.0 to 5.0 mm, and preferably 1.0 to 2.0 mm.

[0056] The device according to the present invention is an industrial free-flow electrophoresis device comprising multiple stages and multiple electrophoresis chambers for each stage, capable of continuously separating and / or continuously purifying solutions of up to 1 to 5 liters per hour, i.e., usable on an industrial scale.

[0057] According to a particular embodiment, the present invention relates to a device as defined above, wherein the height h of the electrophoresis chamber is 650 μm to 20 mm, particularly 650 μm to 10.0 mm, preferably 650 μm to 5.0 mm, and more preferably 650 μm to 2.0 mm.

[0058] The range 650 μm to 2.0 mm includes the following ranges: 650 μm to 700 μm, 700 to 750 μm, 750 to 800 μm, 800 to 850 μm, 850 to 900 μm, 900 to 950 μm, 950 μm to 1.0 mm, 1.0 to 1.1 mm, 1.1 to 1.2 mm, 1.2 to 1.3 mm, 1.3 to 1.4 mm, 1.4 to 1.5 mm, 1.5 to 1.6 mm, 1.6 to 1.7 mm, 1.7 to 1.8 mm, 1.8 to 1.9 mm, and 1.9 to 2.0 mm.

[0059] The range from 650 μm to 5.0 mm includes the following: 650 μm to 2.0 mm, 2.0 to 2.1 mm, 2.1 to 2.2 mm, 2.2 to 2.3 mm, 2.3 to 2.4 mm, 2.4 to 2.5 mm, 2.5 to 2.6 mm, 2.6 to 2.7 mm, 2.7 to 2.8 mm, 2.8 to 2.9 mm, 2.9 to 3.0 mm, 3.0 to 3.1 mm, 3.1 to 3.2 mm, 3.2 to 3.3 mm, and 3.3 to 3.4 mm. This includes the ranges of 3.4-3.5mm, 3.5-3.6mm, 3.6-3.7mm, 3.7-3.8mm, 3.8-3.9mm, 3.9-4.0mm, 4.0-4.1mm, 4.1-4.2mm, 4.2-4.3mm, 4.3-4.4mm, 4.4-4.5mm, 4.5-4.6mm, 4.6-4.7mm, 4.7-4.8mm, 4.8-4.9mm, and 4.9-5.0mm.

[0060] The range of 650 μm to 10.0 mm includes the following ranges: 650 μm to 5.0 mm, 5.0 to 5.5 mm, 5.5 to 6.0 mm, 6.0 to 6.5 mm, 6.5 to 7.0 mm, 7.0 to 7.5 mm, 7.5 to 8.0 mm, 8.0 to 8.5 mm, 8.5 to 9.0 mm, 9.0 to 9.5 mm, and 9.5 to 10.0 mm.

[0061] The range 650 μm to 20.0 mm includes the following ranges: 650 μm to 10.0 mm, 10.0 to 11.0 mm, 11.0 to 12.0 mm, 12.0 to 13.0 mm, 13.0 to 14.0 mm, 14.0 to 15.0 mm, 15.0 to 16.0 mm, 16.0 to 17.0 mm, 17.0 to 18.0 mm, 18.0 to 19.0 mm, and 19.0 to 20.0 mm.

[0062] According to a particular embodiment, the present invention relates to a device as defined above, wherein the height h of the electrophoresis chamber is 25 μm to 200 μm or 1.0 to 5.0 mm.

[0063] The range of 25 μm to 200 μm includes the ranges of 25 to 50 μm, 50 to 75 μm, 75 to 100 μm, 100 to 125 μm, 125 to 150 μm, 150 to 175 μm, and 175 to 200 μm.

[0064] The range from 1 to 5.0 mm includes 1.0 to 1.1 mm, 1.1 to 1.2 mm, 1.2 to 1.3 mm, 1.3 to 1.4 mm, 1.4 to 1.5 mm, 1.5 to 1.6 mm, 1.6 to 1.7 mm, 1.7 to 1.8 mm, 1.8 to 1.9 mm, 1.9 to 2.0 mm, 2.0 to 2.1 mm, 2.1 to 2.2 mm, 2.2 to 2.3 mm, 2.3 to 2.4 mm, 2.4 to 2.5 mm, 2.5 to 2.6 mm, 2.6 to 2.7 mm, 2.7 to 2.8 mm, 2.8 to 2.9 mm, and 2.9 to 3.0 mm. This range includes mm, 3.0-3.1mm, 3.1-3.2mm, 3.2-3.3mm, 3.3-3.4mm, 3.4-3.5mm, 3.5-3.6mm, 3.6-3.7mm, 3.7-3.8mm, 3.8-3.9mm, 3.9-4.0mm, 4.0-4.1mm, 4.1-4.2mm, 4.2-4.3mm, 4.3-4.4mm, 4.4-4.5mm, 4.5-4.6mm, 4.6-4.7mm, 4.7-4.8mm, 4.8-4.9mm, and 4.9-5.0mm.

[0065] The use of devices including electrophoresis chambers, particularly those with a single electrophoresis chamber with a height of 25–200 μm or 1.0–5.0 mm, is advantageous for conducting preliminary tests to identify the effects of various parameters and optimize the device's characteristics.

[0066] The height h of the electrophoresis chamber is constant throughout the entire system. "Height of 25-200 μm" means a constant height, selected from between 25 and 200 μm. Similarly, "Height of 1.0-5.0 mm" means a constant height selected from between 1.0 and 5.0 mm.

[0067] According to a particular embodiment, the present invention relates to a device as defined above, wherein the height h of the electrophoresis chamber is 1.0 mm to 5.0 mm, particularly 1.0 to 2.0 mm.

[0068] The use of devices with heights on the order of millimeters is advantageous for achieving industrial-scale product purification. For example, a device with 10 to 30 chambers, each 2 mm high, can yield 100 to 300 kg of purified product per year.

[0069] According to a particular embodiment, the present invention relates to a device as defined above, wherein the height h of the electrophoresis chamber is 650 μm to 20 mm, particularly 650 μm to 10.0 mm, preferably 650 μm to 5.0 mm, more preferably 650 μm to 2.0 mm, or the height h of the electrophoresis chamber is 25 μm to 200 μm or 1.0 to 5.0 mm.

[0070] According to a particular embodiment, the present invention relates to a device as defined above, wherein i electrophoresis chambers of each plate X are adjacent to each other by the surfaces (c) or (d) of each recess. Therefore, in the two adjacent electrophoresis chambers of plane (c) and (d), there is a common wall between the two chambers. Adjacent chambers along faces (c) and (d) of the same electrophoresis plate X constitute a row of electrophoresis chambers. This row configuration of chambers optimizes the use of plate X in terms of surface area and facilitates its manufacturing and machining. It also allows for optimization of the layout by, for example, sharing supply channels and cooling systems.

[0071] According to a particular embodiment, the present invention relates to a device as defined above, wherein the inlets E(1) for two adjacent chambers are supplied from the same supply channel.

[0072] According to certain embodiments, the present invention relates to a device as defined above, wherein the entrances to two adjacent chambers are symmetrical. They are symmetrical with respect to a wall, i.e., surface (c) or (d), that separates them.

[0073] According to a particular embodiment, the present invention relates to a device as defined above, wherein the ratio of the length Loe to the width Lae of the recess is 2 to 15. The ratio of the recess length (Loe) to the recess width (Lae) is selected to allow electrophoresis of the product to be purified. Advantageously, since it is selected independently of the electrophoresis of the product to be purified or separated, the device can be used for several types of products.

[0074] According to a particular embodiment, the present invention relates to a device as defined above, wherein the width Lae of the recess of the electrophoresis chamber is 1.0 to 8.0 cm, preferably 1.0 to 5.0 cm. The range of 1.0 to 8.0 cm includes the ranges of 1.0 to 2.0 cm, 2.0 to 3.0 cm, 3.0 to 4.0 cm, 4.0 to 5.0 cm, 5.0 to 6.0 cm, 6.0 to 7.0 cm, and 7.0 to 8.0 cm.

[0075] According to a particular embodiment, the present invention relates to a device as defined above, wherein the length Loe of the recess of the electrophoresis chamber is 5.0 to 20.0 cm, preferably 5.0 to 15.0 cm.

[0076] The range of 5.0 to 20.0 cm includes the following ranges: 5.0 to 6.0 cm, 6.0 to 7.0 cm, 7.0 to 8.0 cm, 8.0 to 9.0 cm, 9.0 to 10.0 cm, 10.0 to 11.0 cm, 11.0 to 12.0 cm, 12.0 to 13.0 cm, 13.0 to 14.0 cm, 14.0 to 15.0 cm, 15.0 to 16.0 cm, 16.0 to 17.0 cm, 17.0 to 18.0 cm, 18.0 to 19.0 cm, and 19.0 to 20.0 cm.

[0077] According to a particular embodiment, the present invention relates to a device as defined above, wherein the electrophoresis plate X is made of a material selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), and fluoroethylene propylene (FEP), particularly Teflon®, Teflon®-PFA, and Teflon®-FEP plates.

[0078] According to a particular embodiment, the present invention relates to a device as defined above, wherein in each electrophoresis plate X, a portion of the plate is configured to provide a flow path for a supply channel and a recovery channel, and the plate X is partially or entirely etched, cut, or drilled.

[0079] According to a particular embodiment, the present invention relates to a device as defined above, wherein the i electrophoresis chambers of each plate X are adjacent to each other by the surface (c) or (d) of each recess, and / or, the width of the recess in the electrophoresis chamber is 1.0 to 8.0 cm, preferably 1.0 to 5.0 cm. and / or, the length Loe of the recess of the electrophoresis chamber is 5.0 to 20.0 cm, preferably 5.0 to 15.0 cm. and / or, electrophoresis plate X is made from a material selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), and fluoroethylene propylene (FEP), particularly Teflon®, Teflon®-PFA, and Teflon®-FEP plates. and / or, in each electrophoresis plate X, a portion of the plate is configured to provide a flow path for supply channels and recovery channels, and is partially or entirely etched, cut, or drilled into the plate X.

[0080] According to a particular embodiment, the present invention relates to a device as defined above, wherein plates X, Y, Z have dimensions La × Lo, and La and Lo are 2.0 to 50.0 cm.

[0081] The range of 2.0 to 50.0 cm includes the following ranges: 2.0 to 5.0 cm, 5.0 to 10.0 cm, 10.0 to 15.0 cm, 15.0 to 20.0 cm, 20.0 to 25.0 cm, 25.0 to 30.0 cm, 30.0 to 35.0 cm, 35.0 to 40.0 cm, 40.0 to 45.0 cm, and 45.0 to 50.0 cm.

[0082] According to a particular embodiment, the present invention relates to a device as defined above, wherein plates X, Y, Z have dimensions La × Lo, where La is 6.0 to 30 cm and Lo is 2.0 to 50.0 cm.

[0083] The range of 6.0 to 30.0 cm includes the ranges of 6.0 to 10.0 cm, 10.0 to 15.0 cm, 15.0 to 20.0 cm, 20.0 to 25.0 cm, and 25.0 to 30.0 cm.

[0084] In the case of a device having a single electrophoresis chamber on plate X, the width "La" of plate X is slightly greater than the width "Lae" of the recess of the electrophoresis chamber, and the length "Lo" of plate X is slightly greater than the length "Loe" of the recess of the electrophoresis chamber.

[0085] In the case of a device consisting of 10 identical electrophoresis chambers adjacent to each other in a row on plate X, the width "La" of plate X is substantially greater than the length "Loe" of the recesses of the electrophoresis chambers, and the length "Lo" of plate X is substantially greater than 10 times the width "Lae" of the recesses of the electrophoresis chambers.

[0086] A slightly larger or substantially larger value is, for example, a value that is 1 to 20 mm larger. Advantageously, to improve the airtightness of the rows of 10 electrophoresis cells, the width of the sides of the electrophoresis plate X is increased by bordering the rows.

[0087] According to a particular embodiment, the present invention relates to a device as defined above, wherein the plate Y has a thickness of 0.5 mm to 5.0 mm.

[0088] The range of 0.5 to 5.0 mm includes the ranges of 0.5 to 1.0 mm, 1.0 to 1.5 mm, 1.5 to 2.0 mm, 2.0 to 2.5 mm, 2.5 to 3.0 mm, 3.0 to 3.5 mm, 3.5 to 4.0 mm, 4.0 to 4.5 mm, and 4.5 to 5.0 mm.

[0089] According to a particular embodiment, the present invention relates to a device as defined above, wherein plate Z is made of a material selected from plexiglass, PTFE, or polyamine.

[0090] According to a particular embodiment, the present invention relates to a device as defined above, wherein the cooling plate Z has a thickness of 1.0 to 10.0 mm, and in particular 1.0 to 5.0 mm.

[0091] The range of 1.0 to 10.0 mm includes the ranges of 1.0 to 2.0 mm, 2.0 to 3.0 mm, 3.0 to 4.0 mm, 4.0 to 5.0 mm, 5.0 to 6.0 mm, 6.0 to 7.0 mm, 7.0 to 8.0 mm, 8.0 to 9.0 mm, and 9.0 to 10.0 mm.

[0092] The range of 1.0 to 5.0 mm includes the ranges of 1.0 to 1.5 mm, 1.5 to 2.0 mm, 2.0 to 2.5 mm, 2.5 to 3.0 mm, 3.0 to 3.5 mm, 3.5 to 4.0 mm, 4.0 to 4.5 mm, and 4.5 to 5.0 mm.

[0093] According to a particular embodiment, the present invention relates to a device as defined above, wherein plates X, Y, Z have dimensions La × Lo, and La and Lo are 2.0 to 50.0 cm. and / or, plate Y has a thickness of 0.5 mm to 5.0 mm, And / or, the cooling plate Z has a thickness of 1.0 to 10.0 mm, especially 1.0 to 5.0 mm.

[0094] According to a particular embodiment, the present invention relates to a device as defined above, wherein the electrophoresis chamber preferably includes channeling means etched into the electrophoresis plate X and communicating with an inlet and / or outlet. These channeling means are configured to direct the flow within the electrophoresis chamber at both the inlet and outlet, thereby better dispersing the flow across the entire width of the recess at the inlet and better concentrating the flow at the outlet.

[0095] Advantageously, these channeling means are integral to the electrophoresis chamber, i.e., fused with the walls of the electrophoresis chamber, and made of the same material as the electrophoresis plate. These channeling means are positioned along the rectangular parallelepiped faces (a) and (b) where the recesses are located.

[0096] According to certain embodiments, the present invention relates to a device as defined above, wherein each electrophoresis chamber is configured to include at least one selectively permeable, preferably size-selective, membrane, which is positioned so that a solution containing the product to be separated or purified during operation of the device can pass through it.

[0097] According to a particular embodiment, the present invention relates to a device as defined above, wherein each electrophoresis chamber includes at least one size-selective and permeable membrane. Arranged parallel to surface (c), adjacent to the buffer inlet, This allows the initial solution containing the product to be separated or purified to pass through during the operation of the device. As a result, any portion of the initial solution that did not pass through the membrane is transported by the buffer solution from the inlet adjacent to the membrane to one of the outlets. The presence of these membranes also enables size selectivity for the products being purified and / or separated.

[0098] According to a particular embodiment, the present invention relates to a device as defined above, wherein each upper and / or lower surface of the electrophoresis chamber includes projections configured to not obstruct the circulation of the product to be purified and / or separated within the chamber during operation of the device, and to improve heat transfer and maintain the electrophoresis chamber at a selected temperature. The projection extends inward from the surface (e) and / or (f) of the recess. The projection may be located on one side only or on both sides.

[0099] According to a particular embodiment, the present invention relates to a device as defined above, wherein the protrusions are made of a thermally conductive material, preferably sapphire or alumina containing 99% α-Al2O3, preferably the same material as that of plate Y, in order to ensure thermal conductivity within the chamber and control the temperature within the cell.

[0100] The presence of these protrusions facilitates heat exchange between cooling plates along the pathway, which in turn allows for the optimization of the separation of desired molecules while maintaining an optimal temperature and avoiding molecular denaturation.

[0101] According to a particular embodiment, the present invention relates to a device as defined above, wherein the projection has a shape configured not to obstruct the circulation of the product to be purified and / or separated within the chamber during the operation of the device. The shape and arrangement of the protrusions can be calculated or simulated to prevent obstruction of laminar flow streamlines around the protrusions and within the electrophoresis chamber, but the shape or arrangement of the protrusions is efficient for heat transfer.

[0102] According to a particular embodiment, the present invention relates to a device as defined above, wherein the projection is a triangular spike, a pillar with a rectangular base, or an oval stud.

[0103] According to a particular embodiment, the present invention relates to a device as defined above, wherein plates X and / or Y are etched to accommodate the protrusions.

[0104] According to a particular embodiment, the present invention relates to a device as defined above, wherein each electrophoresis chamber is configured to include at least one selectively permeable, preferably size-selective, membrane arranged so that a solution containing the product to be separated or purified during operation of the device can pass through it. and / or, each upper and / or lower surface of the electrophoresis chamber includes protrusions configured to not obstruct the circulation of the product to be purified and / or separated within the chamber during the operation of the device, and to improve heat transfer and maintain the electrophoresis chamber at a selected temperature. In particular, the protrusions are made of a thermally conductive material, preferably sapphire or alumina containing 99% α-Al2O3.

[0105] According to a particular embodiment, the present invention relates to a device as defined above, wherein the heat transfer system within plate Z is a channeling network configured to allow the circulation of one or more heat transfer fluids, the channeling network being in direct contact with a portion of plate Y adjacent to plate Z, and the portion being thermally connected to the electrophoresis chamber to allow temperature control within the electrophoresis chamber.

[0106] According to a particular embodiment, the present invention relates to a device as defined above, wherein the channeling network of the heat transfer system is formed by recesses in the plate Z.

[0107] According to a particular embodiment, the present invention relates to a device as defined above, wherein the channeling network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers.

[0108] According to a particular embodiment, the present invention relates to a device as defined above, wherein the channeling network of the heat transfer system includes channels parallel to side C1 for each of the electrophoretic chambers, and the channels may contain heat transfer fluids of varying temperatures to generate the temperature gradient.

[0109] According to a particular embodiment, the present invention relates to a device as defined above, wherein the inlet of the heat transfer system is located on the (a) side of the electrophoresis chamber.

[0110] According to a particular embodiment, the present invention relates to a device as defined above, wherein the channeling network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers. The heat transfer system is a channeling network configured to allow the circulation of one or more heat transfer fluids, the channeling network being in direct contact with a portion of plate Y adjacent to plate Z, and the portion being thermally connected to the electrophoresis chamber to allow temperature control within the electrophoresis chamber. Preferably, the channeling network of the heat transfer system comprises channels parallel to side C1 for each of the electrophoresis chambers, and the channels may contain heat transfer fluids of various temperatures to generate the temperature gradient.

[0111] According to a particular embodiment, the present invention relates to a device as defined above, wherein the channeling network is configured to obtain a selected and controlled temperature in the electrophoresis chamber.

[0112] According to a particular embodiment, the present invention relates to a device as defined above, wherein the inlet and outlet flows of each channeling network of the heat transfer system are perpendicular to the side C1 of each electrophoresis chamber.

[0113] According to a particular embodiment, the present invention relates to a device as defined above, wherein the inlet of the heat transfer system is located on the face (c) side of the recess of the electrophoresis chamber.

[0114] According to a particular embodiment, the present invention relates to a device as defined above, wherein the fastening means for ensuring the airtightness of the device consists of two external fastening plates surrounding the device, and the means is particularly removable plate by plate.

[0115] Another object of the present invention relates to the use of devices as defined above in preparative electrophoresis for the purification and / or separation of molecules, particularly proteins, under continuous flow. The device of the present invention is advantageous in that it allows for continuous use for the purification and / or separation of products.

[0116] According to a particular embodiment, the present invention relates to the use of a device as defined above, wherein the solution to be purified or separated is processed at a rate of 1 to 5 L / hour, and in particular, the device comprises 100 electrophoresis chambers, preferably comprising 10 stages, with 10 electrophoresis chambers per stage.

[0117] According to a particular embodiment, the present invention relates to the use of the device as defined above, wherein the productivity of the purified or separated product is 100 to 300 kg / year of purified or separated product, and in particular, the device includes 10 to 50 electrophoresis chambers.

[0118] According to a particular embodiment, the present invention relates to the use of a device as defined above, wherein the device includes 100 electrophoresis chambers operating for 300 days / year.

[0119] Another object of the present invention relates to a method for purifying and / or separating products contained in a solution by free-flow electrophoresis. - Connecting a supply channel of the device according to the present invention as defined above to a supply circuit of a liquid cathode, a liquid anode, an initial solution containing the product to be purified and / or separated, and at least one buffer, wherein the channel and circuit are controlled by a central processing device (UC1), - The step of connecting the cooling system to a cooling circuit controlled by a central processing device (UC2), - The step of generating an electric field along side A1 via a liquid anode and a liquid cathode, -The central processing device (UC1) generates fluid circulation in each of the electrophoresis chambers, thereby, The liquid cathode is circulated from the inlet E(1) to the outlet S(1). The liquid anode is circulated from the inlet E(n) to the outlet S(m). The steps include: circulating an initial solution containing the product to be purified and / or separated between the liquid cathode and liquid anode in an electrophoresis chamber, and at least one buffer, from the inlet E(2) to E(n-1) to the outlets S(2) and S(m-1); -The process includes the step of selecting and recovering a separation product or a purification product from at least one of the outlets S(2) to S(m-1) of the electrophoresis chamber of the device.

[0120] The fluid flow is controlled by the central processing device (UC1) using, for example, flow meters and pumps in the supply and recovery circuits. For instance, each inlet of the electrophoresis chamber can be connected to a flow meter. The electric field along side A1 in the electrophoresis chamber can be generated by the circulation of the liquid cathode from E(1) to S(1) and the circulation of the liquid anode from E(n) to S(m).

[0121] Advantageously, the same central processing device may control the fluid flow in the electrophoresis chamber and the heat transfer fluid flow in the cooling system.

[0122] According to certain embodiments, the present invention relates to a method as defined above, which is carried out under a continuous flow of an initial solution containing the product to be purified and / or separated. In particular, at a flow rate of 1-5 L / hour, the device includes 100 electrophoresis chambers. Preferably, it operates for 300 days / year.

[0123] According to a particular embodiment, the present invention relates to the method defined above, wherein the liquid cathode and liquid anode are electrolytes of the same composition.

[0124] "Liquid electrodes" refer to liquid cathodes and liquid anodes. It is important to note that electrodes are electron or ion conductors that capture or release electrons.

[0125] In this embodiment, the cathode and anode are referred to as liquid electrodes. These liquid electrodes are electrolytes with high ionic conductivity. For example, the liquid electrode consists of 40% methanol, 10 mM HEPES, 0.2% HPMC, 0.1% Tween 20, and 1.5 M pH=7.5% KCl, with an ionic conductivity of 250 mS / cm.

[0126] The liquid electrode may contain species of ions in the form of physiological saline. Advantageously, the liquid electrode may contain chloride or fluoride salts.

[0127] In one embodiment, the liquid electrode comprises 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), or its citrate, or 2-(N-morpholino) ethanesulfonic acid (TSS), or its acetate.

[0128] In one embodiment, the liquid electrode also includes hydroxypropyl methylcellulose (HPMC), Tween20 (polyoxyethylene (20) sorbitan monolaurate), methanol, ethanol, and / or KCl.

[0129] Advantageously, the liquid electrode has a composition of 10 mM HEPES, 0.2% (m / v) HPMC, 0.1% (m / v) Tween20, 40% methanol, 0.5-1.5 M KCl, and water.

[0130] Advantageously, the pH of the liquid electrode can be adjusted with a NaOH solution.

[0131] Advantageously, the ionic conductivity of the liquid electrode is 0.01 to 250 mS / cm.

[0132] Advantageously, the liquid electrode is charged before being introduced into the device chamber, particularly using a carbon electrode introduced into an electrolyte consisting of, for example, 40% methanol, 10 mM HEPES, 0.2% HPMC, 0.1% Tween20, and 0.1% 1.5 M pH=7.5 KCl.

[0133] The use of liquid cathodes and liquid anodes means that no metal is used within the device, preventing the electrolysis of water and the formation of bubbles in the electrophoresis chamber.

[0134] According to a particular embodiment, the present invention relates to a method as defined above, wherein the buffer has a pH suitable for the purification and / or separation of the product to be purified and / or separated, which is contained in the initial solution.

[0135] Generally, the pH of a buffer solution is between 5.8 and 7.5, depending on the molecules being purified in the medium.

[0136] According to a particular embodiment, the present invention relates to the method defined above, wherein the fluid circulation of the liquid cathode in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 10,000 μL / min.

[0137] According to a particular embodiment, the present invention relates to the method defined above, wherein the fluid circulation of the liquid anode in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 10,000 μL / min.

[0138] According to a particular embodiment, the present invention relates to a method as defined above, wherein the fluid circulation of the solution to be purified and / or separated in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 30,000 μL / min.

[0139] According to a particular embodiment, the present invention relates to the method defined above, wherein the fluid circulation of the buffer in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 50,000 μL / min.

[0140] The range of 10 to 10,000 μL / min includes 10 to 20 μL / min, 20 to 50 μL / min, 50 to 80 μL / min, 80 to 100 μL / min, 100 to 150 μL / min, 150 to 200 μL / min, 200 to 300 μL / min, 300 to 400 μL / min, 400 to 500 μL / min, 500 to 600 μL / min, 600 to 800 μL / min, 800 to 1,000 μL / min, 1,000 to 1,500 μL / min, 1,500 to 2,000 μL / min, 2,000 to 2,500 μL / min, and 2,500 to 3,000 μL / min. This includes the ranges of 3,500-4,000 μL / min, 4,000-4,500 μL / min, 4,500-5,000 μL / min, 5,000-5,500 μL / min, 5,500-6,000 μL / min, 6,000-6,500 μL / min, 6,500-7,000 μL / min, 7,000-7,500 μL / min, 7,500-8,000 μL / min, 8,000-8,500 μL / min, 8,500-9,000 μL / min, 9,000-9,500 μL / min, and 9,500-10,000 μL / min.

[0141] The range of 10-20,000 μL / min includes the following ranges: 10-10,000 μL / min, 10,000-11,000 μL / min, 11,000-12,000 μL / min, 12,000-13,000 μL / min, 13,000-14,000 μL / min, 14,000-15,000 μL / min, 15,000-16,000 μL / min, 16,000-17,000 μL / min, 17,000-18,000 μL / min, 18,000-19,000 μL / min, and 19,000-20,000 μL / min.

[0142] The range of 10-50,000 μL / min includes the ranges of 10-20,000 μL / min, 20,000-25,000 μL / min, 25,000-30,000 μL / min, 30,000-35,000 μL / min, 35,000-40,000 μL / min, 40,000-45,000 μL / min, and 45,000-50,000 μL / min.

[0143] According to a particular embodiment, the present invention relates to the method defined above, wherein the fluid circulation of the liquid cathode in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 10,000 μL / min. and / or, the fluid flow of the liquid anode in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 10,000 μL / min. and / or the fluid circulation of the solutions to be purified and / or separated in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 30,000 μL / min. and / or, the fluid circulation of the buffer in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 50,000 μL / min.

[0144] According to a particular embodiment, the present invention relates to a method as defined above, wherein the device includes at least 100 electrophoresis chambers arranged in parallel.

[0145] An electrophoresis cell arranged in parallel or in parallel means an electrophoresis cell in which the supply of fluid through channels at different inlets occurs in parallel, i.e., simultaneously with the inlets from which the fluid (liquid electrode, buffer, solution to be purified and / or separated) is introduced from the same source. Cells 1 and 2 are simultaneously supplied at their respective inlets E(1) by the liquid electrode from the same container, for example, the liquid electrode. Therefore, the supply circuit for the solutions purified and / or separated from the electrophoresis chamber is a parallel circuit.

[0146] According to a particular embodiment, the present invention relates to the method defined above, wherein at least two buffers of different pH values ​​are used during fluid circulation in each of the electrophoresis chambers to cause a pH fluctuation along side A1 within each electrophoresis chamber.

[0147] According to a particular embodiment, the present invention relates to a method as defined above, wherein the device comprises at least one size-selective membrane and is configured to separate the product to be purified and / or separated from the initial solution during fluid circulation in each of the electrophoresis chambers.

[0148] According to a particular embodiment, the present invention relates to a method as defined above, wherein each electrophoresis chamber comprises at least one size-selective membrane positioned parallel to side C1 and adjacent to the buffer inlet, During fluid circulation in each electrophoresis chamber, the initial solution containing the products to be separated and / or purified passes through the membrane, and the portion of the initial solution that does not pass through the membrane is carried to one of the outlets S(2) to S(m-1) by the buffer flowing in from the inlet adjacent to the membrane.

[0149] According to a particular embodiment, the present invention relates to the method defined above, wherein during fluid circulation in each of the electrophoresis chambers, In each electrophoresis chamber, In particular, a temperature gradient is applied along the edge A1 of the recess by a cooling system of the device, which includes a heat transfer system consisting of a channeling network configured to allow the circulation of one or more heat transfer fluids parallel to each face (C) of the electrophoresis chamber.

[0150] According to a particular embodiment, the present invention relates to a method as defined above, wherein the device includes at least 100 electrophoresis chambers arranged in parallel. and / or, the device comprises at least one size-selective membrane and is configured to separate products to be purified and / or separated from the initial solution during fluid circulation in each of the electrophoresis chambers. and / or, during fluid circulation in each electrophoresis chamber, use at least two buffers of different pH values ​​to create a pH variation along side A1 within each electrophoresis chamber. and / or, during fluid circulation in each electrophoresis chamber, a temperature gradient along the side A1 of the recess is applied in each electrophoresis chamber.

[0151] According to a particular embodiment, the present invention relates to the method defined above, wherein a uniform temperature, particularly 10-40°C, is applied in each of the electrophoresis chambers during fluid circulation in each of the electrophoresis chambers.

[0152] The 10-40°C range includes the ranges of 10-15°C, 15-20°C, 20-25°C, 25-30°C, 30-35°C, and 35-40°C, particularly the values ​​of 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C.

[0153] According to a particular embodiment, the present invention relates to the method defined above, wherein the electric field generated during fluid circulation in each of the electrophoretic chambers is 200 to 4,000 V.

[0154] The 200-4000V range includes the following ranges: 200-500V, 500-1,000V, 1,000-1,500V, 1,500-2,000V, 2,000-2,500V, 2,500-3,000V, 3,000-3,500V, and 3,500-4,000V.

[0155] According to a particular embodiment, the present invention relates to the method defined above, wherein a uniform temperature, particularly 10-40°C, is applied in each of the electrophoresis chambers during fluid circulation in each of the electrophoresis chambers. And / or, the electric field generated during fluid circulation in each of the electrophoresis chambers is 200 to 4,000 V.

[0156] According to certain embodiments, the present invention relates to the methods defined above and is carried out for the purification and / or separation of proteins.

[0157] According to certain embodiments, the present invention relates to methods as defined above and is carried out for the purification and / or separation of isomers, particularly optical isomers.

[0158] According to certain embodiments, the present invention relates to methods as defined above and is used for the purification and / or separation of proteins, or for the purification and / or separation of isomers, particularly optical isomers.

[0159] Another object of the present invention is, with respect to the use of the device according to the present invention as defined above, comprising a single electrophoresis chamber, in particular, having a recess height h of 25 to 200 μm or 1.0 to 5.0 mm, thereby enabling the installation of the industrial device according to the present invention as defined above, comprising 10 to 100 electrophoresis chambers, and identifying and optimizing the fluid circulation of the products to be purified and / or separated.

[0160] Another object of the present invention relates to a method for developing an industrial device for purifying and / or separating a solution containing a product to be purified and / or separated by electrophoresis. a) A first step of implementing a device according to the present invention as defined above, comprising a single electrophoresis chamber, and in particular for identifying and optimizing the fluid circulation of the product to be purified and / or separated, wherein the height h of the recess is 25 to 200 μm or 1.0 to 5.0 mm, b) a second step of mounting the industrial device, which consists of 10 to 100 electrophoresis chambers. [Brief explanation of the drawing]

[0161] [Figure 1] This is an exploded perspective view of an electrophoresis microcell device in which 30 electrophoresis cells are arranged in 3 rows of 10 columns. [Figure 2] This figure shows the recess (6) in the chamber of the electrophoresis plate (1). [Figure 3] This is an exploded perspective view of a single-stage device having a single electrophoresis chamber. [Figure 4] Figure a) shows a single-stage device having a row of 10 electrophoresis chambers, and figure b) shows a two-stage device having 10 electrophoresis chambers in each stage. [Figure 5] This diagram shows a row of four electrophoresis chambers. [Figure 6] Figure a) shows a device having a cooling system that allows for the establishment of a temperature gradient along the width of the electrophoresis chamber, and figure b) shows the length of the chamber. [Figure 7] This figure shows a device having a fastening mechanism. [Figure 8] This is a diagram of the electrophoresis chamber of the electrophoresis chip to be used. [Figure 9] This is a photograph of the KPLE-100-008 device, which has a 100 μm thick electrophoresis chamber, taken during a fluid dynamics test. [Figure 10] These are a series of photographs taken during a fluid dynamics test of a 1 mm thick device, including an electrophoresis chamber with five inlets and seven outlets. [Figure 11] This is a photograph taken during a fluid dynamics test of a device with a thickness of h2mm, having an electrophoresis chamber with 5 inlets and 7 outlets. [Figure 12] This is a photograph of a 100 μm thick device having an electrophoresis chamber with five inlets and five outlets, taken during the separation of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 13] This figure shows the HPLC spectra of the products at outlets S(2), S(3), and S(4) of a device having five inlets and five outlets and an electrophoresis chamber with a thickness of 100 μm, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 14] This is a photograph of a device with a 100 μm thick electrophoresis chamber having 5 inlets and 7 outlets, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 15]This figure shows the HPLC spectra of the products at outlets S(2), S(3), S(4), S(5), and S(6) of a device having five inlets and seven outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 16] This is a photograph of a device with an electrophoresis chamber having 5 inlets and 7 outlets and a thickness of 1.0 mm, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 17] This is a photograph of a device with an electrophoresis chamber having 5 inlets and 7 outlets and a thickness of 1.0 mm, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 18] This is a photograph of a device with an electrophoresis chamber having 5 inlets and 7 outlets and a thickness of 2.0 mm, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). [Figure 19] This figure shows the HPLC spectra of the product at outlets S(1) to S(7) of a device having five inlets and seven outlets and an electrophoresis chamber with a thickness of 100 μm, during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP). [Figure 20] This figure shows the HPLC spectra of the product at outlets S(1) to S(7) of a device having five inlets and seven outlets and an electrophoresis chamber with a thickness of 100 μm, during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP). [Figure 21] This figure shows the HPLC spectra of the product at outlets S(1) to S(7) of a device having five inlets and seven outlets and an electrophoresis chamber with a thickness of 100 μm, during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP). [Figure 22]This figure shows the species electrophoresis of a device with a 100 μm thick electrophoresis chamber having five inlets and five outlets, during a separation test of a protein and its linker mixture. [Figure 23] This figure shows the HPLC spectrum of the product at outlet S(3) of a device having five inlets and five outlets and an electrophoresis chamber with a thickness of 100 μm, during a separation test of a protein and its linker mixture. [Figure 24] This figure shows the HPLC spectrum of the product at outlet S(4) of a device having five inlets and five outlets and an electrophoresis chamber with a thickness of 100 μm, during a protein mixture separation test. [Modes for carrying out the invention]

[0162] Drawings and Examples

[0163] Figure 1 is an exploded perspective view of an electrophoresis microcell device in which 30 electrophoresis cells are arranged in 3 rows of 10 columns, and does not show all plate clamping mechanisms.

[0164] (1) represents an electrophoresis plate comprising a row of 10 electrophoresis chambers, each containing a rectangular recess (6), with each chamber having an inlet or outlet (7) and a supply channel or recovery channel (8), and two adjacent recesses separated by the same wall. (2) represents a synthetic sapphire plate, enabling heat exchange while ensuring fluid separation between the two plates. (3) represents a cooling plate comprising a cooling system (4), which includes a recess for circulating heat transfer fluid from the inlet to the outlet. (5) represents a stage consisting of a continuous plate YZYXYZY. The device comprises three stages, consisting of the following sequence YZYXYZYXYZYXYZY, with two central YZY sequences common to two consecutive stages, the first and second stages, and the second and third stages, respectively.

[0165] Figure 2 shows the recess (6) of the chamber of the electrophoresis plate (1). The recess is a rectangular parallelepiped portion with width Lae, length Loe, and height h, enclosed by faces (a, b, c, d, e, f). Faces (a, b, c, d) form the side walls between the recess and the plate X, and faces (a, b) and (c, d) are parallel to each other. Face (a) is enclosed by edges (A1, A2) of dimension Lae, and face (b) is enclosed by edges (B1, B2) of dimension Lae. Face (c) is enclosed by edges (C1, C2) of dimension Loe, and face (d) is enclosed by edges (D1, D2) of dimension Loe. Edges (A1, B1, C1, D1) demarcate face (e), and edges (A2, B2, C2, D2) demarcate face (f).

[0166] Figure 3 is an exploded perspective view of a single-stage device with a single electrophoresis chamber, and does not show all plate clamping mechanisms. (1) represents an electrophoresis plate comprising a single chamber containing a rectangular recess (6), and including an inlet or outlet (7) and a supply channel or recovery channel (8). (2) represents a synthetic sapphire plate that allows heat exchange while ensuring fluid separation between the two plates. (3) represents a cooling plate comprising a cooling system (4), which includes a recess for circulating heat transfer fluid from inlet to outlet, with the heat transfer fluid inlet on the same side as the inlet of the electrophoresis chamber. The device comprises a single stage (5) containing a series of plates YZYXYZY.

[0167] Figure 4 shows a) a single-stage device with a row of 10 electrophoresis chambers and b) a two-stage device with 10 electrophoresis chambers in each stage. (1) represents an electrophoresis plate comprising a row of 10 electrophoresis chambers, each containing a rectangular recess (6), with each chamber having an inlet or outlet (7) and a supply channel or recovery channel (8), and two adjacent recesses separated by the same wall. (2) represents a synthetic sapphire plate that allows heat exchange while ensuring fluid separation between the two plates. (3) represents a cooling plate comprising a cooling system (4) that includes recesses for circulating heat transfer fluid from the inlet to the outlet. (5) represents a stage consisting of a continuous plate YZYXYZY. a) The device consists of one stage and comprises the following sequence YZYXYZY. b) The device consists of two stages and is composed of the following sequence YZYXYZYXYZY, where the middle sequence YZY is common to both stages.

[0168] Figure 5 shows a row of four electrophoresis chambers, where (a) the inlets to each electrophoresis chamber are identically aligned, and (b) the inlets to adjacent chambers are symmetrical with respect to a partition. The inlets and outlets of the electrophoresis chambers include channeling means (9).

[0169] Figure 6 shows a device having a cooling system that allows for the establishment of a temperature gradient along the width of the electrophoresis chamber (a) and the length of the chamber (b). In this device, the cooling system includes a cooling system having recesses that form channels (10) parallel to the flow of the electrophoresis chamber (a) and channels (10) perpendicular to the flow (b).

[0170] Figure 7 shows a device having a fastening means. In this particular embodiment, the fastening means consists of two plates (11) that surround the entire continuous plates X, Y, Z, and the distance between the two plates (11) can be adjusted by a mounting means (12) connecting them. The mounting means (12) is, for example, a screw.

[0171] Figure 8 shows the electrophoresis chamber of the electrophoresis chip to be used, where a) represents the KPLE-100-008 chip with 5 inlets and 7 outlets, the outlets numbered 1 to 7 from top to bottom, the central inlet is for the sample, inlets E(2) and E(4) are for the buffer solution (TS), and inlets E(1) and E(5) are for the liquid electrode, and b) represents the KPLE-100-009 chip with 5 inlets and 5 outlets, the outlets numbered 1 to 5 from top to bottom, the central inlet is for the sample, inlets E(2) and E(4) are for the buffer solution (TS), and inlets E(1) and E(5) are for the liquid electrode.

[0172] The recess of the electrophoresis chamber has a width Lae and a length Loe. The electrophoresis plate X has a width La and a length Lo. The electrophoresis chamber includes channeling means (9) for inflow and outflow. The channeling means is, for example, a chisel point (92) shaped triangular element (91) located between two inlets or two outlets.

[0173] Figure 9 is a photograph of the KPLE-100-008 device with a 100 μm thick electrophoresis chamber, taken during a hydrodynamic test. The sample flow and electrode flow were colored yellow to allow visualization of the different flows.

[0174] Figure 10 is a series of photographs taken during a hydrodynamic test of a 1 mm thick device including an electrophoresis chamber with five inlets and seven outlets, with sample / buffer / electrode flow rates of a) 160 / 1600 / 1000 μL / min, b) 320 / 3200 / 800 μL / min, c) 400 / 4000 / 1000 μL / min, and d) 400 / 2000 / 500 μL / min.

[0175] Figure 11 is a photograph of a device with a thickness of h2 mm, having an electrophoresis chamber with 5 inlets and 7 outlets, taken during a hydrodynamic test, with sample / buffer / electrode flow rates of 400 / 4000 / 1000 μL / min.

[0176] Figure 12 is a photograph of a 100 μm thick device with an electrophoresis chamber having five inlets and five outlets, taken during the separation of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The procedure was performed at 1500 V with sample / buffer / electrode solution flow rates of 10 / 80 / 20 μL / min.

[0177] Figure 13 shows the HPLC spectra of the products at outlets S(2), S(3), and S(4) of a device with five inlets and five outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The test was performed at 1500 V with sample / buffer / electrode solution flow rates of 10 / 80 / 20 μL / min, respectively.

[0178] Figure 14 is a photograph of a device with a 100 μm thick electrophoresis chamber having 5 inlets and 7 outlets, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G) at 2500 V, with sample / buffer / electrode solution flow rates of 10 / 100 / 20 μL / min, respectively.

[0179] Figure 15 shows the HPLC spectra of products at outlets S(2), S(3), S(4), S(5), and S(6) of a device with five inlets and seven outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The test was performed at 2500 V with sample / buffer / electrode solution flow rates of 10 / 100 / 20 μL / min, respectively.

[0180] Figure 16 is a photograph of a device with a 1.0 mm thick electrophoresis chamber having 5 inlets and 7 outlets, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The test was performed at 2000 V with sample / buffer / electrode solution flow rates of 10 / 600 / 20 μL / min. a) corresponds to a photograph taken without annotation, and b) shows the same photograph with annotations indicating the pathways of the colored compounds.

[0181] Figure 17 is a photograph of a device with a 1.0 mm thick electrophoresis chamber having 5 inlets and 7 outlets, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The test was performed at 3000 V, with sample / buffer / electrode solution flow rates of 20 / 3000 / 50 μL / min, respectively. a) corresponds to a photograph taken without annotation, and b) shows the same photograph with annotations indicating the pathways of the colored compounds.

[0182] Figure 18 is a photograph of a device with a 2.0 mm thick electrophoresis chamber having 5 inlets and 7 outlets, taken during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B, and rhodamine 6G). The test was performed at 3000 V, with sample / buffer / electrode solution flow rates of 20 / 3000 / 50 μL / min. a) corresponds to a photograph taken without annotation, and b) shows the same photograph with annotations indicating the pathways of the colored compounds.

[0183] Figure 19 shows the HPLC spectra of the products at outlets S(1) to S(7) of a device with five inlets and seven outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP), performed at 0 V with sample / buffer / electrode solution flow rates of 10 / 100 / 25 μL / min, respectively.

[0184] Figure 20 shows the HPLC spectra of the products at outlets S(1) to S(7) of a device with five inlets and seven outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP). The test was performed at 1000 V with sample / buffer / electrode solution flow rates of 10 / 100 / 25 μL / min, respectively.

[0185] Figure 21 shows the HPLC spectra of the products at outlets S(1) to S(7) of a device with five inlets and seven outlets and a 100 μm thick electrophoresis chamber during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP). The test was performed at 2,000 V with sample / buffer / electrode solution flow rates of 10 / 100 / 25 μL / min, respectively.

[0186] Figure 22 shows the electrophoresis of a 100 μm thick electrophoresis chamber with five inlets and five outlets, photographed during a separation test of a protein and its linker mixture, performed at 1500 V with sample / buffer / electrode solution flow rates of 10 / 80 / 20 μL / min, respectively.

[0187] Figure 23 shows the HPLC spectrum of the product at outlet S(3) of a device with five inlets and five outlets and a 100 μm thick electrophoresis chamber during a separation test of a protein and its linker mixture, performed at 1500 V with sample / buffer / electrode solution flow rates of 10 / 80 / 20 μL / min, respectively.

[0188] Figure 24 shows the HPLC spectrum of the product at outlet S(4) of a device with five inlets and five outlets and a 100 μm thick electrophoresis chamber during a protein mixture separation test, performed at 1,500 V with sample / buffer / electrode solution flow rates of 10 / 80 / 20 μL / min, respectively. [Examples]

[0189] Example 1: Materials and Method

[0190] Electrophoresis devices Two devices known as electrophoresis chips were used in the experiment: two electrophoresis chambers with different dimensions for the recess (so-called separation chamber) and plate X, and two devices with different numbers of outlets. The dimensions of the two chips are shown in Table 1 below. Three different heights were used: h100μm, 1mm, and 2mm.

[0191] The KPLE-100-009 chip is an electrophoresis chamber with five inlets and five outlets. The inlets and outlets are symmetrical. The inlets are numbered 1 through 5 from top to bottom, E(1) through E(5).

[0192] The KPLE-100-008 tip is an electrophoresis chamber with five inlets and seven outlets. The recovery rate is increased by repeatedly discharging the samples. These outlets are numbered from 1 to 7 from top to bottom, S(1) to S(7), i.e., S1 to S7.

[0193] Each inlet was connected to a flow meter that controlled the flow rate supplied to the electrophoresis chip. Each flow meter itself was connected to an independent liquid supply container, i.e., sample, buffer, or liquid electrode. All parameters, including inlet flow rates, product recovery rates at the outlet, and analytical results, were controlled and automatically recorded by a computer.

[0194] [Table 1]

[0195] Example 2: Study of hydrodynamic flow within a device

[0196] The hydrodynamic flow studies were set up using a free-flow electrophoresis device that included electrophoresis chambers with five inlets and seven outlets, and thicknesses h of 100 μm, 1 mm, and 2 mm.

[0197] The purpose of these tests was to analyze the electrophoresis of sample flow, electrolyte flow (anode and cathode), and buffer flow while the device was operating. No electric field was applied during these tests.

[0198] Test 1: KPLE-100-008 chip with thickness h=100μm The KPLE-100-008 chip was connected to the flow meter at each inlet under the conditions shown in Table 2 below.

[0199] [Table 2]

[0200] Figure 9 is a photograph of the KPLE-100-008 device with a 100 μm thick electrophoresis chamber, taken during a hydrodynamic test. The sample flow and electrode flow were colored yellow to allow visualization of the different flows.

[0201] Without applying an electric field, the sample flow path from the inlet E(3) to the outlet S(4) facing E(3) was observed.

[0202] A band-shaped area colored yellow was observed from E(1) to S(1). Another band-shaped area colored yellow was observed from E(1) to S(6) and S(7). These two bands represent the two flow paths of the liquid electrode, respectively.

[0203] Tests 2-5: Five inlet chips and seven outlet chips with a thickness of h=1mm. Under the conditions shown in Table 3 below, a tip containing an electrophoresis chamber with 5 inlets, 7 outlets, and a thickness h of 1 mm was connected to a flow meter at each inlet.

[0204] [Table 3]

[0205] Figure 10 is a photograph of a 1 mm thick device, including an electrophoresis chamber with five inlets and seven outlets, taken during a hydrodynamic test. The sample / buffer / electrode flow rates were a) 160 / 1600 / 100 μL / min, b) 320 / 3200 / 800 μL / min, c) 400 / 4000 / 1000 μL / min, and d) 400 / 2000 / 500 μL / min.

[0206] By coloring the buffer solution yellow and the electrode flow blue, it became possible to visualize the different flow paths.

[0207] Without an applied electric field, the buffer solution was stained blue while the sample solution was not, indicating that the sample flow path was almost linear from inlet E(3) to outlet S(4) facing E(3). In fact, the sample flow was bordered on both sides by two dark bands representing the paths of the injected buffer, from inlet E(2) to outlets S(2)-S(4) and from inlet E(4) to outlets S(5)-S(6).

[0208] Two yellow-colored bands were observed, one from E(1) to S(1) and the other from E(1) to S(7). These two bands represent the two flow paths of the liquid electrode, respectively.

[0209] Test 6: Five inlet chips and seven outlet chips with a thickness of h=2mm. Under the conditions shown in Table 4 below, a tip containing an electrophoresis chamber with 5 inlets, 7 outlets, and a thickness h of 2 mm was connected to a flow meter at each inlet.

[0210] [Table 4]

[0211] Figure 11 is a photograph of a device with a thickness of h2 mm, including an electrophoresis chamber with five inlets and seven outlets, taken during a hydrodynamic test, with flow rates of 400 / 4000 / 1000 μL / min for the sample / buffer / electrode solutions, respectively.

[0212] Visualization of different flows was enabled by coloring the sample flow blue and the electrode flow yellow.

[0213] The path of the sample flow from inlet E(3) to outlets S(4) and S(5) was shown without applying an electric field. The path of the sample was visualized by coloring the buffer blue.

[0214] Regions colored yellow in a band shape were observed from E(1) to outlets S(1) and S(2), and another region colored yellow in a band shape was observed from E(5) to outlets S(6) and S(7). These two bands represent the paths of the two flows of the liquid electrodes, respectively.

[0215] As a conclusion, through hydrodynamic tests 1 - 6, the laminar flow of the injection solution at various flow rates in the chamber was confirmed, enabling the implementation of free-flow electrophoresis.

[0216] Example 3: Separation of a mixture of fluorescein, rhodamine B, and rhodamine 6G

[0217] Assays 7 - 11 were set up on a free-flow electrophoresis device to separate a mixture of three molecules, fluorescein, rhodamine B, and rhodamine 6G, which exhibit fluorescence. The chemical structures of the three molecules are shown below.

[0218]

Chemical formula

[0219] These three compounds are of similar sizes below nanometers and have significantly different charges. The zeta potential of each compound was measured in advance and presented in Table 5. At pH 7.49, the zeta potential was -23.5 mV for fluorescein, -0.3 mV for rhodamine B, and +36.2 mV for rhodamine 6G.

[0220]

Table 5

[0221] Test 7 - Chamber with 5 inlets and 5 outlets - h=100μm - V=1500V The main features of Test 7 are as follows: - Chamber with 5 inlets and 5 outlets -h=100μm, -V=1500V -Flow rate (μl / min): Sample / buffer / electrode: 10 / 80 / 20

[0222] In this test, a KPLE-100-009 tip, which has five inlets and five outlets and an electrophoresis chamber height h of 100 μm, was connected to a flow meter at each inlet.

[0223] Electrolytes for the anode and cathode were supplied to inlets E(1) and E(5), respectively. The two electrolytes for the cathode and anode had the same composition. The liquid electrolyte solution consisted of 10 mM HEPES, 0.2% (w / v) HPMC, 0.1% (m / v) Tween 20, 40% methanol, and 1.5 M KCl. The flow rate was set to 20 μL / min. The pH of these electrolyte solutions was 7.49 (adjusted with NaOH solution), and the conductivity was 92.33 mS / cm. The electric field was generated by the electrolyte in the electrophoresis chamber and was obtained from the solutions containing the carbon electrode, anode electrode, and cathode electrode, respectively.

[0224] The central inlet E(3) was supplied with the sample to be purified, which was a mixture of three dyes: fluorescein, rhodamine B, and rhodamine 6G. The sample to be purified consisted of 0.175 g / L of fluorescein, 0.176 g / L of rhodamine 6G, and 0.185 g / L of rhodamine B, and the sample flow rate during purification was 10 μL / min.

[0225] The last two inlets, E(2) and E(4), were supplied with a buffer solution consisting of 10 mM HEPES in water, 0.2% (m / v) HPMC, and 0.1% (m / v) Tween20 at a flow rate of 80 μL / min. The buffer solution had a pH of 7.5 (adjusted with NaOH solution) and a conductivity of 600 μS / cm.

[0226] The electric field is variable from 0 to 3000V, but it was set to 1500V for these tests. After the flow stabilized, the product that flowed out of the electrophoresis chamber was collected in a tube and analyzed by HPLC to determine the proportion of each compound at each outlet.

[0227] Figure 12 is a photograph of the device during electrophoresis, made possible by using a transparent sapphire plate and colored separation products.

[0228] Hydrodynamic monitoring was performed to demonstrate that none of the compounds in the sample migrated to the liquid electrode. Therefore, only the outlets S(2), S(3), and S(4) of the electrophoresis chamber were analyzed by HPLC.

[0229] result Figure 12 shows how yellow fluorescein electrophores toward the liquid cathode and reaches outlet S(2). Rhodamine B, which has a low charge, is unaffected during electrophoresis and flows out from outlet S(3). Rhodamine 6G (PZ=+36.2mV) electrophores toward the liquid anode and flows out from outlet S(4).

[0230] Figure 13 shows the HPLC spectra of the products leaching out from outlets S(2), S(3), and S(4).

[0231] HPLC analysis of this test, conducted at 1500V, confirmed that fluorescein was present in outlet S(2), rhodamine B was mainly present in outlet S(3), and rhodamine 6G was present in outlet S(4).

[0232] It is estimated that the calculated migration rate of fluorescein is 92.98% with respect to outlet S(2), and that of rhodamine 6G is 62.75% with respect to outlet S(4).

[0233] Test 8 The main features of Test 8 are as follows. - A chamber with five inlets and seven outlets - h = 100 μm, - V = 2,500 V - Flow rate (μl / min): sample / buffer / electrode: 10 / 100 / 20

[0234] A separation test was set up using a second device, the KPLE-100-008 chip, which has five inlets and seven outlets. The height h of the electrophoresis chamber is 100 μm.

[0235] The arrangement of the solutions at the inlets was the same as in Test 7, but the separation method in this test was that the flow rates of the sample, buffer, and electrode were 10 / 100 / 20 μL / min respectively, and the applied electric field voltage was different. In Test 8, it was fixed at 2500 V.

[0236] Therefore, in this test, the KPLE-100-008 chip was connected to a flow meter. An electrolytic solution for the anode and a cathodic electrolytic solution were supplied to inlets E(1) and E(5) respectively. The two electrolytic solutions for the cathode and anode had the same composition respectively. The liquid electrolyte solution had a composition of 10 mM HEPES, 0.2% (w / v) HPMC, 0.1% (m / v) Tween 20, 40% methanol, and 1.5 M KCl. The flow rate was set at 20 μL / min. The pH of these electrolyte solutions was 7.49 (adjusted with NaOH solution), and the conductivity was 92.33 mS / cm. The electric field was generated by the electrolytic solution in the electrophoresis chamber and was obtained from the solutions containing a carbon electrode, an anode electrode, and a cathode electrode respectively.

[0237] A purified sample composed of 0.175 g / L fluorescein, 0.176 g / L rhodamine 6G, and 0.185 g / L rhodamine B was supplied to the central inlet E(3), and the sample flow rate was 10 μL / min during purification.

[0238] The last two inlets, E(2) and E(4), were supplied with a buffer solution consisting of 10 mM HEPES in water, 0.2% (m / v) HPMC, and 0.1% (m / v) Tween20 at a flow rate of 100 μL / min. The buffer solution had a pH of 7.5 (adjusted with NaOH solution) and a conductivity of 600 μS / cm.

[0239] The electric field was set to 2,500V in this test. After the flow stabilized, the product that flowed out of the electrophoresis chamber was collected in a tube and analyzed by HPLC to determine the proportion of each compound at each outlet.

[0240] Figure 14 is a photograph of the device during electrophoresis, made possible by using a transparent sapphire plate and colored separation products.

[0241] Hydrodynamic monitoring was performed to demonstrate that none of the compounds in the sample migrated to the liquid electrode. Therefore, only the outlets S(2) to S(6) of the electrophoresis chamber were analyzed by HPLC.

[0242] result Figure 14 shows how yellow fluorescein electrophores toward the liquid cathode and reaches outlet S(2). Rhodamine B was unaffected by the presence of the electric field during electrophoresis and flowed out from outlet S(4) facing inlet E(3). Rhodamine 6G migrated to the liquid anode in a smaller amount than fluorescein and flowed out at S(5).

[0243] Figure 15 shows the HPLC spectra of the products leaching out from outlets S(2) to S(6).

[0244] HPLC analysis in the 8-2,500V assay showed no signals from rhodamine B or rhodamine 6G, confirming the presence of fluorescein mainly at exits S(2) and S(3). The spectrum of the product obtained from S(4) mainly shows rhodamine B. The spectrum of the product derived from S(5) mainly shows rhodamine 6G, and the product does not contain rhodamine B.

[0245] At 2500V, fluorescein was 100% purified and recovered at outlets S(2) and S(3). Rhodamine B did not show electrophoresis, but rhodamine 6G showed partial electrophoresis toward outlet S(5) on the order of 83.49%.

[0246] Test 9 The main features of Test 9 are as follows: - Chamber with 5 inlets and 7 outlets -h=1mm -V=2000V -Flow rate (μl / min): Sample / buffer / electrode: 10 / 600 / 10

[0247] Separation test 9 was set up in a third device, which included an electrophoresis chamber with five inlets and seven outlets, and a height h of 1 mm.

[0248] The arrangement of the inlet solution was the same as in Tests 7 and 8, but the separation method in Test 9 differed in that the volume of the electrophoresis chamber, which was 1 mm high, the flow rates of the sample, buffer, and electrode were 10 / 600 / 10 μL / min, respectively, and the applied electric field voltage was 2,000 V.

[0249] Figure 16 is a photograph of the device during electrophoresis. Figure 16 shows the electrophoresis of charged fluorescein (yellow) to the cathode and rhodamine 6G to the anode in an electrophoresis chamber with a height of 1 mm.

[0250] Rhodamine B was unaffected by the presence of the electric field during electrophoresis and flowed out from outlet S(4) facing inlet E(3).

[0251] Under the conditions set for this test, the electrophoresis of fluorescein and rhodamine 6G was not significant compared to the previous test; therefore, fluorescein reached between exit S(3) and exit S(4), and rhodamine 6G reached between exit S(4) and exit S(5).

[0252] Test 10 The characteristics of Test 10 are as follows: - Chamber with 5 inlets and 7 outlets -h=1mm -V=3,000V -Flow rate (μl / min): Sample / buffer / electrode: 20 / 3000 / 50

[0253] Separation test 10 was set up in a separate device having an electrophoresis chamber with five inlets and seven outlets, and a height h of 1 mm.

[0254] The arrangement of the solutions at the inlet was the same as in Test 9, but the separation method in Test 10 differed in that the flow rates of the sample, buffer, and electrode were 20 / 3,000 / 50 μL / min, respectively, and the applied electric field voltage was set to 3,000 V.

[0255] Figure 17 is a photograph of the device during electrophoresis. Figure 17 shows the electrophoresis of charged species in a device containing an electrophoresis chamber with a height of 1 mm.

[0256] In this experiment 10, by increasing the electric field voltage and flow rate compared to experiment 9, the electrophoretic transfer of fluorescein and rhodamine 6G is greater than in experiment 9, so that fluorescein moves to outlet S(3) and the main flow of rhodamine 6G moves to outlet S(5). This made it possible to change the experimental conditions to optimize the separation of the products.

[0257] Test 11 The main features of Test 11 are as follows: - Chamber with 5 inlets and 7 outlets -h=2mm -V=2000V -Flow rate (μl / min): Sample / buffer / electrode: 20 / 3000 / 50

[0258] The same device used in Experiment 9 was employed, but the height of the electrophoresis chamber was 2 mm.

[0259] The arrangement of the solutions at the inlet was the same as in Test 11, but the separation method in this test differed in that the flow rates of the sample, buffer, and electrode were 20 / 3000 / 50 μL / min, respectively, and the applied electric field voltage was set to 3,000 V, the same conditions as in Test 11.

[0260] Figure 18 is a photograph of the device during electrophoresis. Figure 18 shows the separation of the product flow and confirms the electrophoresis of charged species in a device including an electrophoresis chamber with a height of 2 mm.

[0261] Example 4: Separation of ATP / MPA mixture

[0262] A separation test was conducted on a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP). The structures of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) are shown below.

[0263] [ka]

[0264] Assays 12-14 were performed using a KPLE-100-008 electrophoresis chip with five inlets and seven outlets, and an electrophoresis chamber thickness of 100 μm. Table 6 below shows the composition of the liquid electrode and separation buffer used, and the samples to be separated.

[0265] [Table 6]

[0266] Table 7 below shows the types of solutions introduced into the device inlet and the respective flow rates applied.

[0267] [Table 7]

[0268] The flow rates of the liquid electrodes at inlets E(1) and E(5) were 25 μL / min, the sample flow rate at inlet E(3) was 10 μL / min, and the buffer flow rates at inlets E(2) and E(4) were 100 μL / min.

[0269] During the test, the electrophoresis chip operated continuously throughout the entire experiment after the system stabilized.

[0270] Three different tests, 12, 13, and 14, were conducted. In these tests, the flow rate at the inlet remained constant throughout. Only the applied electric field value was increased from 0V to 3000V.

[0271] The tests were conducted at 0V (Test 12), i.e., no electric field was applied, 1000V (Test 13), and 2000V (Test 14). The products recovered at exits S(1) to S(7) were analyzed by HPLC during the experiment. Figure 19 shows the HPLC spectra of outlet S(1) to S(7) at 0V for test 12, Figure 20 shows the spectra at 13-1000V, and Figure 21 shows the spectra at 14-2000V.

[0272] result At 0V, the entire sample is visible at S(4), which corresponds to the central exit of the tip, i.e., S(4) facing the sample inlet E(3). Therefore, the system is stable. At 1000V, the entire sample was still visible in S(4), but this voltage was not sufficient to cause one of the compounds to migrate. Therefore, the electric field strength was increased. At 2000V, partial migration from ATP (first peak) to S(3) is observed, but AMP does not show migration to a different exit. Approximately 40% of ATP is biased towards exit S(3).

[0273] Therefore, the isolation of ATP / AMP biomolecules was confirmed by these tests, demonstrating proof of concept.

[0274] To increase the electrophoretic migration rate, the electric field within the chip can be increased. However, the separation can be optimized by adjusting parameters such as the composition of the separation buffer (pH, concentration, viscosity), the optimal electric field, the sample inlet position, and the sample preparation.

[0275] Example 5: Purification of protein / linker

[0276] In these 15 tests, a KPLE-100-009 tip was connected to a flow meter. Liquid electrodes were provided at the inlets E(1) and E(5) of the electrophoresis chamber. The liquid anode and liquid cathode had the same composition. The liquid electrode solution consisted of 10 mM HEPES, 0.2% (m / v) HPMC, 0.1% (m / v) Tween20, 40% methanol, and 1.5 M KCl, and was introduced at a flow rate of 20 μL / min. An electric field was generated inside the liquid electrode container using a carbon electrode. These liquid electrodes had a pH of 7.5 (adjusted with NaOH solution) and a conductivity of 86.66 μS / cm.

[0277] The central inlet E(3) was supplied with the sample to be purified, i.e., the reaction mixture obtained from the reaction of the protein and linker. During purification, the sample flow rate at E(3) was set to 10 μL / min.

[0278] The last two inlets, E(2) and E(4), were supplied with a buffer consisting of 10 mM HEPES in water, 0.2% (m / v) HPMC, and 0.1% (m / v) Tween20 at a flow rate of 80 μL / min. The pH of the separation buffer was 7.5 (adjusted with NaOH solution).

[0279] The applied electric field was 1500V. After stabilizing the flow, the product at the outlet of the electrophoresis tip was collected in a tube and analyzed by HPLC to measure the proportion of each compound at each outlet.

[0280] The hydrodynamic monitoring performed showed that no elements of the system migrated to the liquid electrode. Only the outlets of the electrophoresis chamber, i.e., outlets S(2) to S(4), were analyzed. Figure 22 shows the electrophoresis of chemical species obtained by HPLC analysis. Figures 23 and 24 show the HPLC spectra of the products at outlets S(2), S(3), and S(4), respectively.

[0281] The analysis revealed that 80% of the linkers were biased towards a different exit point than the majority of the protein, with only 10.43% of the protein biased towards the same exit point as the linkers.

Claims

1. A free-flow electrophoresis microcell device comprising vertically continuous plates X, Y, and Z, wherein the surface of the plates is YZY (XYZY) ｐ Stacked according to sequence, X represents an electrophoresis plate (1) made of an inert substance, Y is 99% α-Al ２ O ３ Sapphire or alumina Al ２ O ３ This represents the waterproof plate (2) that is manufactured by Z represents the cooling plate (3) including the heat transfer system (4), p is an integer from 1 to 100, representing the number of stages in the device and the number of plates X. Each section (5) is: - Defined by the plate YZYXYZY in the following sequence, - Plate X is located between the two plates Y, - The two plates Z are each adjacent to plate Y, - Each of the two plates Y located at the ends of the YZYXYZY sequence covers plate Z, thereby positioning each plate Z between the two plates Y. The device also includes means for fastening all plates that enable the waterproofing of the device, The aforementioned device has each plate X, It comprises i electrophoresis chambers (Fi), where i is an integer from 1 to 100, particularly 50, preferably 10. Each electrophoresis chamber (6) is ○ Including the recessed part, It has the shape of a rectangular prism with four sides (a, b, c, d) and two top and bottom faces (e, f). The recess has a length Loe and a width Lae. The height h, depending on the thickness of the electrophoresis plate X, is 25 μm to 20 mm, particularly 50 to 200 μm, or 1.0 mm to 5.0 mm. The sides (a, b) are parallel to each other, face (a) is enclosed by two sides (A1, A2) of dimension Lae, and face (b) is enclosed by two sides (B1, B2) of dimension Lae. The sides (c, d) are parallel to each other, face (c) is enclosed by two sides (C1, C2) of dimension Loe, and face (d) is enclosed by two sides (D1, D2) of dimension Loe. ○ Includes n consecutive entrances E(1), E(2) to E(n-1), En, where n is an integer from 4 to 9, preferably 5 or 6, distributed on the plane (a) between A1 and A2, and aligned in a direction parallel to A1 and A2. ○ Includes m consecutive exits S(1), S(2) to S(m-1), S(m), where m is an integer from 4 to 12, preferably 5 or 7, distributed on the plane (b) between B1 and B2, aligned in a direction parallel to B1 and B2, so that in a direction parallel to C1 and D1, S(1) faces E(1) and S(m) faces E(n). The plate Y ensures the watertightness of the electrophoresis chamber, The chamber (Fi) is arranged such that the sides C1 of each recess are parallel to each other. The device described above, - A supply channel is configured to connect the inlet E(1) of each electrophoresis chamber to the micro / millimeter flow supply circuit of the liquid cathode, - A supply channel is configured to connect the inlet E(n) of each electrophoresis chamber to a micro / millimeter flow supply circuit that supplies liquid cathodes, - A supply channel is configured to connect at least one of the inlets E(2) to E(n-1) of each electrophoresis chamber to a micro / millimeter flow supply circuit for an initial solution containing the product to be purified and / or separated, - The remaining inlets of each electrophoresis chamber are configured to be connected to a micro / millimeter flow supply circuit of at least one buffer, - Includes a recovery channel configured to connect each outlet S(1) to S(m) of each electrophoresis chamber to a micro / millimeter flow recovery circuit, The device is parallel to A1 and perpendicular to C1, and is operating in the presence of an electric field generated between the liquid cathode and the liquid anode. - The liquid cathode is circulated from the inlet E(1) to the outlet S(1), - The liquid anode is circulated from the inlet E(n) to the outlet S(m), - In the electrophoresis chamber (Fi), a solution containing the separated and / or purified products and at least one buffer solution are circulated between the liquid cathode and the liquid anode, from inlet E(2) to E(n-1) to outlet S(2) to S(m-1). - A device for recovering the purified and / or separated products contained in the initial solution at one of the outlets S(2) to S(m-1) of each electrophoresis chamber in the recovery circuit.

2. - The outlet S(1) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for collecting the liquid cathode. - The outlet S(m) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for collecting the liquid anode. - One of the outlets S(2) to S(m-1) of each electrophoresis chamber is connected to a micro / millimeter flow circuit for recovering the purified and / or separated products contained in the initial solution. - The device according to claim 1, comprising a recovery channel configured to connect the other remaining outlets of each electrophoresis chamber to at least one micro / millimeter flow recovery circuit of at least one buffer.

3. The device according to claim 1 or 2, comprising a single electrophoresis plate containing a single electrophoresis chamber in which p is equal to 1, i is equal to 1, and in particular the height h is 25 to 200 μm or 1.0 to 5.0 mm.

4. The device according to claim 1 or 2, comprising a single electrophoresis plate X having 2 to 10, preferably 10, electrophoresis chambers, where p is equal to 1 and i is between 2 and 10.

5. The device according to claim 1 or 2, wherein p is 2 to 10, i is 2 to 10, and comprises 2 to 10 electrophoresis plates X, each electrophoresis plate X comprising 2 to 10 electrophoresis chambers, in particular p is equal to 10 and i is equal to 10.

6. The height h of the electrophoresis chamber is 650 μm to 20 mm, particularly 650 μm to 10.0 mm, preferably 650 μm to 5.0 mm, and more preferably 650 μm to 2.0 mm. Alternatively, the device according to any one of claims 1 to 5, wherein the height h of the electrophoresis chamber is 25 μm to 200 μm or 1.0 to 5.0 mm.

7. The i electrophoresis chambers of each plate X are adjacent to each other by the surfaces (c) or (d) of each recess, and / or, the width of the recess of the electrophoresis chamber is 1.0 to 8.0 cm, preferably 1.0 to 5.0 cm. The device according to any one of claims 1 to 6, wherein the length Loe of the recess of the electrophoresis chamber is 5.0 to 20.0 cm, preferably 5.0 to 15.0 cm.

8. The device according to any one of claims 1 to 7, wherein the electrophoresis plate X is made of a substance selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), and fluoroethylene propylene (FEP), particularly Teflon®, Teflon®-PFA, and Teflon®-FEP plates.

9. The device according to any one of claims 1 to 8, wherein in each electrophoresis plate X, a portion of the plate is configured to provide a flow path for a supply channel and a recovery channel, and the plate X is partially or entirely etched, cut, or drilled.

10. Plates X, Y, and Z are rectangular parallelepipeds with widths La and Lo, where La and Lo are in the range of 2.0 to 50.0 cm. Furthermore, plate Y has a thickness of 0.5 mm to 5.0 mm. The device according to any one of claims 1 to 9, wherein the cooling plate Z has a thickness of 1.0 to 10.0 mm, particularly 1.0 to 5.0 mm.

11. The device according to any one of claims 1 to 10, wherein the electrophoresis chamber preferably includes channeling means etched into the electrophoresis plate X and communicating with an inlet and / or outlet.

12. Each electrophoresis chamber is configured to include at least one selectively permeable, preferably size-selective, membrane positioned so that a solution containing the product to be separated or purified during device operation passes through it. and / or, each upper and / or lower surface of the electrophoresis chamber includes protrusions configured to not obstruct the circulation of the product being purified and / or separated within the chamber during the operation of the device, and to improve heat transfer and maintain the electrophoresis chamber at a selected temperature, In particular, the protrusions are made of a thermally conductive material, preferably 99% α-Al ２ O ３ The device according to any one of claims 1 to 11, comprising sapphire or alumina.

13. The heat transfer system (4) is a channeling network configured to allow the circulation of one or more heat transfer fluids, the channeling network being in direct contact with a portion of plate Y adjacent to plate Z, and the portion being thermally connected to the electrophoresis chamber to allow temperature control within the electrophoresis chamber. The channeling network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers. Preferably, the channeling network of the heat transfer system comprises channels parallel to side C1 for each of the electrophoresis chambers, and the channels can contain heat transfer fluids of various temperatures to generate the temperature gradient, according to any one of claims 1 to 12.

14. The device according to any one of claims 1 to 13, wherein the fastening means for ensuring the airtightness of the device consists of two external fastening plates surrounding the device, and the means is particularly removable for each plate.

15. A method for purifying and / or separating products contained in a solution by free-flow electrophoresis, by implementing an electrophoretic microcell device according to any one of claims 1 to 14, - Connecting the supply channel of the device to a supply circuit for a liquid cathode, a liquid anode, an initial solution containing the product to be purified and / or separated, and at least one buffer, wherein the channel and circuit are controlled by a central processing device (UC1), - The step of connecting the cooling system to a cooling circuit controlled by a central processing device (UC2), - A step of generating an electric field along edge A1 via a liquid anode and a liquid cathode, - The central processing device (UC1) generates fluid circulation in each of the electrophoresis chambers, thereby, The liquid cathode is circulated from the inlet E(1) to the outlet S(1). The liquid anode is circulated from the inlet E(n) to the outlet S(m). The steps include: circulating an initial solution containing the product to be purified and / or separated between the liquid cathode and liquid anode in an electrophoresis chamber, and at least one buffer solution, from inlets E(2) to E(n-1) to outlets S(2) and S(m-1); A method comprising the steps of selecting and recovering the separated or purified product from at least one of the outlets S(2) to S(m-1) of the electrophoresis chamber of the device.

16. The above method is carried out under a continuous flow of an initial solution containing the product to be purified and / or separated. In particular, at a flow rate of 1 to 5 L / hour, the device includes 100 electrophoresis chambers. The method according to claim 15, preferably operating for 300 days / year.

17. The method according to claim 15 or 16, wherein the liquid cathode and liquid anode are electrolytes of the same composition.

18. The fluid flow of the liquid cathode in each electrophoresis chamber is controlled by a central processing device (UC1) and is performed at a flow rate of 10 to 10,000 μL / min. and / or, the fluid flow of the liquid anode in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 10,000 μL / min. And / or, the fluid circulation of the solutions to be purified and / or separated in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 30,000 μL / min. The method according to any one of claims 15 to 17, wherein the fluid circulation of the buffer in each of the electrophoresis chambers is controlled by a central processing device (UC1) and carried out at a flow rate of 10 to 50,000 μL / min.

19. The device includes at least 100 electrophoresis chambers arranged in parallel. and / or, the device comprises at least one size-selective membrane and is configured to separate products to be purified and / or separated from the initial solution during fluid circulation in each of the electrophoresis chambers, and / or, during fluid circulation in each electrophoresis chamber, use at least two buffers of different pH values ​​to create a pH variation along side A1 within each electrophoresis chamber. and / or, the method according to any one of claims 15 to 17, wherein a temperature gradient along the side A1 of the recess is applied in each electrophoresis chamber during fluid circulation in each of the electrophoresis chambers.

20. During fluid circulation in each electrophoresis chamber, a uniform temperature, particularly 10-40°C, is applied in each electrophoresis chamber. The method according to any one of claims 15 to 19, wherein the electric field generated during fluid circulation in each of the electrophoresis chambers is 200 to 4,000 V.

21. The method according to any one of claims 15 to 20, used for the purification and / or separation of proteins, or for the purification and / or separation of isomers, particularly optical isomers.

22. A method for developing an industrial device for purifying and / or separating a solution containing a product to be purified and / or separated by electrophoresis, a) A first step of mounting the device according to claim 3, comprising a single electrophoresis chamber, wherein the height h of the recess is 25 to 200 μm or 1.0 to 2.0 mm in order to identify and optimize the fluid circulation of the product to be purified and / or separated, b) A method comprising a second step of mounting the industrial device according to claim 4 or 5, which comprises 10 to 100 electrophoresis chambers.

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