Free-flow electrophoresis microcell device and its uses

Abstract

The invention relates to a free-flow electrophoresis microcell device comprising a succession of electrophoresis plates X, sealing plates Y, and cooling plates Z containing a heat transfer system, said plates being stacked in tiers comprising electrophoresis chambers, each electrophoresis chamber comprising a hollowed-out portion of height h corresponding to the thickness of the electrophoresis plate X, from 25 µm to 200 mm, with 4 to 9 successive aligned inlets and 4 to 12 successive aligned outlets, such that the inlets and outlets face each other, and supply and return channels connected to each of the inlets and outlets. (No figure)

Description

Title of the invention: Free-flow electrophoresis microcell device and its uses

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

[0002] High-performance liquid chromatography (HPLC) and zone electrophoresis using a support are techniques for the analytical and / or preparative separation of molecules, in particular biomolecules, present in a mixture. However, the need for a stationary phase for chromatography used in industrial processes for separation and / or purification leads to significant costs, and the need for support for zone electrophoresis limits its use on an industrial scale as a preparative method in terms of cost and quantity.

[0003] There is therefore a need to develop an adaptable, robust device without the need for restrictive maintenance, operating in flow, designed for easy and modular operation to implement, in order to allow purification and / or separation of the expected products at volumes and quantities on an industrial scale.

[0004] One of the aims of the invention is to provide a device for purifying and / or separating molecules by free-flow electrophoresis which can operate in continuous flow and on an industrial scale.

[0005] Another aim of the invention is a method for purifying and / or separating molecules, in particular biomolecules, adaptable to industrial scale.

[0006] Another object of the invention is a method of setting up such a device on an industrial scale.

[0007] A first object of the present invention is a free-flow electrophoresis microcell device comprising a vertical succession of plates X, Y, Z whose surfaces are stacked according to the sequence YZY(XYZY)p,

[0008] in which

[0009] X represents an electrophoresis plate (1) made of inert material,

[0010] Y represents a sealed plate (2) made of inert, electrically insulating material and thermal conductor, made of sapphire or alumina A12O3 with 99% a-Al2O3,

[0011] Z represents a cooling plate (3) comprising a heat transfer system (4),

[0012] p integer from 1 to 100, represents both the number of stages of said device and the number of plates X

[0013] each stage (5) being defined:

[0014] - by the following sequence of plates YZYXYZY, in which:

[0015] - plate X is located between two plates Y,

[0016] - each of the two Z plates being respectively adjacent to a Y plate,

[0017] - and each of the two Y plates located at the ends of the sequence YZYXYZY, respectively covers a Z plate so that each Z plate is located between two Y plates,

[0018] said device further comprising means for clamping all the plates allowing the sealing of said device,

[0019] said device being such that each X plate comprises:

[0020] a number i of electrophoresis chamber(s) (Fi), i being an integer from 1 to 100, in particular 50, preferably 10,

[0021] each electrophoresis chamber (6) comprising: • a hollowed-out part • in the shape of a rectangular parallelepiped with 4 side faces (a, b, c, d) and 2 upper and lower faces (e, f), • said hollowed out part being of length Loe and width Lae, • of height h corresponding to the thickness of the X-ray electrophoresis plate, from 25 pm to 20 mm, in particular from 50 pm to 200 pm or from 1.0 mm to 5.0 mm, • the lateral faces (a, b) being parallel to each other, the face (a) being delimited by two edges (Al, A2) of dimension Lae and the face (b) being delimited by two edges (Bl, B2) of dimension Lae, • the lateral faces (c, d) being parallel to each other, the face (c) being delimited by two edges (Cl, C2) of dimension Loe and the face (d) being delimited by two edges (Dl, D2) of dimension Loe, • n successive entries E(l), E(2) to E(nl), En, n being an integer from 4 to 9, preferably 5 or 6, distributed on the face (a) between Al and A2 and aligned in a direction parallel to Al and A2, • m successive outputs from S(l), S(2) to S(ml), S(m), m being an integer from 4 to 12, preferably 5 or 7, distributed on the face (b) between Bl and B2 and aligned in a direction parallel to Bl and B2, so that S(l) faces E(l) and S(m) faces E(n) in a direction parallel to Cl and Dl,

[0022] said Y plates ensuring the sealing of the electrophoresis chamber,

[0023] said chambers (Fi) being arranged so that the edges Cl of each hollowed-out part are parallel to each other,

[0024] said device comprising: - supply channels configured to connect the E( 1 ) inlets of each electrophoresis chamber to a micro / millifluidic circuit supply of a liquid cathode, - supply channels configured to connect the inputs E(n) of each electrophoresis chamber to a micro / millifluidic circuit for supplying a liquid anode, - supply channels configured to connect at least one of the inlets E(2) to E(nl) of each electrophoresis chamber to a micro / millifluidic circuit for supplying an initial solution containing a product to be purified and / or separated, - supply channels configured to connect the other remaining inlets of each electrophoresis chamber to micro / millifluidic circuits for supplying at least one buffer solution, - recovery channels configured to connect each of the outputs S(l) to S(m) of each electrophoresis chamber to micro / millifluidic recovery circuits,

[0025] said device being configured for, in the presence of an electric field generated between the liquid cathode and the liquid anode parallel to Al and perpendicular to Cl, and in operation - circulate the liquid cathode from the inlet E(l) to the outlet S(l), - circulate the liquid anode from the inlet E(n) to the outlet S(m), - circulate in the electrophoresis chamber (Fi), between the liquid cathode and the liquid anode, from the inlets E(2) to E(nl) to the outlets S(2) to S(ml), the solution containing the product to be separated and / or purified and at least one buffer solution, - recover at one of the outlets S(2) to S(ml) of each electrophoresis chamber in a recovery circuit the purified and / or separated product contained in said initial solution.

[0026] The term "free-flow electrophoresis" means electrophoresis not using a stationary phase, i.e. without the use of a solid phase serving as a support for the migration of species during electrophoresis.

[0027] By "plate" is meant a rigid element, generally in the form of a rectangular parallelepiped, in which at least two faces are parallel to each other and the latter mainly represent the total area of ​​this element, namely Making said two faces parallel to each other represents more than half of this total area. These two faces are called "surfaces" or "lower or upper surfaces" of the plate.

[0028] The distance between these two surfaces of the plate defines the thickness of the plate.

[0029] By "vertical succession of plates" is meant a stack of plates, in which the surfaces of the different plates are in contact.

[0030] By "electrophoresis plate" is meant the plate, named X, containing the i electrophoresis chambers (Fi). The plate X is made of a material inert with respect to electrophoresis.

[0031] By "electrophoresis chamber" is meant a part of the X electrophoresis plate comprising:

[0032] - a hollowed-out part in the X-ray electrophoresis plate, delimited by walls lateral, and

[0033] - the inlets and outlets located in the side walls of said hollowed-out part.

[0034] The electrophoresis chamber is the part of the X-ray plate in which the electrophoresis takes place free-flow electrophoresis.

[0035] The "electrophoresis cell" is a constituent unit of the device. The electrophoresis cell comprises an electrophoresis chamber, the walls of the Y plates closing the electrophoresis chamber and the supply and recovery channels connected to the inlets and outlets of said electrophoresis chamber. The electrophoresis cell comprises the recessed portion closed by the side walls in the X plate and those of the Y plates, the inlets and outlets of the recessed portion and the supply and recovery channels.

[0036] A device consisting of an electrophoresis cell is called an “electrophoresis chip”.

[0037] By "cooling plate" is meant the plate, called Z, containing a heat transfer system. The heat transfer system has the function of transporting a heat transfer fluid in order to allow temperature control. The heat transfer system is for example made up of a network of channels formed by recesses in the plate Z.

[0038] By "watertight plate", named Y, we mean a plate intended to ensure the fluidic sealing of the device.

[0039] In fact, each X plate is located between two Y plates, which ensure the sealing of the electrophoresis chambers of the X plate.

[0040] Similarly, each Z plate is located between two Y plates, which ensure the sealing of the heat transfer system of the Z plate.

[0041] The Y plate is made of inert, electrically insulating and thermally conductive material in order to ensure the chemical and electrical inertia of the device necessary for the free-flow electrophoresis process and in order to ensure thermal conductivity between the heat transfer systems and the electrophoresis chambers to allow control of the temperature of the device.

[0042] The plate Y is made of sapphire or alumina A12O3 containing 99% a-Al2O3.

[0043] By “sapphire” is meant a material consisting of corundum or an alumina A12O3 comprising 99% by weight of the a-Al2O3 phase.

[0044] Advantageously, the Y plate has a Mohs hardness of 9 (Coridon), a thermal conductivity from 30 W / m / K to 50 W / m / K. The Y plate has high mechanical resistance.

[0045] As a non-limiting example, the sapphire Y plate is supplied by Saint-Gobain (Luxium Solutions).

[0046] The sapphire plate has the advantage of being transparent.

[0047] The device consists of a succession of stacked plates clamped by clamping means, in which the surfaces of the adjacent plates are in direct contact. The adjacent plates are contiguous plates. Each plate in the device has at most two adjacent plates, i.e. two neighboring plates, arranged on either side of said plate.

[0048] A stage of the present device consists of a succession of plates stacked according to the following sequence of plates YZYXYZY. The number of plates X defines the number of stages of the device. Each stage comprises a plate X surrounded by two cooling plates Z, each of the plates X and Z being adjacent to two plates Y in order to ensure the sealing of the heat transfer systems of the plates Z and of the i electrophoresis chambers of the plate X and the thermal conductivity between the plate X and the cooling plates. The plates X and Z are separated by a plate Y so that the fluid of the heat transfer system of the plates Z and the fluids circulating in the electrophoresis chamber of the plate X are separated and do not communicate fluidically, the exchanges taking place being solely of a thermal nature.

[0049] In a device consisting of several stages, the YZY sequence plates located between two X plates are common to two successive stages.

[0050] In a two-stage device of sequence YZYXYZYXYZY, the central sequence YZY is common to both stages.

[0051] In a three-stage device of sequence YZYXYZYXYZYXYZY, the two central sequences YZY are common to two successive stages, respectively the first and second stages and the second and third stages.

[0052] The device advantageously comprises from 1 to 100 electrophoresis chambers per electrophoresis plate distributed in the stages. In the plate X of each stage, the chambers are advantageously organized in rows.

[0053] The range of "1 to 100" includes whole numbers in the following ranges: 1 to 10; 10 to 20; 20 to 30; 30 to 40; 40 to 50; 50 to 60; 60 to 70; 70 to 80; 80 to 90; 90 to 100, in particular the following 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.

[0054] The electrophoresis chamber in which the free-flow electrophoresis takes place comprises a hollowed-out portion of a plate X, closed by the side walls in the plate X, said hollowed-out portion being in the general shape of a rectangular parallelepiped.

[0055] By "in the shape of a rectangular parallelepiped" is meant that this hollowed-out part generally fits into a rectangular parallelepiped of length Loe, width Lae and height h corresponding to the thickness of the plate X ([Fig.2]). No account is taken in defining the general shape of the hollowed-out part of any means possibly present in the electrophoresis chamber such as the channeling means present at the inlets and outlets and the protuberances.

[0056] The rectangular parallelepiped comprises four lateral faces (a, b, c, d) and two faces (e, f), respectively lower and upper ([Fig.2]).

[0057] The lateral faces (a, b) are parallel to each other and of the same dimensions.

[0058] The face (a) is delimited by two edges (Al, A2) of dimension Lae, spaced h apart.

[0059] The face (b) is delimited by two edges (Bl, B2) of dimension Lae, spaced h apart.

[0060] The lateral faces (c, d) are parallel to each other and of the same dimensions.

[0061] The face (c) is delimited by two edges (Cl, C2) of dimension Loe, spaced h apart.

[0062] The face (d) is delimited by two edges (Dl, D2) of dimension Loe, spaced h apart.

[0063] The upper (e) and lower (f) faces are parallel to each other and of the same dimensions. They constitute the surfaces framing the hollowed-out part, dug into the plate X.

[0064] The face (e) is delimited by the edges (A1, B1, C1, D1) forming a rectangle of width Lae and length Loe.

[0065] The face (f) is delimited by the edges (A2, B2, C2, D2) forming a rectangle of width Lae and length Loe.

[0066] The height h corresponds to the thickness of the plate X, and to the height of the electrophoresis chamber and the hollowed-out part of the electrophoresis chamber.

[0067] The height h is from 100 pm to 20 mm.

[0068] The range of 25 pm to 20 mm includes the following ranges: 25 to 50 pm; 50 to 75 pm; 75 to 100 pm; 100 to 200 pm; 200 to 300 pm; 300 to 400 pm; 400 to 500 pm; 500 to 600 pm; 600 to 700 pm; 700 to 800 pm; 800 to 900 pm; 900 pm to 1.0 mm; 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; 9.0 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; 19.0 to 20.0 mm.

[0069] Advantageously the height h is from 25 to 200 pm.

[0070] Advantageously the height h is from 1.0 to 5.0 mm.

[0071] By "inlet" is meant a passage allowing the circulation of the flow of a liquid fluid, from the outside to the inside of a system containing a closed hollow part, configured to contain said fluid, for example such as the electrophoresis chamber or the heat transfer system.

[0072] By "outlet" we mean a passage allowing the circulation of the flow of a fluid liquid, from the inside to the outside of a system containing a closed hollow part, configured to contain said fluid, for example such as the electrophoresis chamber or the heat transfer system.

[0073] On face (a), the n inlets in the hollowed-out part of the electrophoresis chamber are distributed successively, respectively referenced E(l) to E(n), i.e. E(l), E(2) to E(nl), E(n). The number of inlets n varies from 4 to 9, i.e. 4, 5, 6, 7, 8 and 9, preferably 5 or 6.

[0074] The inputs E(l) to E(n) are located between the edges Al and A2 and aligned in a direction parallel to Al and A2, advantageously at a substantially equal distance from each other.

[0075] By "aligned" is meant that the inlets, respectively the outlets, are close to the same straight line. The inlets can therefore be positioned slightly in front of or behind the face (a) and / or slightly above or below one another. The positioning of the inlets is configured to introduce the different flows into the electrophoresis chamber so as to allow their circulation in the chamber parallel to the edge CL

[0076] On face (b), the m outputs are distributed successively, respectively referenced S(l) to S(m), i.e. S(l), S(2) to S(ml), S(m). The number of outputs m varies from 4 to 12, i.e. 4, 5, 6, 7, 8, 9, 10, 11 and 12, preferably 5 or 7.

[0077] The inputs S(l) to S(m) are located between the edges B1 and B2 and aligned in a direction parallel to B1 and B2, advantageously at a substantially equal distance from each other.

[0078] The inputs E(l) and E(n) are located near the two ends of the edges Al and A2, that is to say as close as possible to the faces (c) or (d).

[0079] The outputs S(l) and S(m) are located near the two ends of the edges B1 and B2, that is to say as close as possible to the faces (c) or (d).

[0080] The inlet E(l) is configured for the introduction of a liquid cathode. The outlet S(l) is configured for the evacuation of the liquid cathode after circulation in the electrophoresis chamber.

[0081] The input E(n) is configured for the introduction of a liquid anode. The output S(m) is configured for the evacuation of the liquid anode after circulation in the electrophoresis chamber.

[0082] The inlets and outlets of a chamber are configured so that E(l) faces E(n) and S(l) faces S(m) so as to induce the electric field in the electrophoresis chamber between the liquid anode and the liquid cathode during the circulation of the fluids in order to allow the implementation of the electrophoresis.

[0083] The liquid anode and the liquid cathode are electrolyte solutions, i.e. solutions comprising ions.

[0084] By “liquid cathode” is meant an electrolyte solution configured to act as a cathode during electrophoresis.

[0085] By "liquid anode" is meant an electrolyte solution configured to act as an anode during electrophoresis.

[0086] The presence of the liquid cathode and the liquid anode makes it possible to generate an electric field in the electrophoresis chamber when the electrolytic solutions are charged.

[0087] The liquid cathode and the liquid anode are electrolytic solutions, capable of generating an electric field, which can be increased or decreased by the action of the generator which charges the liquid anode and the liquid cathode and / or with an increase in the concentration of the ions.

[0088] One of the inputs E(2) to E(nl) is configured for the introduction into the electrophoresis chamber of the solution to be purified and / or separated.

[0089] One of the outputs S(2) to S(ml) is configured for the recovery of the purified and / or separated solution after the electrophoresis process carried out in the electrophoresis chamber.

[0090] At least one of the inlets E(2) to E(nl), distinct from the inlet of the solution to be purified and / or separated, is configured for the introduction of a buffer solution into the electrophoresis chamber.

[0091] The inlets and outlets of the electrophoresis chamber are connected by channels to supply or recovery circuits, configured for micro or millifluidic circulation of the fluid flows.

[0092] The term “microfluidic circuit” means a set of channels with a cross-section of dimensions of the order of a micrometer.

[0093] The term “millifluidic circuit” means a set of channels with a cross-section of dimensions of the order of a millimeter.

[0094] The inputs E( 1 ) to E(n) are connected by supply channels to supply circuits.

[0095] The outputs S(l) to S(m) are connected by recovery channels to recovery circuits.

[0096] The device of the present invention comprises channels connecting:

[0097] - the inputs E(l) of each electrophoresis chamber (Fi) to a micro / milli circuit fluidic supply of a liquid cathode,

[0098] - the inputs E(n) of each electrophoresis chamber (Fi) to a micro / milli circuit fluidic supply of a liquid anode,

[0099] - at least one of the inputs E(2) to E(nl) of each electrophoresis chamber (Fi) to a micro / millifluidic circuit for supplying an initial solution containing a product to be purified and / or separated,

[0100] - the other remaining inputs, from each electrophoresis chamber to micro / circuits millifluidics supplying at least one buffer solution,

[0101] - the outputs S(l) of each electrophoresis chamber (Fi) to a micro / milli circuit fluidic recovery of the liquid cathode, after circulation in the electrophoresis chamber,

[0102] - the outputs S(m) of each electrophoresis chamber (Fi) to a micro / milli circuit fluidic recovery of the liquid anode, after circulation in the electrophoresis chamber,

[0103] - at least one of the outputs S(2) to S(ml) of each electrophoresis chamber (Fi) to a micro / millifluidic circuit for recovering the purified and / or separated product contained in said initial solution to be purified and / or separated;

[0104] - the other outputs to at least one micro / millifluidic recovery circuit.

[0105] Thus, the device of the present invention is configured to, in the presence of a electric field applied parallel to Al and perpendicular to Cl, and in operation for each electrophoresis chamber (Fi) - circulate the liquid cathode from the inlet E(l) to the outlet S(l), along the face (c) - circulate the liquid anode from the inlet E(n) to the outlet S(m), along the face (d) - circulate in the electrophoresis chamber (Fi), between the liquid cathode and the liquid anode, from the inlets E(2) to E(nl) to the outlets S(2) to S(ml), the solution containing the product to be separated and / or purified and at least one buffer solution, - recover at one of the outlets S(2) to S(ml) of each electrophoresis chamber in a recovery circuit the purified and / or separated product contained in said initial solution.

[0106] The inventors have surprisingly found that the introduction of a cooling circuit with the use of sapphire or alumina A12O3 plates containing 99% a-Al2O3 as a means of separation between the heat transfer system and the electrophoresis chambers induces excellent temperature control and its homogeneity in such a way that it allows: - a thickness h of the electrophoresis chambers constituting the device which can vary from micrometer to millimeter and thus increase the processing capacity of the solutions to be purified or separated, - purification and / or separation of heat-sensitive molecules, - an introduction of a temperature gradient in the i chambers electrophoresis.

[0107] In addition, the use of the sapphire Y plate makes it possible to have a device robust and waterproof which allows disassembly and reassembly, facilitating the washing of the device parts and its maintenance. The modularity of the device allows reuse of the Y and Z plates.

[0108] According to a particular embodiment, the number m of outlets is equal to or greater than the number of inlets n in order to induce a finer separation of the product to be separated or purified.

[0109] Advantageously, the number m of outputs is greater than the number n of inputs. The multiplication of outputs refines the recovery possibilities.

[0110] According to a particular embodiment, n is equal to 5 and m is equal to 5.

[0111] According to a particular embodiment, n is equal to 5 and m is equal to 7.

[0112] According to a particular embodiment, the present invention relates to a device as defined above, including recovery channels configured to connect: - the output S(l) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the liquid cathode, - the output S(m) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the liquid anode, - one of the outputs S(2) to S(ml) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the purified and / or separated product contained in said initial solution, - the other remaining outputs of each electrophoresis chamber to at least one micro / millifluidic circuit for recovering at least one buffer solution.

[0113] The recovery channels of the device are configured to recover, after circulation in the electrophoresis chambers, the liquid cathode, the liquid anode, the buffer solution(s) and the purified and / or separated product contained in said initial solution. These recoveries take place in four separate recovery circuits, so that, in the electrophoresis chambers of the device, the liquid anode and the liquid cathode are not in contact to maintain the electric field before or during the recovery of the purified and / or separated product at the outlet. These recovery channels are also configured to separately recover the purified and / or separated product and the at least one buffer solution used.

[0114] By "separation" is meant the separation from one another of at least two products present in the initial solution which can be recovered separately, said two products each being able to be recovered at different outlets of the device of the present invention.

[0115] By “purification” is meant the separation of a product from the other species present in the initial solution.

[0116] According to a particular embodiment, the present invention relates to a device as defined above, wherein p is 1 and i is 1, comprising a single electrophoresis plate having a single electrophoresis chamber, in particular of height h of 25 to 200 pm or 1.0 to 5.0 mm.

[0117] This device of the invention in which p is equal to 1 and i is equal to 1, consists of the following sequence of plates YZYXYZY, comprising a single electrophoresis chamber, advantageously with a height h of 25 to 200 pm or 1.0 to 5.0 mm.

[0118] It constitutes a laboratory tool. It advantageously makes it possible to determine the influence of different parameters such as the nature of the buffer, the electric field, the temperature, the number and distribution of the inlets and outlets of the electrophoresis chamber, in order to optimize the conditions of purification and separation of a solution to be purified or separated.

[0119] According to a particular embodiment, the present invention relates to a device as defined above, in which p is equal to 1 and i varies from 2 to 10, comprising a single electrophoresis plate X comprising from 2 to 10 electrophoresis chambers, preferably 10 electrophoresis chambers.

[0120] This device according to the invention in which p is equal to 1 and i varies from 2 to 10, consists of the following sequence of plates YZYXYZY, namely a single stage comprising at least two electrophoresis chambers, preferably 10 electrophoresis chambers.

[0121] According to a particular embodiment, the present invention relates to a device as defined above, in which p varies from 2 to 10 and i varies from 2 to 10, comprising from 2 to 10 X electrophoresis plates and each X electrophoresis plate comprising from 2 to 10 electrophoresis chambers, in particular p is equal to 10 and i is equal to 10.

[0122] Advantageously, the height of the electrophoresis chambers is from 1.0 to 20 mm, in particular from 1.0 to 5.0 mm, preferably from 1.0 to 2.0 mm.

[0123] This device according to the invention is an industrial free-flow electrophoresis device consisting of several stages and several electrophoresis chambers per stage which makes it possible to continuously separate and / or purify a solution which can reach from 1 to 5 liters per hour of solution to be purified or separated, namely allowing use on an industrial scale.

[0124] According to a particular embodiment, the present invention relates to a device as defined above, in which the height h of the electrophoresis chamber is from 650 pm to 20 mm, in particular from 650 pm to 10.0 mm, preferably from 650 to 5.0 mm, preferentially from 650 pm to 2.0 mm.

[0125] The range of 650 pm to 2.0 mm includes the following ranges: 650 to 700 pm; 700 to 750 pm; 750 to 800 pm; 800 to 850 pm; 850 to 900 pm; 900 to 950 pm; 950 pm 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; from 1.8 to 1.9 mm; from 1.9 to 2.0 mm.

[0126] The range of 650 pm to 5.0 mm includes the following ranges: 650 to 2.0 mm;

[0127] 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;

[0128] from 3.0 to 3.1 mm; from 3.1 to 3.2 mm; from 3.2 to 3.3 mm; from 3.3 to 3.4 mm; from 3.4 to 3.5 mm; from 3.5 to 3.6 mm; from 3.6 to 3.7 mm; from 3.7 to 3.8 mm; from 3.8 to 3.9 mm; from 3.9 to 4.0 mm;

[0129] from 4.0 to 4.1 mm; from 4.1 to 4.2 mm; from 4.2 to 4.3 mm; from 4.3 to 4.4 mm; from 4.4 to 4.5 mm; from 4.5 to 4.6 mm; from 4.6 to 4.7 mm; from 4.7 to 4.8 mm; from 4.8 to 4.9 mm; from 4.9 to 5.0 mm.

[0130] The range from 650 pm to 10.0 mm includes the following ranges: from 650 to 5.0 mm;

[0131] from 5.0 to 5.5 mm; from 5.5 to 6.0 mm; from 6.0 to 6.5 mm; from 6.5 to 7.0 mm; from 7.0 to 7.5 mm; from 7.5 to 8.0 mm; from 8.0 to 8.5 mm; from 8.5 to 9.0 mm; from 9.0 to 9.5 mm; from 9.5 to 10.0 mm.

[0132] The range of 650 pm to 20.0 mm includes the following ranges: 650 to 10.0 mm;

[0133] from 10.0 to 11.0 mm; from 11.0 to 12.0 mm; from 12.0 to 13.0 mm; from 13.0 to 14.0 mm; from 14.0 to 15.0 mm; from 15.0 to 16.0 mm; from 16.0 to 17.0 mm; from 17.0 to 18.0 mm; from 18.0 to 19.0 mm; from 19.0 to 20.0 mm.

[0134] According to a particular embodiment, the present invention relates to a device as defined above, in which the height h of the electrophoresis chamber is from 25 pm to 200 pm or from 1.0 to 5.0 mm.

[0135] The range of “25 pm to 200 pm” includes the following ranges: from 25 to 50 pm; from 50 to 75 pm; from 75 to 100 pm; from 100 to 125 pm; from 125 to 150 pm; from 150 to 175 pm; from 175 to 200 pm.

[0136] The range of “1.0 to 5.0 mm” includes the following ranges:

[0137] from 1.0 to 1.1 mm; from 1.1 to 1.2 mm; from 1.2 to 1.3 mm; from 1.3 to 1.4 mm; from 1.4 to 1.5 mm; from 1.5 to 1.6 mm; from 1.6 to 1.7 mm; from 1.7 to 1.8 mm; from 1.8 to 1.9 mm; from 1.9 to 2.0 mm;

[0138] from 2.0 to 2.1 mm; from 2.1 to 2.2 mm; from 2.2 to 2.3 mm; from 2.3 to 2.4 mm; from 2.4 to 2.5 mm; from 2.5 to 2.6 mm; from 2.6 to 2.7 mm; from 2.7 to 2.8 mm; from 2.8 to 2.9 mm; from 2.9 to 3.0 mm;

[0139] from 3.0 to 3.1 mm; from 3.1 to 3.2 mm; from 3.2 to 3.3 mm; from 3.3 to 3.4 mm; from 3.4 to 3.5 mm; from 3.5 to 3.6 mm; from 3.6 to 3.7 mm; from 3.7 to 3.8 mm; from 3.8 to 3.9 mm; from 3.9 to 4.0 mm;

[0140] from 4.0 to 4.1 mm; from 4.1 to 4.2 mm; from 4.2 to 4.3 mm; from 4.3 to 4.4 mm; from 4.4 to 4.5 mm; 4.5 to 4.6 mm; 4.6 to 4.7 mm; 4.7 to 4.8 mm; 4.8 to 4.9 mm; 4.9 to 5.0 mm.

[0141] The use of a device comprising electrophoresis chambers, in particular a device comprising a single electrophoresis chamber, having a height of 25 pm to 200 pm or of 1.0 to 5.0 mm, is advantageous for carrying out preliminary tests making it possible to determine the influence of the various parameters and to optimize the characteristics of the device.

[0142] The height h of the electrophoresis chamber is constant throughout the system. “A height of 25 pm to 200 pm” means a height of constant value, said value being chosen between 25 pm and 200 pm. Similarly, “A height of 1.0 to 5.0 mm” means a constant height chosen between 1.0 and 5.0 mm.

[0143] According to a particular embodiment, the present invention relates to a device as defined above, in which the height h of the electrophoresis chamber is from 1.0 mm to 5.0 mm, in particular from 1.0 to 2.0 mm.

[0144] The use of a device having a height of the order of millimeters is advantageous for achieving industrial quantities for the purification of products. For example, a device with 10 to 30 chambers with heights of 2 mm makes it possible to obtain 100 to 300 kg / year of purified product.

[0145] According to a particular embodiment, the present invention relates to a device as defined above, in which the height h of the electrophoresis chamber is from 650 pm to 20 mm, in particular from 650 pm to 10.0 mm, preferably from 650 to 5.0 mm, preferentially from 650 pm to 2.0 mm,

[0146] or wherein the height h of the electrophoresis chamber is 25 pm to 200 pm or 1.0 to 5.0 mm.

[0147] According to a particular embodiment, the present invention relates to a device as defined above, in which the i electrophoresis chambers of each plate X are adjacent to each other by the faces (c) or (d) of each hollowed-out part.

[0148] Two adjacent electrophoresis chambers along faces (c) and (d) therefore have a common wall between the two chambers.

[0149] The chambers, adjacent along faces (c) and (d), in the same X electrophoresis plate constitute a row of electrophoresis chambers. The row configuration of the chambers optimizes the use of the X plates in terms of surface area and facilitates their manufacture and machining. It also makes it possible to optimize the arrangement of the distribution channels and the cooling systems, for example by pooling them.

[0150] According to a particular embodiment, the present invention relates to a device as defined above, in which the inputs E( 1 ) for two adjacent chambers are supplied by the same distribution channel.

[0151] According to a particular embodiment, the present invention relates to a device as defined above, in which the inlets of two adjacent chambers are symmetrical. They are symmetrical with respect to the wall separating them, namely face (c) or (d).

[0152] According to a particular embodiment, the present invention relates to a device as defined above, in which the ratio between the length Loe and the width Lae of the hollowed-out part is from 2 to 15

[0153] The ratio between the length Loe and the width Lae of the hollowed-out part is chosen to allow the migration of the product to be purified. Advantageously, it is chosen independently of the electrophoretic mobility of the product to be purified or separated so that the device can be used for several types of products.

[0154] According to a particular embodiment, the present invention relates to a device as defined above, in which the width Lae of the hollowed-out part of the electrophoresis chamber is from 1.0 to 8.0 cm, preferably from 1.0 to 5.0 cm.

[0155] The range of "1.0 to 8.0 cm" includes the ranges: 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; 7.0 to 8.0 cm.

[0156] According to a particular embodiment, the present invention relates to a device as defined above, in which the length Loe of the hollowed-out part of the electrophoresis chamber is from 5.0 to 20.0 cm, preferably from 5.0 to 15.0 cm.

[0157] The range of "5.0 to 20.0 cm" includes the 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; 19.0 to 20.0 cm.

[0158] According to a particular embodiment, the present invention relates to a device as defined above, in which the X-ray electrophoresis plates are made of a material chosen from polytetrafluoroethylene (PTFE), perfloroalkoxy (PFA) and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP plates.

[0159] According to a particular embodiment, the present invention relates to a device as defined above, in which in each X-ray electrophoresis plate a part of said plate is configured to leave room for a fluid circuit of supply and recovery channels, which is partially or totally etched, cut or pierced in the X-ray plate.

[0160] According to a particular embodiment, the present invention relates to a device as defined above, in which the i electrophoresis chambers of each plate X are adjacent to each other by the faces (c) or (d) of each hollowed-out part,

[0161] and / or in which the width Lae of the hollowed-out part of the electrophoresis chamber is from 1.0 to 8.0 cm, preferably from 1.0 to 5.0 cm,

[0162] and / or wherein the length Loe of the recessed portion of the electrophoresis chamber is 5.0 to 20.0 cm, preferably 5.0 to 15.0 cm,

[0163] and / or wherein the X-ray electrophoresis plates are made of a material chosen from polytetrafluoroethylene (PTFE), perfloroalkoxy (PFA) and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP plates,

[0164] and / or wherein in each X-ray electrophoresis plate a portion of said plate is configured to leave space for a fluid circuit of supply and recovery channels, which is partially or totally etched, cut or pierced in the X-ray plate.

[0165] According to a particular embodiment, the present invention relates to a device as defined above, in which the plates X, Y, Z have a dimension La x Lo, in which La and Lo vary from 2.0 to 50.0 cm.

[0166] The range of "2.0 to 50.0 cm" includes the ranges: from 2.0 to 5.0 cm; from 5.0 to 10.0 cm; from 10.0 to 15.0 cm; from 15.0 to 20.0 cm; from 20.0 to 25.0 cm; from 25.0 to 30.0 cm; from 30.0 to 35.0 cm; from 35.0 to 40.0 cm; from 40.0 to 45.0 cm; from 45.0 to 50.0 cm.

[0167] According to a particular embodiment, the present invention relates to a device as defined above, in which the plates X, Y, Z have a dimension La x Lo, in which La is from 6.0 to 30 cm and Lo is from 2.0 to 50.0 cm.

[0168] The range of “6.0 to 30.0 cm” includes the ranges: from 6.0 to 10.0 cm; from 10.0 to 15.0 cm; from 15.0 to 20.0 cm; from 20.0 to 25.0 cm; from 25.0 to 30.0 cm.

[0169] In the case of a device comprising a single electrophoresis chamber on a plate X, the width “La” of the plate X is slightly greater than the width “Lae” of the recessed portion of the electrophoresis chamber and the length “Lo” of the plate X is slightly greater than the length “Loe” of the recessed portion of the electrophoresis chamber.

[0170] In the case of a device comprising 10 identical electrophoresis chambers adjacent in a row on a plate X, the width “La” of the plate X is substantially greater than the length “Loe” of the hollowed-out portion of an electrophoresis chamber and the length “Lo” of the plate X is substantially greater than 10 times the width “Lae” of the hollowed-out portion of an electrophoresis chamber.

[0171] A slightly higher or significantly higher value is, for example, a value 1 to 20 mm higher. Advantageously, to increase the sealing of a row of 10 electrophoresis cells, the width of the edges of the X electrophoresis plate is increased by framing said row.

[0172] According to a particular embodiment, the present invention relates to a device as defined above, in which the plates Y have a thickness of 0.5 mm to 5.0 mm.

[0173] The range of "0.5 to 5.0 mm" includes the following ranges: 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; 4.5 to 5.0 mm.

[0174] According to a particular embodiment, the present invention relates to a device as defined above, in which the Z plates are made of a material chosen from plexiglass, PTFE or polyamines.

[0175] According to a particular embodiment, the present invention relates to a device as defined above, in which the cooling plates Z have a thickness of 1.0 to 10.0 mm, in particular of 1.0 to 5.0 mm.

[0176] The range of "1.0 to 10.0 mm" includes the following ranges: 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; 9.0 to 10.0 mm.

[0177] The range of “1.0 to 5.0 mm” includes the following ranges: 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; 4.5 to 5.0 mm.

[0178] According to a particular embodiment, the present invention relates to a device as defined above, in which the plates X, Y, Z have a dimension La x Lo, in which La and Lo vary from 2.0 to 50.0 cm,

[0179] and / or wherein the Y plates have a thickness of 0.5 mm to 5.0 mm,

[0180] and / or in which in which the cooling plates Z have a thickness of 1.0 to 10.0 mm, in particular 1.0 to 5.0 mm.

[0181] According to a particular embodiment, the present invention relates to a device as defined above, in which the electrophoresis chambers comprise channeling means opening onto the inlets and / or onto the outlets, preferably etched in the X-ray electrophoresis plate.

[0182] These channeling means are configured to allow the orientation of the flows in the electrophoresis chamber at each of the inlets and outlets, in order to better distribute the flows in the case of the inlets over the entire width of the hollowed-out part and to better concentrate the flows in the case of the outlets.

[0183] Advantageously, these channeling means are an integral part of the electrophoresis chamber, i.e. fused with the walls of the electrophoresis chamber and are made of the same material as the electrophoresis plate. These channeling means are located along the faces (a) and (b) of the rectangular parallelepiped in which the hollowed-out part is inscribed.

[0184] According to a particular embodiment, the present invention relates to a device as defined above, in which each electrophoresis chamber is configured to each contain at least one membrane of selective permeability, preferably selective in size, positioned so as to be crossed by the solution containing the product to be separated or purified during operation of the device.

[0185] According to a particular embodiment, the present invention relates to a device as defined above, in which each electrophoresis chamber comprises at least one size-selective permeability membrane,

[0186] positioned parallel to face (c) and adjacent to an inlet of a buffer solution,

[0187] so as to be crossed by the initial solution containing the product to be separated or purified during operation of the device and

[0188] so that the part of the initial solution which has not passed through said membrane is conveyed towards one of the outlets by said buffer solution coming from said inlet adjacent to said membrane.

[0189] The presence of these membranes also allows size selectivity of the product to be purified and / or separated.

[0190] According to a particular embodiment, the present invention relates to a device as defined above, in which the upper face and / or the lower face of each of the electrophoresis chambers comprises protrusions configured so as not to disturb, during operation of the device, the circulation in the chamber of the product to be purified and / or separated, and configured to improve heat transfer and to maintain the electrophoresis chambers at a selected temperature.

[0191] The protrusions extend from the faces (e) and / or (f) of the recessed portion towards the inside of said recessed portion. They may be located only on one of the faces or on both faces.

[0192] According to a particular embodiment, the present invention relates to a device as defined above, in which said protuberances are made of thermally conductive materials, preferably sapphire or 99% a-Al2O3 alumina, preferably in the same material as that of the Y plate, in order to ensure thermal conductivity in the chambers and to control the temperature in the cell.

[0193] The presence of these protuberances makes it possible to promote thermal exchanges between the cooling plates along the path and thus to optimize the separation of the desired molecule while avoiding denaturation of the molecules, while maintaining the optimal temperature.

[0194] According to a particular embodiment, the present invention relates to a device as defined above, in which said protuberances have a shape configured so as not to disturb, during operation of the device, the circulation in the chamber of the product to be purified and / or separated.

[0195] The shape and arrangement of the protrusions can be calculated or simulated in order to to avoid a disturbance of the streamlines of the laminar flows around the protuberance and in the electrophoresis chamber, said shape and said arrangement being however effective for heat transfer.

[0196] According to a particular embodiment, the present invention relates to a device as defined above, in which said protuberances are triangular-shaped spikes, rectangular-based pillars or pads or ovoid shapes.

[0197] According to a particular embodiment, the present invention relates to a device as defined above, in which the X and / or Y plates are etched to be able to accommodate said protuberances.

[0198] According to a particular embodiment, the present invention relates to a device as defined above, in which each electrophoresis chamber is configured to each contain at least one membrane of selective permeability, preferably selective in size, positioned so as to be crossed by the solution containing the product to be separated or purified during operation of the device,

[0199] and / or wherein the upper face and / or the lower face of each of the electrophoresis chambers comprises protrusions configured so as not to disturb, during operation of the device, the circulation in the chamber of the product to be purified and / or separated, and configured to improve heat transfer and to maintain the electrophoresis chambers at a selected temperature,

[0200] in particular in which said protuberances are made of thermally conductive materials, preferably sapphire or 99% a-Al2O3 alumina.

[0201] According to a particular embodiment, the present invention relates to a device as defined above, in which the heat transfer system in the Z plates is a pipe network configured to allow the circulation of one or more heat transfer fluids, said network being placed in direct contact with a part of the Y plates adjacent to Z, said part being thermally connected to the electrophoresis chambers, in order to allow the temperature in said electrophoresis chambers to be controlled.

[0202] According to a particular embodiment, the present invention relates to a device as defined above, in which the pipe network of the heat transfer system is formed by recesses in the plate Z.

[0203] According to a particular embodiment, the present invention relates to a device as defined above, in which said pipe network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers.

[0204] According to a particular embodiment, the present invention relates to a device as defined above, in which said pipe network of the heat transfer system comprises, for each of the electrophoresis chambers, channels parallel to the edge Cl, said channels being able to contain heat transfer fluids of different temperatures in order to generate said temperature gradient.

[0205] According to a particular embodiment, the present invention relates to a device as defined above, in which the inlet of the heat transfer system is located on the side of the face (a) of the electrophoresis chamber.

[0206] According to a particular embodiment, the present invention relates to a device as defined above, in which said pipe network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers,

[0207] in particular, said heat transfer system is a pipe network configured to allow the circulation of one or more heat transfer fluids, said network being placed in direct contact with a part of the plates Y adjacent to Z, the above-mentioned part being thermally connected to the electrophoresis chambers, in order to allow the temperature in said electrophoresis chambers to be controlled,

[0208] preferably in which said pipe network of the heat transfer system comprises, for each of the electrophoresis chambers, channels parallel to the edge C1, said channels being able to contain heat transfer fluids of different temperatures in order to generate said temperature gradient.

[0209] According to a particular embodiment, the present invention relates to a device as defined above, in which said pipeline network is configured to obtain a selected and controlled temperature in the electrophoresis chambers.

[0210] According to a particular embodiment, the present invention relates to a device as defined above, in which the respective inlet and outlet flow of the pipe network of the heat transfer system is perpendicular to the edge C1 of each electrophoresis chamber.

[0211] According to a particular embodiment, the present invention relates to a device as defined above, in which the inlet of the heat transfer system is located on the side of the face (c) of the hollowed-out part of the electrophoresis chamber.

[0212] According to a particular embodiment, the present invention relates to a device as defined above, in which the clamping means for ensuring the sealing of said device comprise two external clamping plates enclosing said device, said means being removable, in particular plate by plate.

[0213] Another object of the present invention relates to the use of a device of the invention as defined above, in a preparative electrophoresis method for the purification and / or separation of a molecule, in particular a protein, under continuous flow.

[0214] The device of the invention advantageously allows continuous use for the purification and / or separation of a product.

[0215] According to a particular embodiment, the present invention relates to the use as defined above, in which from 1 to 5 L / hour of solution to be purified or separated are treated, in particular in which said device comprises 100 electrophoresis chambers, preferably comprising 10 stages and 10 electrophoresis chambers per stage.

[0216] According to a particular embodiment, the present invention relates to the use as defined above, in which the productivity of the purified or separated product is 100 to 300 kg / year of purified or separated product, in particular in which said device comprises 10 to 30 electrophoresis chambers.

[0217] According to a particular embodiment, the present invention relates to the use as defined above, said device comprising 100 electrophoresis chambers in operation for 300 days / year.

[0218] Another object of the present invention relates to a method of purification and / or separation by free-flow electrophoresis of a product contained in a solution comprising the following steps:

[0219] - connecting the supply channels of a device according to the invention such as defined above to the supply circuits of the liquid cathode, the liquid anode, an initial solution containing a product to be purified and / or separated, and at least one buffer solution, said channels and circuits being controlled by a central unit (UC1)

[0220] - connect the cooling systems to a cooling circuit, controlled by a central unit (UC2)

[0221] - generate an electric field along the Al edges, via the anode liquid and liquid cathode,

[0222] - generate fluid circulation in each of the electrophoresis chambers, by the central unit (UC1), so as to: • circulate a liquid cathode from the inlet E(l) to the outlet S(l), • circulate a liquid anode from the inlet E(n) to the outlet E(m), • circulate in the electrophoresis chamber, between the liquid cathode and the liquid anode, the initial solution containing the product to be purified and / or separated and said at least one buffer solution, from the inlets E(2) to E(nl) to the outlets S(2) and S(ml), - select and recover in at least one of the outlets S(2) to S(ml), of each of the electrophoresis chambers of said device, the separated or purified product.

[0223] The control of the circulation of fluids by the central unit (UC1) is carried out for example using flow meters and pumps present in the supply and recovery circuits. For example, each inlet of the chamber electrophoresis can be connected to a flow meter.

[0224] The electric field along the edge Al in the electrophoresis chamber can be generated by the circulation of the liquid cathode from E(l) to S(l) and by the circulation of the liquid anode from E(n) to S(m).

[0225] Advantageously, the same central unit can control the fluid circulation in the electrophoresis chambers and the circulation of the heat transfer fluid in the cooling circuit.

[0226] According to a particular embodiment, the present invention relates to the process as defined above, implemented under continuous flow of the initial solution containing the product to be purified and / or separated,

[0227] in particular at a flow rate of 1 to 5 L / hour, in particular said device comprising 100 electrophoresis chambers,

[0228] preferably in operation 300 days / year.

[0229] According to a particular embodiment, the present invention relates to the method as defined above, in which the liquid cathode and the liquid anode are electrolytic solutions of the same composition.

[0230] By "liquid electrode" we mean a liquid cathode or anode. Let us specify that an electrode is an electronic or ionic conductor capturing or releasing electrons.

[0231] In this embodiment, the cathode and the anode are called liquid electrodes. These liquid electrodes are electrolytic solutions having a high ionic conductivity. For example, the liquid electrode which is composed of 40% Methanol, 10 mM HEPES, 0.2% HPMC, 0.1% Tween 20, 1.5M KCl pH=7.5, has an ionic conductivity of 250 mS / cm.

[0232] The liquid electrode may comprise species in the form of ions such as in saline solutions. Advantageously, the liquid electrode comprises chloride or fluoride salts.

[0233] In one embodiment, the liquid electrode comprises 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES), or citrate, or 2-(N-morpholino) ethanesulfonic acid (MES), or acetate.

[0234] In one embodiment, the liquid electrode further comprises hydroxypropyl methyl cellulose (HPMC), Tween 20 (Polyoxyethylene (20) sorbitan monolaurate), methanol, ethanol and / or KC1.

[0235] Advantageously, the liquid electrode has the following composition: HEPES at 10mM, HPMC at 0.2% (m / v), Tween 20 at 0.1% (m / v), methanol at 40% and KCL from 0.5 to 1.5M and water.

[0236] Advantageously the pH of the liquid electrode is adjusted with a NaOH solution.

[0237] Advantageously the ionic conductivity of the liquid electrode is from 0.01 to 250mS / cm.

[0238] Advantageously, the liquid electrode is previously charged before introduction into the chambers of the device, in particular using a carbon electrode introduced into the electrolytic solution, for example composed of 40% methanol, 10mM HEPES, 0.2% HPMC, 0.1% Tween 20, 1.5M KCl pH=7.5.

[0239] The use of a liquid cathode and anode implies the non-use of metal in the device and makes it possible to avoid the electrolysis of water and therefore the formation of bubbles in the electrophoresis chamber(s).

[0240] According to a particular embodiment, the present invention relates to the process as defined above, in which the buffer solution has a pH suitable for the purification and / or separation of the product to be purified and / or separated contained in the initial solution.

[0241] In general, the pH of the buffer solution is between 5.8 and 7.5 and depends on the molecule to be purified in its medium.

[0242] According to a particular embodiment, the present invention relates to the method as defined above, in which said fluidic circulation of the liquid cathode in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 10,000 pL / min.

[0243] According to a particular embodiment, the present invention relates to the method as defined above, in which said fluidic circulation of the liquid anode in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 10,000 pL / min.

[0244] According to a particular embodiment, the present invention relates to the method as defined above, in which said fluid circulation of the solution to be purified and / or separated in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 30,000 pL / min.

[0245] According to a particular embodiment, the present invention relates to the method as defined above, in which said fluidic circulation of the buffer solution in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 50,000 pL / min.

[0246] The range of “10 to 10,000 pL / min” includes the following ranges: 10 to 20 pL / min; 20 to 50 pL / min; 50 to 80 pL / min; 80 to 100 pL / min; 100 to 150 pL / min; 150 to 200 pL / min; 200 to 300 pL / min; 300 to 400 pL / min; 400 to 500 pL / min; 500 to 600 pL / min; 600 to 800 pL / min; 800 to 1,000 pL / min; 1,000 to 1,500 pL / min; from 1,500 to 2,000 pL / min; from 2,000 to 2,500 pL / min; from 2,500 to 3,000 pL / min; from 3,500 to 4,000 pL / min; from 4,000 to 4,500 pL / min; from 4,500 to 5,000 pL / min; from 5,000 to 5,500 pL / min; from 5,500 to 6,000 pL / min; from 6,000 to 6,500 pL / min; from 6,500 to 7,000 pL / min; from 7,000 to 7,500 pL / min; from 7,500 to 8,000 pL / min; from 8,000 to 8,500 pL / min; 8,500 to 9,000 pL / min; 9,000 to 9,500 pL / min; 9,500 pL / min to 10,000 pL / min.

[0247] The range of “10 to 20,000 pL / min” includes the following ranges: from 10 to 10,000 pL / min; from 10,000 to 11,000 pL / min; from 11,000 to 12,000 pL / min; from 12,000 to 13,000 pL / min; 13,000 to 14,000 pL / min; 14,000 to 15,000 pL / min; 15,000 to 16,000 pL / min; 16,000 to 17,000 pL / min; 17,000 to 18,000 pL / min; 18,000 to 19,000 pL / min; from 19,000 to 20,000 pL / min.

[0248] The range of “10 to 50,000 pL / min” includes the following ranges: from 10 to 20,000 pL / min; from 20,000 to 25,000 pL / min; from 25,000 to 30,000 pL / min; from 30,000 to 35,000 pL / min; 35,000 to 40,000 pL / min; 40,000 to 45,000 pL / min; 45,000 to 50,000 pL / min.

[0249] According to a particular embodiment, the present invention relates to the method as defined above, in which said fluidic circulation of the liquid cathode in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 10,000 pL / min,

[0250] and / or wherein said fluidic circulation of the liquid anode in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 10,000 pL / min,

[0251] and / or in which said fluid circulation of the solution to be purified and / or separated in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 30,000 pL / min,

[0252] and / or wherein said fluid circulation of the buffer solution in each of the electrophoresis chambers, controlled by the central unit (UC1), is implemented at a flow rate of 10 to 50,000 pL / min.

[0253] According to a particular embodiment, the present invention relates to the method as defined above, in which said device comprises at least 100 electrophoresis chambers positioned in parallel.

[0254] The term “electrophoresis cells positioned or placed in parallel” means electrophoresis cells in which the supply of fluid through the channels of the different inlets is carried out in parallel, that is to say simultaneously at the inlets with introduced fluids (liquid electrodes, buffer solution and the solution to be purified and / or separated) coming from the same sources. Cell 1 and cell 2 are supplied simultaneously at the inlet E(l) of each cell by a liquid electrode coming from the same container containing the liquid electrode, for example.

[0255] Thus the supply circuit of the solution to be purified and / or separated from the electrophoresis chambers is a parallel circuit.

[0256] According to a particular embodiment, the present invention relates to the method as defined above, in which during the fluid circulation in each of the electrophoresis chambers, a pH variation along the edge Al in each electrophoresis chamber is generated, using at least two buffers of different pH.

[0257] According to a particular embodiment, the present invention relates to the method as defined above, in which said device comprises in each electrophoresis chamber at least one size-selective membrane, configured to separate the product to be purified and / or separated from the initial solution during fluid circulation in each of the electrophoresis chambers.

[0258] According to a particular embodiment, the present invention relates to the method as defined above, in which each electrophoresis chamber comprises at least one size-selective membrane, positioned parallel to the edge C1 and adjacent to an inlet of a buffer solution,

[0259] in which during the fluid circulation in each of the electrophoresis chambers the initial solution containing the product to be separated and / or purified passes through said membrane, the part of the initial solution not having passed through said membrane being conveyed towards one of the outlets S(2) to S(ml) by said buffer solution coming from said inlet adjacent to said membrane.

[0260] According to a particular embodiment, the present invention relates to the method as defined above, in which during the fluid circulation in each of the electrophoresis chambers,

[0261] a temperature gradient along the edge Al of the recessed portion is applied in each electrophoresis chamber,

[0262] in particular through the cooling systems of said device comprising a heat transfer system consisting of a network of pipes configured to allow the circulation of one or more heat transfer fluids parallel to the face (c) of each of the electrophoresis chambers.

[0263] According to a particular embodiment, the present invention relates to the method as defined above, in which said device comprises at least 100 electrophoresis chambers positioned in parallel,

[0264] and / or wherein said device comprises in each electrophoresis chamber at least one size-selective membrane, configured to separate the product to be purified and / or separated from the initial solution during fluid circulation in each of the electrophoresis chambers,

[0265] and / or wherein during the fluid circulation in each of the electrophoresis chambers, a pH variation along the edge A1 in each electrophoresis chamber is generated, using at least two buffers of different pH,

[0266] and / or in which during the fluid circulation in each of the electrophoresis chambers, a temperature gradient along the edge Al of the hollowed-out portion is applied in each electrophoresis chamber.

[0267] According to a particular embodiment, the present invention relates to the method as defined above, in which during the fluid circulation in each of the electrophoresis chambers, a homogeneous temperature, in particular from 10°C to 40°C, is applied in each of the electrophoresis chambers.

[0268] The range of "10°C to 40°C" includes the following ranges: from 10 to 15°C; from 15 to 20°C; from 20 to 25°C; from 25 to 30°C; from 30 to 35°C; from 35 to 40°C, in particular the values ​​of 35°C, 36°C, 37°C, 38°C, 39°C and 40°C.

[0269] According to a particular embodiment, the present invention relates to the method as defined above, in which, during the fluid circulation in each of the electrophoresis chambers, the electric field generated is from 200V to 4,000V.

[0270] The range of “200 V to 4000 V” includes the following ranges: from 200 to 500 V; from 500 to 1000 V; from 1000 to 1500 V; from 1500 to 2000 V; from 2000 to 2500 V; from 2500 to 3000 V; from 3000 to 3500 V; from 3500 to 4000 V.

[0271] According to a particular embodiment, the present invention relates to the method as defined above, in which during the fluid circulation in each of the electrophoresis chambers, a homogeneous temperature, in particular from 10°C to 40°C, is applied in each of the electrophoresis chambers,

[0272] and / or wherein, during fluid circulation in each of the electrophoresis chambers, the electric field generated is from 200V to 4,000V.

[0273] According to a particular embodiment, the present invention relates to the method as defined above, implemented to purify and / or separate a protein.

[0274] According to a particular embodiment, the present invention relates to the method as defined above, implemented to purify and / or separate isomers, in particular enantiomers.

[0275] According to a particular embodiment, the present invention relates to the method as defined above, implemented to purify and / or separate a protein or implemented to purify and / or separate isomers, in particular enantiomers.

[0276] Another object of the present invention relates to a use of a device according to the invention as defined above, comprising a single electrophoresis chamber, in particular the height h of the hollowed-out part of which is from 25 to 200 μm or from 1.0 to 5.0 mm, to determine and optimize the fluid circulation of the product to be purified and / or separated in order to set up an industrial device according to the invention as defined above comprising from 10 to 100 electrophoresis chambers.

[0277] Another subject of the present invention relates to a method for developing an industrial device for purifying and / or separating a solution comprising the product to be purified and / or separated by electrophoresis comprising the following steps: a. a first study step implementing a device according to the invention as defined above comprising a single electrophoresis chamber, in particular the height h of the hollowed-out part of which is 25 to 200 pm or 1.0 to 5.0 mm, to determine and optimize the fluid circulation of the product to be purified and / or separated, b. a second stage of setting up said industrial device comprising 10 to 100 electrophoresis chambers.

[0278] Figures and Example

[0279] [Fig.l] represents an exploded view diagram of an electrophoresis microcell device comprising 30 electrophoresis cells distributed in rows of 10 cells on 3 levels, without representation of the clamping means of all the plates.

[0280] (1) represents an electrophoresis plate comprising a row of 10 chambers electrophoresis apparatus each comprising a hollowed-out portion (6) in the shape of a rectangular parallelepiped, each chamber comprising inlets or outlets (7) and supply or recovery channels (8), two adjacent hollowed-out portions are separated by the same wall. (2) represents a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchanges. (3) represents a cooling plate comprising a cooling system (4) which comprises a recess allowing the circulation of a heat transfer fluid from an inlet to an outlet. (5) represents a stage consisting of a succession of plates YZYXYZY. The device comprises 3 stages and consists of the following sequence YZYXYZYXYZYXYZY in which the two central sequences YZY are common to two successive stages, respectively the first and second stages and the second and third stages.

[0281] [Fig.2] represents the hollowed-out part (6) of a chamber of an electrophoresis plate (1). The hollowed-out part is inscribed in a rectangular parallelepiped of width Lae, length Loe and height h, delimited by the faces (a, b, c, d, e, f), the faces (a, b, c, d) form the side walls between the hollowed-out part and the plate X, the faces (a, b) being parallel to each other and the faces (c, d) being parallel to each other. The face (a) is delimited by the edges (Al, A2) of dimension Lae, the face (b) by the edges (Bl, B2) of dimension Lae. The face (c) is delimited by the edges (Cl, C2) of dimension Loe, the face (d) by the edges (Dl, D2) of dimension Loe. The edges (A1, B1, C1, D1) delimit the face (e) and the edges (A2, B2, C2, D2) delimit the face (f).

[0282] [Fig.3] represents an exploded view diagram of a single-stage device comprising a single electrophoresis chamber, without representation of the clamping means of all the plates.

[0283] (1) represents an electrophoresis plate comprising a single chamber comprising a hollow part (6) in the shape of a rectangular parallelepiped, comprising inlets or outlets (7) and supply or recovery channels (8). (2) re has a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchanges. (3) represents a cooling plate comprising a cooling system (4) which comprises a recess allowing the circulation of a heat transfer fluid from an inlet to an outlet, the inlet of the heat transfer fluid being on the same side as that of the inlets of the electrophoresis chamber. The device consists of a single stage (5) comprising a succession of YZYXYZY plates.

[0284] [Fig.4] partly represents a) a one-stage device comprising a row of 10 electrophoresis chambers and in part b) a two-stage device, each stage comprising a row of 10 electrophoresis chambers.

[0285] (1) represents an electrophoresis plate comprising a row of 10 chambers electrophoresis chamber each comprising a hollowed-out part (6) in the shape of a rectangular parallelepiped, each chamber comprising inlets or outlets (7) and supply or recovery channels (8), two adjacent hollowed-out parts are separated by the same wall. (2) represents a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchanges. (3) represents a cooling plate comprising a cooling system (4) which comprises a recess allowing the circulation of a heat transfer fluid from an inlet to an outlet. (5) represents a stage consisting of a succession of YZYXYZY plates.

[0286] The device in part a) comprises 1 stage and consists of the following sequence YZYXYZY.

[0287] The device in part b) comprises 2 stages and consists of the following sequence YZYXYZYXYZY in which the central sequence YZY is common to both stages.

[0288] [Fig.5] represents a diagram of a row of four electrophoresis chambers, in which the inlets of each electrophoresis chamber follow one another in an identical manner in part a) or the inlets of the adjacent chambers are symmetrical with respect to the wall separating them (part b). The inlets and outlets of the electrophoresis chambers comprise channeling means (9).

[0289] [Fig.6] shows in exploded view a device with a cooling system allowing the establishment of a temperature gradient, either along the width of the electrophoresis chamber (part a), or along the length of the chamber (part b). In the device the cooling plate comprises a heat transfer system comprising recesses forming channels (10) which are parallel to the flows of the electrophoresis chamber in part a) or which are perpendicular in part b).

[0290] [Fig.7] represents a device comprising clamping means. In this embodiment particular embodiment, the clamping means consist of two plates (11) which enclose the entire succession of plates X, Y and Z, using attachment means (12) connecting the two plates (11) whose distance can be adjusted. The attachment means (12) are for example screws.

[0291] [Fig-8] represents the diagram of the electrophoresis chamber of the electrophoresis chips used; part a) represents that of the KPLE-100-008 chip which has 5 inlets and 7 outlets, said outlets being numbered from top to bottom from 1 to 7, the central inlet is intended for the sample, the inlets E(2) and E(4) for the buffer solution (TS) and the inlets E(1) and E(5) for the liquid electrodes; part b) represents that of the KPLE-100-009 chip which has 5 inlets and 5 outlets, said outlets being numbered from top to bottom from 1 to 5, the central inlet is intended for the sample, the inlets E(2) and E(4) for the buffer solution (TS) and the inlets E(1) and E(5) for the liquid electrodes.

[0292] The hollowed-out portion of the electrophoresis chambers is of width Lae and length Loe. The electrophoresis plate X is of width La and length Lo. The electrophoresis chambers comprise channeling means (9) for the inlet and outlet flows. The channeling means are, for example, triangular-shaped elements (91) in the form of a beveled point (92) located between two inlets or two outlets.

[0293] [Fig.9] is a photograph taken of the KPLE-100-008 device comprising a 100 pm thick electrophoresis chamber, during the hydrodynamic test. The visualization of the different flows was made possible by coloring the sample flow and the electrode flows in yellow.

[0294] [Fig. 10] shows a series of photographs taken of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 1 mm thick, during hydrodynamic tests at flow rates (pL / min) sample / buffer solution / electrode respectively of 160 / 1600 / 100 for part a), 320 / 3200 / 800 for part b), 400 / 4000 / 1000 for part c) and 400 / 2000 / 500 for part d).

[0295] [Fig. 11] is a photograph taken of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, with a thickness h of 2 mm, during hydrodynamic tests at flow rates in pL / min of the sample / buffer solution / electrode solutions of 400 / 4000 / 1000 respectively.

[0296] [Fig. 12] is a photograph taken of the device comprising an electrophoresis chamber with 5 inlets and 5 outlets, 100 pm thick, during a separation of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 1500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 80 / 20.

[0297] [Fig. 13] represents the HPLC spectra of the products at the outputs S(2), S(3) and S(4) of a device comprising an electrophoresis chamber with 5 inlets and 5 outlets, 100 pm thick, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 1500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 80 / 20

[0298] [Fig. 14] is a photograph taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 100 pm thick, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 2500 V and at flow rates in pL / min of the sample / buffer solution / electrode solutions of 10 / 100 / 20 respectively.

[0299] [Fig. 15] represents the HPLC spectra of the products at the outputs S(2), S(3), S(4), S(5) and S(6) of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, with a thickness of 100 pm, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 2500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 100 / 20

[0300] [Fig. 16] represents photographs taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 1.0 mm thick, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 2000 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 600 / 20, part a) corresponds to a photograph taken without annotations, part b) represents the same photograph with annotations on the path of travel of the colored compounds.

[0301] [Fig. 17] represents photographs taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 1.0 mm thick, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 3000 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 20 / 3000 / 50, part a) corresponds to a photograph taken without annotations, part b) represents the same photograph with annotations on the path of the colored compounds.

[0302] [Fig. 18] represents photographs taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 2.0 mm thick, during a separation test of a mixture of three colored compounds (fluorescein, rhodamine B and rhodamine 6G), carried out at 3000 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 20 / 3000 / 50, part a) corresponds to a photograph taken without annotations, part b) represents the same photograph with annotations on the path of the colored compounds.

[0303] [Fig. 19] represents the HPLC spectra of the output products S(l) to S(7) of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 100 pm thick, during a separation test of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 0 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 100 / 25.

[0304] [Fig.20] represents the HPLC spectra of the products at the outputs S(l) to S(7) of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, with a thickness of 100 pm, during a test for the separation of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 1000 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 100 / 25.

[0305] [Fig.21] represents the HPLC spectra of the output products S(l) to S(7) of a device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 100 μm thick, during a test for the separation of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 2,000 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 100 / 25.

[0306] [Fig.22] represents a diagram of the migration of species from a device comprising an electrophoresis chamber with 5 inlets and 5 outlets, 100 μm thick, during a test for the separation of a mixture of protein and its linker, carried out at 1500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 80 / 20.

[0307] [Fig.23] represents the HPLC spectra of the products at the outlet of S(3) of a device comprising an electrophoresis chamber with 5 inlets and 5 outlets, with a thickness of 100 pm, during a test for the separation of a mixture of protein and its linker, carried out at 1500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 80 / 20.

[0308] [Fig.24] represents the HPLC spectra of the products at the outlet of S(4) of a device comprising an electrophoresis chamber with 5 inlets and 5 outlets, with a thickness of 100 pm, during a test for the separation of a mixture of protein and its linker, carried out at 1500 V and at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 10 / 80 / 20.

[0309] Examples

[0310] Example 1: Material and method

[0311] Electrophoresis device

[0312] Two devices, called electrophoresis chips, comprising an electrophoresis chamber having different characteristics of dimensions of the hollowed-out part (called separation chamber) and of the X plate and a distinct number of outlets, were used in the trials.

[0313] The dimensions of the two chips are reported in Table 1 below.

[0314] Three heights h were used: 100 pm, 1 mm and 2 mm.

[0315] The KPLE-100-009 chip has an electrophoresis chamber with 5 inputs and 5 outputs. The inputs and outputs are symmetrical. The inputs are numbered from top to bottom from 1 to 5, or E(1) to E(5) respectively.

[0316] The KPLE-100-008 chip has an electrophoresis chamber with 5 inputs and 7 outputs. Multiplying the outputs refines the recovery possibilities. These outputs are also numbered from top to bottom from 1 to 7, respectively S(1) to S(7), or S1 to S7.

[0317] Each inlet was connected to a flow meter controlling the flow introduced into the electrophoresis chip. Each flow meter was itself connected to an independent liquid supply container, namely either the sample, a buffer solution, or a liquid electrode. All parameters, including the flow rate of the inlets, the recovery of the products at the outlets and their analysis are controlled by computer and automatically recorded.

[0318] [Tables 1] Hollowed-out part of the electrophoresis chamber Length Loe x width Lae Electrophoresis plate (separation plate) Length Lo x width La KPLE-100-008 6.4 cm x 3.0 cm 10.2 cm x 5.2 cm KPLE-100-009 4.5 cm x 1.8 cm 7.5 cm x 5.0 cm

[0319] Table 1: Dimensions of the devices

[0320] Example 2: Study of the hydrodynamic flow in the device

[0321] Hydrodynamic flow studies were carried out in a free-flow electrophoresis device comprising an electrophoresis chamber with 5 inlets and 7 outlets and having a thickness h of 100 μm, 1 mm and 2 mm.

[0322] They aim to analyze the movement of the sample flow, the electrolyte flows (anode and cathode) and the buffer solution flows when the device is in operation. No electric field was applied during these tests.

[0323] Test 1: KPLE-100-008 chip with thickness h = 100 pm.

[0324] The KPLE-100-008 chip was connected to flow meters at each input according to the conditions indicated in the following Table 2.

[0325] [Tables2] Inputs Injected solution Composition Flow rate (pL / min) E(l) Liquid electrode (cathode) Water / methanol (60 / 40), 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20, 1.5M KCl pH 7.45 adjusted with NaOH Yellow dye 20 E(2) Buffer solution Water 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20 pH 7.45 80 E(3) Sample Yellow dye 10 E(4) Buffer solution Water 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20 pH 7.45 80 E(5) Liquid electrode (anode) Water / methanol (60 / 40), 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20, 1.5M KCL pH 7.45 adjusted with NaOH Yellow 20 dye

[0326] Table 2: Operating conditions of test 1.

[0327] [Fig.9] is a photograph taken of the KPLE-100-008 device comprising a 100 pm thick electrophoresis chamber, during the hydrodynamic test. The visualization of the different flows was made possible by coloring the sample flow and the electrode flows in yellow.

[0328] It was observed, without application of an electric field, a path of the sample flow from the inlet E(3) to the outlet S(4), outlet facing E(3).

[0329] A yellow colored area in the form of a band was observed from E(l) to S(l). Another yellow colored area in the form of a band was observed from E(1) to S(6) and S(7).

[0330]

[0331] These two bands represent the paths of the two flows of the liquid electrodes respectively. Tests 2 to 5: Chip with 5 inputs and 7 outputs with a thickness h = 1 mm. A chip comprising an electrophoresis chamber with 5 inputs, 7 outputs and a thickness h of 1 mm was connected to flow meters at each input according to the conditions indicated in the following table 3.

[0332] [Tables3] Inputs Injected solution Composition Test 2 Flow rate (pL / min) Test 3 Flow rate (pL / min ) Test 4 Flow rate (pL / min ) Test 5 Flow rate (pL / min) E(l) Liquid electrode (cathode) Water / methanol (60 / 40), 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20, 1.5M KCL pH 7.45 adjusted with NaOH Yellow dye 1,000 800 1,000 500 E(2) Buffer solution Water 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20 pH 7.45 Blue dye 1,600 3,200 4,000 2,000 E(3) Sample Water 160 320 400 400 E(4) Buffer solution Water HEPES at 10mM, HPMC at 0.2%(m / v), Tween 20 at 0.1%(m / v) pH at 7.45 blue dye 1600 3200 4000 2000 E(5) Liquid electrode Water / methanol (60 / 40), 1000 800 1000 500 (anode) 10 mM HEPES, 0.2% (w / v) HPMC, 0.1% (w / v) Tween 20, 1.5 M KCL pH 7.45 adjusted with NaOH Yellow dye

[0333] Table 3: Operating conditions of test 2

[0334] [Fig. 10] is a photograph taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 1 mm thick, during hydrodynamic tests at flow rates (pL / min) sample / buffer solution / electrode of 160 / 1600 / 100 for part a), 320 / 3200 / 800 for part b), 400 / 4000 / 1000 for part c) and 400 / 2000 / 500 for part d).

[0335] The visualization of the path of the different flows was made possible by coloring the buffer solution in blue and coloring the electrode flows in yellow.

[0336] It was highlighted, without application of an electric field, a path of the sample flow in a quasi-straight line from the inlet E(3) to the outlet S(4), outlet facing E(3), due to the blue coloration of the buffer solutions which contrasts with an absence of coloration of the sample solution. Indeed, the flow of the sample was bordered on both sides by two darker bands representing the paths of the buffer solutions injected from the inlet E(2) to the outlets S(2) to S(4) and from the inlet E(4) to the outlets S(5) to S(6).

[0337] A yellow colored area in the form of a band from E(l) to S(l) and another yellow colored area in the form of a band from E(l) to S(7) were observed. These two bands represent the paths of the two flows of the liquid electrodes respectively.

[0338] Test 6: 5-input and 7-output chip with a thickness h = 2 mm.

[0339] A chip comprising an electrophoresis chamber, comprising 5 inputs, 7 outputs, with a thickness h of 2 mm, was connected to flow meters at each inlet according to the conditions indicated in the following table 4.

[0340] [Tables4] Inputs Injected solution Composition Test 6 Flow rate (pL / min) E(l) Liquid electrode (cathode) Water / methanol (60 / 40), 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20, 1.5M KCL pH 7.45 adjusted with NaOH Yellow dye 1000 E(2) Buffer solution Water 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20 pH 7.45 Blue dye 4000 E(3) Sample Water 400 E(4) Buffer Water 10mM HEPES, 0.2%(w / v) HPMC, 0.1%(w / v) Tween 20 pH 7.45 Blue dye 4000 E(5) Liquid electrode (anode) Water / methanol (60 / 40), 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20, 1.5M KCL pH 7.45 adjusted with NaOH Yellow dye 1000

[0341] Table 4: Operating conditions of test 3

[0342] [Fig. 11] is a photograph taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, with a thickness h of 2 mm, during hydrodynamic tests at flow rates in pL / min of the solutions respectively of the sample / buffer solution / electrode of 400 / 4000 / 1000.

[0343] The visualization of the path of the different flows is made possible by the blue coloring of the buffer solution and the yellow coloring of the electrode flows.

[0344] A sample flow path from inlet E(3) to outlets S(4) and S(5) was demonstrated without applying an electric field. The sample flow path was made visible by the blue coloration of the buffer solutions.

[0345] A yellow colored area in the form of a band was observed from E(l) to the outlets S(l) and S(2), another yellow colored area in the form of a band was observed from E(5) to the outlets S(6) and S(7). These two bands represent the paths of the two flows of the liquid electrodes respectively.

[0346] In conclusion, hydrodynamic tests 1 to 6 confirm laminar flow of the solutions injected at different flow rates into the chamber, which allows the implementation of the free-flow electrophoresis process.

[0347] Example 3: Separation of a mixture of Fluorescein, Rhodamine B and Rhodamine 6G

[0348] Tests 7 to 11 in a free-flow electrophoresis device were set up for the separation of a mixture of three molecules: fluorescein, rhodamine B and rhodamine 6G, exhibiting fluorescence. The chemical structures of the three molecules are presented below.

[0349] [Chem.l] Rhodamine B Rhodamine 6G Fluorescein Mm = 479.01 g / mol (Purple) Mm = 479.01 g / mo (Red) Mm = 332.3 g / mo (Yellow) CzsHmCINzOs C2gHMCIN2O3 C;+ù-O.

[0350] These 3 compounds have a similar size less than a nanometer and significantly different charges.

[0351] The zeta potential of each compound was measured beforehand and reported in Table 5.

[0352] Fluorescein at pH 7.49 exhibited a zeta potential of -23.5 mV, rhodamine B -0.3 mV and rhodamine 6G +36.2 mV.

[0353] [Tables5] Compound Zeta Potential at pH 7.49 Fluorescein -23.5 mV Rhodamine B -0.3 mV Rhodamine 6G +36.2 mV

[0354] Table 5: Zeta potential measured at pH 7.49

[0355] Test 7 - Chamber with 5 inlets and 5 outlets -h = 100 jim - V=1500 V

[0356] The main characteristics of test 7 are as follows:

[0357] - Room with 5 entrances and 5 exits

[0358] - h = 100 pm,

[0359] - V = 1500 V

[0360] - Flow rate (pl / min): Sample / Buffer / Electrode: 10 / 80 / 20

[0361] During this test, the KPLE-100-009 chip, comprising 5 inputs and 5 outputs, with an electrophoresis chamber height h of 100 pm, was connected to flow meters at each input.

[0362] The inlets E(1) and E(5) were each supplied with an electrolytic solution for the anode and the cathode respectively. The two electrolytic solutions for the cathode and the anode respectively are of the same composition. The liquid electrolytic solution has the following composition: 10 mM HEPES, 0.2% (m / v) HPMC, 0.1% (m / v) Tween 20, 40% methanol and 1.5M KCL. The flow rate was set at 20 pL / min. These electrolytic solutions had a pH = 7.49 (which was adjusted by a NaOH solution) and a conductivity = 92.33 mS / cm. The electric field is generated by the electrolytic solutions in the electrophoresis chamber, originating from solutions containing the carbon electrodes, anode and cathode respectively.

[0363] The central inlet E(3) was supplied with the sample to be purified, namely a mixture of the 3 dyes: fluorescein, rhodamine B and rhodamine 6G. The sample to be purified was composed of 0.175 g / L of fluorescein, 0.176 g / L of Rhodamine 6G and 0.185 g / L of Rhodamine B. The flow rate set for the sample was 10 pL / min throughout the purification.

[0364] The last two inlets E(2) and E(4) were supplied with a buffer solution composed of 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20 in water, at a flow rate set at 80 pL / min. The buffer solution had a pH = 7.5 (adjusted by a NaOH solution) and a conductivity = 600 ps / cm.

[0365] The electric field, variable from 0 V to 3000 V, was fixed in these tests at 1500 V. After stabilization of the flows, the products leaving the electrophoresis chamber were recovered in tubes and analyzed by HPLC in order to de- complete the percentage of each compound at each exit.

[0366] [Fig. 12] is a photograph taken of the device during electrophoresis made possible by the use of transparent sapphire plates and colored separation products.

[0367] Hydrodynamic monitoring was carried out to show that there was no migration towards the liquid electrodes of any compound in the sample. Thus, only the outputs of the electrophoresis chamber S(2), S(3) and S(4) were analyzed by HPLC.

[0368] Results

[0369] [Fig. 12] shows an electrophoretic migration of fluorescein, in yellow, towards the liquid cathode, arriving at outlet S(2). Rhodamine B, with a low charge, is not influenced during electrophoresis and leaves at outlet S(3). Rhodamine 6G (PZ = +36.2 mV) migrates towards the liquid anode and leaves at outlet S(4).

[0370] The HPLC spectra of the products leaving the outlets S(2), S(3) and S(4) are shown in [Fig. 13],

[0371] The results of the HPLC analysis for this test carried out at 1500 V confirm the presence of fluorescein at the S(2) outlet, mainly that of rhodamine B at the S(3) outlet and that of rhodamine 6G at the S(4) outlet.

[0372] The calculated percentage of fluorescein migrated is estimated at 92.98% towards the S(2) exit, while Rhodamine 6G migrates at 62.75% towards the S(4) exit.

[0373] Test 8

[0374] The main characteristics of test 8 are as follows:

[0375] - Room with 5 entrances and 7 exits

[0376] - h = 100 pm,

[0377] - V = 2,500 V

[0378] - Flow rate (pl / min): Sample / Buffer / Electrode: 10 / 100 / 20

[0379] A separation test was set up with a second device which has 5 inputs and 7 outputs, the KPLE-100-008 chip, having an electrophoresis chamber height h of 100 pm.

[0380] The installation of the inlet solutions was identical to that of test 7, but the separation process of this test is distinguished by the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 10 / 100 / 20 and by the voltage of the applied electric field, fixed at 2,500 V in test 8.

[0381] Thus during this test, the KPLE-100-008 chip was connected to flow meters. The inputs E(1) and E(5) were each supplied with an electrolytic solution respectively for the anode and the cathode. The two electrolytic solutions respectively for the cathode and the anode are of the same composition. The liquid electrolytic solution has the following composition: HEPES at 10nM, HPMC at 0.2% (m / v), Tween 20 at 0.1%(m / v), 40% methanol and 1.5M KCL. The flow rate was set at 20 pL / min. These electrolytic solutions presented a pH = 7.49 (which was adjusted by NaOH solution) and a conductivity = 92.33 mS / cm. The electric field is generated by the electrolytic solutions in the electrophoresis chamber, originating from solutions containing the carbon electrodes, anode and cathode respectively.

[0382] The central inlet E(3) was fed with the sample to be purified, composed of 0.175 g / L of fluorescein, 0.176 g / L of Rhodamine 6G and 0.185 g / L of Rhodamine B. The flow rate set for the sample was 10 pL / min throughout the purification.

[0383] The last two inlets E(2) and E(4) were supplied with a buffer solution composed of 10 mM HEPES, 0.2% (m / v) HPMC, 0.1% (m / v) Tween 20 in water, at a flow rate set at 100 pL / min. The buffer solution had a pH = 7.5 (adjusted by a NaOH solution) and a conductivity = 600 pS / cm.

[0384] The electric field was set in this test at 2,500 V. After stabilization of the flows, the products at the outlet of the electrophoresis chamber were recovered in tubes and analyzed by HPLC in order to determine the percentage of each compound at each outlet.

[0385] [Fig. 14] is a photograph taken of the device during electrophoresis made possible by the use of transparent sapphire plates and colored separation products.

[0386] Hydrodynamic monitoring was carried out to show that there was no migration towards the liquid electrodes of any compound in the sample. Thus, only the outputs from the electrophoresis chamber S(2) to S(6) were analyzed by HPLC.

[0387] Results

[0388] [Fig. 14] shows an electrophoretic migration of fluorescein, in yellow, towards the liquid cathode, arriving at outlet S(2). Rhodamine B was not influenced by the presence of the electric field during electrophoresis and exited at S(4), the outlet facing the inlet E(3). Rhodamine 6G migrated towards the liquid anode, with a less significant displacement than that of fluorescein, and exited at S(5).

[0389] The HPLC spectra of the products leaving the outlets S(2) to S(6) are shown in [Fig.15].

[0390] The results of the HPLC analysis for test 8 at 2500 V confirm the presence of fluorescein at the S(2) and S(3) outlets, mainly at the S(2) outlet, with no signal from rhodamines B and 6G. The spectrum of the product from S(4) indicates mainly rhodamine B. The spectrum of the product from S(5) indicates mainly rhodamine 6G, with no rhodamine B in the product.

[0391] At 2500 V, Fluorescein was 100% purified and was recovered towards the S(2) and S(3) outlets. Rhodamine B showed no migration while rhodamine 6G showed showed a partial migration of the order of 83.49% towards the S(5) output.

[0392] Test 9.

[0393] The main characteristics of test 9 are as follows:

[0394] - Room with 5 entrances and 7 exits

[0395] - h = 1 mm

[0396] - V= 2000 V

[0397] - Flow rate (pl / min): Sample / Buffer / Electrode: 10 / 600 / 10

[0398] Separation test 9 was set up with a third device comprising 5 inlets and 7 outlets, including an electrophoresis chamber height h of 1 mm.

[0399] The installation of the inlet solutions was identical to that of tests 7 and 8, but the separation process of test 9 is distinguished by the volume of the electrophoresis chamber having a height of 1 mm and by the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 10 / 600 / 10 and by the voltage of the applied electric field, fixed at 2000 V.

[0400] [Fig. 16] is a photograph taken of the device during electrophoresis.

[0401] [Fig. 16] shows in a 1 mm high electrophoresis chamber, an electrophoretic migration of the charged species, fluorescein, in yellow, migrating towards the cathode and rhodamine 6G migrating towards the anode.

[0402] Rhodamine B was not influenced by the presence of the electric field during electrophoresis and exited at S(4), namely the exit facing the entrance E(3).

[0403] Under the conditions set up in this test, the electrophoretic migrations of fluorescein and rhodamine 6G were less significant compared to the previous tests, so that fluorescein arrives between the outlets S(3) and S(4) and rhodamine 6G between the outlets S(4) and S(5).

[0404] Test 10

[0405] The characteristics of test 10 are as follows:

[0406] - room with 5 entrances and 7 exits

[0407] - h = 1mm

[0408] - V= 3000 V

[0409] - Flow rate (pl / min): Sample / Buffer / Electrode: 20 / 3000 / 50

[0410] Separation test 10 was set up with another device comprising 5 inlets and 7 outlets, with an electrophoresis chamber height h of 1 mm.

[0411] The installation of the inlet solutions was identical to that of test 9, but the separation process of this test 10 is distinguished by the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 20 / 3,000 / 50 and by the voltage of the applied electric field, fixed at 3,000 V.

[0412] [Fig. 17] is a photograph taken of the device during electrophoresis.

[0413] [Fig. 17] confirms electrophoretic migration of charged species in a device comprising a 1 mm high electrophoresis chamber.

[0414] Under the conditions set up in this test 10, by increasing the electric field voltage and the flow rates compared to test 9, the electrophoretic migrations of fluorescein and rhodamine 6G were greater than those observed in test 9, so that the fluorescein arrives at outlet S(3) and the main flow of rhodamine 6G arrives at outlet S(5).

[0415] It was thus possible to vary the experimental conditions in order to optimize the separation of the products.

[0416] Test 11

[0417] The main characteristics of test 11 are as follows: - chamber with 5 inlets and 7 outlets - h = 2 mm V= 2000 V - Flow rate (pl / min): Sample / Buffer / Electrode: 20 / 3000 / 50

[0418] A device, similar to that used in test 9, but having a height of 2 mm for the electrophoresis chamber was used.

[0419] The installation of the inlet solutions was identical to that of test 9, but the separation process of this test 11 is distinguished by the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 20 / 3,000 / 50 and by the voltage of the applied electric field, fixed at 3,000 V, conditions identical to test 11.

[0420] [Fig. 18] is a photograph taken of the device during electrophoresis.

[0421] [Fig. 18] shows a separation of product flows and confirms a migration electrophoretic of charged species in a device comprising a 2 mm high electrophoresis chamber.

[0422] Example 4: Separation of an ATP / AMP mixture

[0423] Attempts to separate a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) were carried out.

[0424] The structures of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) are reported below.

[0425] [Chem.2] Adenosine triphosphate Cyclic monophosphate adenosine

[0426] Tests 12 to 14 were implemented with a KPLE-100-008 electrophoresis chip, having 5 inputs and 7 outputs, and an electrophoresis chamber thickness of 100 pm.

[0427] Table 6 below shows the compositions of the liquid electrodes and the separation buffer solutions and the sample to be separated which were used.

[0428] [Tableauxô] Compounds Liquid Electrode Separation Buffer Sample HEPES 10 mM 10 mM / HPMC 0.2% 0.2% / Tween 20 0.1% 0.1% / KC1 1.5 M / / Solvent 60% water / 40% methanol 100% water 100% water ATP / / 0.804 g / L AMP / / 0.792 g / L

[0429] Table 6: Details of the operating conditions of the experiments

[0430] Table 7 below shows the nature of the solutions introduced at the inputs of the device and the respective flow rates that were applied.

[0431] [Tables?] Inputs Composition Flow Rates 1 Liquid Electrode 25 pL / min 2 Separation Buffer 100 pL / min 3 Sample 10 pL / min 4 Separation Buffer 100 pL / min 5 Liquid Electrode 25 pL / min

[0432] Table 7: composition and flow rates of the different chip inputs

[0433] The liquid electrode flow rates at inlets E(1) and E(5) were 25 pL / min, the sample flow rate at inlet E(3) was 10 pL / min and the buffer flow rates at inlets E(2) and E(4) were 100 pL / min.

[0434] During the tests, the electrophoresis chip operated continuously throughout the experiments after stabilization of the system.

[0435] Three different tests 12, 13 and 14 were carried out. In these tests the flow rates at the inlets remained unchanged for all the tests. Only the value of the applied electric field was increased from 0 to 3000 V.

[0436] The tests were carried out at 0V (test 12), i.e. without an applied electric field, at 1000 V (test 13) and at 2000 V (test 14).

[0437] The products recovered at the outlets S(l) to S(7) were analyzed by HPLC during the tests.

[0438] The HPLC spectra of the outputs S(l) to S(7) at 0V of test 12 are reported in [Fig.19], those of test 13 at 1000 V in [Fig.20] and those of test 14 at 2000V in [Fig.21],

[0439] Results

[0440] At 0 V, the entire sample is visible at S(4) corresponding to the central output of the chip, namely the one facing the sample input E(3). The system is therefore stable.

[0441] At 1000 V, the entire sample is still visible in S(4), the voltage was not sufficient to allow migration of one of the compounds. The intensity of the electric field was therefore increased.

[0442] At 2000 V, a partial migration of ATP (first peak) towards S(3), while T AMP shows no migration towards the different outputs. About 40% of the ATP is diverted towards the S(3) output.

[0443] The separation of ATP / AMP biomolecules was therefore verified by these tests, demonstrating proof of concept.

[0444] In order to increase this proportion of migration, the electric field in the chip can be increased. However, optimization of the separation is possible by modulating parameters, such as the composition of the separation buffer (pH, concentration and viscosity), as well as the optimal electric field, and the position of the sample inlet and sample preparation.

[0445] Example 5: Protein / linker purification

[0446] During these tests 15, the KPLE-100-009 chip was connected to flow meters. The electrophoresis chamber inlets E(1) and E(5) were supplied by liquid electrodes. The liquid anode and the liquid cathode were of the same composition. The liquid electrode solutions were composed of 10 mM HEPES, 0.2% (m / v) HPMC, 0.1% (m / v) Tween 20, 40% methanol and 1.5M KCL, and were introduced at a flow rate set at 20 pL / min. The electric field was generated in the container of the liquid electrodes using a carbon electrode. These liquid electrodes used had a pH = 7.5 (adjusted by a NaOH solution) and a conductivity = 86.66 mS / cm.

[0447] The central inlet E(3) was fed with the sample to be purified, i.e. a reaction mixture resulting from the reaction of a protein and a linker. The flow rate in E(3) for the sample was set at 10 pL / min throughout the purification.

[0448] For the last two inlets E(2) and E(4), a buffer solution (TS), composed of 10mM HEPES, 0.2%(m / v) HPMC, 0.1%(m / v) Tween 20 in water, was introduced at a flow rate set at 80 pL / min. The separation buffer had a pH equal to 7.5 (adjusted by a NaOH solution).

[0449] The applied electric field was 1500 V.

[0450] After stabilization of the flows, the products at the outlet of the electrophoresis chip were recovered in tubes in order to analyze them by HPLC and to determine the percentage of each compound at each outlet.

[0451] The hydrodynamic monitoring carried out makes it possible to show that there is no migration towards the liquid electrodes of any element of the system.

[0452] Only the outputs of the electrophoresis chamber, i.e. outputs S(2) to S(4), were analyzed.

[0453] The diagram in [Fig.22] represents the migration of species according to HPLC analysis.

[0454] The HPLC spectra of the output products S(3) and S(4) are shown in Figures 23 and 24 respectively.

[0455] The analyses show that 80% of the linker is diverted to another exit than the majority of the protein, only 10.43% of the protein is diverted to the same exit as the linker.

Claims

Claims

1. A free-flow electrophoresis microcell device comprising a vertical succession of plates X, Y, Z whose surfaces are stacked according to the sequence YZY(XYZY)p, wherein X represents an electrophoresis plate (1) made of inert material, Y represents a sealed plate (2) made of inert, electrically insulating and thermally conductive material, sapphire or alumina A12O3 containing 99% a-Al2O3, Z represents a cooling plate (3) comprising a heat transfer system (4), p integer from 1 to 100, represents both the number of stages of said device and the number of plates X each stage (5) being defined: - by the following sequence of plates YZYXYZY, in which: - plate X is located between two plates Y, - each of the two Z plates being respectively adjacent to a Y plate, - and each of the two Y plates located at the ends of the sequence YZYXYZY, respectively covers a Z plate so that each Z plate is located between two Y plates, said device further comprising means for clamping all the plates allowing the sealing of said device, said device being such that each X plate comprises: a number i of electrophoresis chamber(s) (Fi), i being an integer from 1 to 100, in particular 50, preferably 10, each electrophoresis chamber (6) comprising: • a hollowed-out part • in the shape of a rectangular parallelepiped with 4 side faces (a, b, c, d) and 2 upper and lower faces (e, f), • said hollowed out part being of length Loe and width Lae, • of height h corresponding to the thickness of the X-ray electrophoresis plate, from 25 pm to 20 mm, in particular from 50 pm to 200 pm or from 1.0 mm to 5.0 mm, • the lateral faces (a, b) being parallel to each other, the face (a) being delimited by two edges (Al, A2) of dimension Lae and face (b) being delimited by two edges (Bl, B2) of dimension Lae, • the lateral faces (c, d) being parallel to each other, the face (c) being delimited by two edges (Cl, C2) of dimension Loe and the face (d) being delimited by two edges (Dl, D2) of dimension Loe, n successive inputs E(l), E(2) to E(nl), En, n being an integer from 4 to 9, preferably 5 or 6, distributed on the face (a) between Al and A2 and aligned in a direction parallel to Al and A2, m successive outputs from S(l), S(2) to S(ml), S(m), m being an integer from 4 to 12, preferably 5 or 7, distributed on the face (b) between Bl and B2 and aligned in a direction parallel to Bl and B2, so that S(l) faces E(l) and S(m) faces E(n) in a direction parallel to Cl and Dl, said plates Y ensuring the sealing of the electrophoresis chamber, said chambers (Fi) being arranged so that the edges Cl of each hollowed-out part are parallel to each other, said device comprising: - supply channels configured to connect the E(l) inlets of each electrophoresis chamber to a micro / millifluidic circuit for supplying a liquid cathode, - supply channels configured to connect the inputs E(n) of each electrophoresis chamber to a micro / millifluidic circuit for supplying a liquid anode, - supply channels configured to connect at least one of the inlets E(2) to E(nl) of each electrophoresis chamber to a micro / millifluidic circuit for supplying an initial solution containing a product to be purified and / or separated, - supply channels configured to connect the other remaining inlets of each electrophoresis chamber to micro / millifluidic circuits for supplying at least one buffer solution, - recovery channels configured to connect each of the outlets S(l) to S(m) of each electrophoresis chamber to micro / millifluidic recovery circuits, said device being configured to, in the presence of an electric field generated between the liquid cathode and the liquid anode parallel to Al and perpendicular to Cl, and in operation - circulate the liquid cathode from the inlet E(l) to the outlet S(l), - circulate the liquid anode from the inlet E(n) to the outlet S(m), - circulate in the electrophoresis chamber (Fi), between the liquid cathode and the liquid anode, from the inlets E(2) to E(nl) to the outlets S(2) to S(ml), the solution containing the product to be separated and / or purified and at least one buffer solution, - recover at one of the outlets S(2) to S(ml) of each electrophoresis chamber in a recovery circuit the purified and / or separated product contained in said initial solution.

2. Device according to claim 1, comprising recovery channels configured to connect: - the outlet S(l) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the liquid cathode, - the outlet S(m) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the liquid anode, - one of the outlets S(2) to S(ml) of each electrophoresis chamber to a micro / millifluidic circuit for recovering the purified and / or separated product contained in said initial solution - the other remaining outlets of each electrophoresis chamber to at least one micro / millifluidic circuit for recovering the at least one buffer solution.

3. Device according to one of claims 1 to 2, wherein p is equal to 1 and i is equal to 1, comprising a single electrophoresis plate having a single electrophoresis chamber, in particular with a height h of 25 to 200 pm or 1.0 to 5.0 mm.

4. Device according to one of claims 1 to 2, in which p is equal to 1 and i varies from 2 to 10, comprising a single electrophoresis plate X comprising from 2 to 10 electrophoresis chambers, preferably 10 electrophoresis chambers.

5. Device according to one of claims 1 to 2, in which p varies from 2 to 10 and i varies from 2 to 10, comprising from 2 to 10 X electrophoresis plates and each X electrophoresis plate comprising from 2 to 10 electrophoresis chambers, in particular p is equal to 10 and i is equal to 10.

6. Device according to one of claims 1 to 5, wherein the height h of the electrophoresis chamber is from 650 pm to 20 mm, in particular from 650 pm to 10.0 mm, preferably from 650 to 5.0 mm, preferably from 650 pm to 2.0 mm, or wherein the height h of the electrophoresis chamber is from 25 pm to 200 pm or from 1.0 to 5.0 mm.

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

8. Device according to one of claims 1 to 7, in which the X-ray electrophoresis plates are made of a material chosen from polytetrafluoroethylene (PTFE), perfloroalkoxy (PFA) and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP plates.

9. Device according to one of claims 1 to 8, in which in each X electrophoresis plate a part of said plate is configured to leave room for a fluid circuit of supply and recovery channels, which is partially or totally etched, cut or pierced in the X plate.

10. Device according to one of claims 1 to 9, wherein the plates X, Y, Z are rectangular parallelepipeds and have a width La and a length Lo, wherein La and Lo vary from 2.0 to 50.0 cm, and wherein the plates Y have a thickness of 0.5 mm to 5.0 mm, and wherein the cooling plates Z have a thickness of 1.0 to 10.0 mm, in particular 1.0 to 5.0 mm.

11. Device according to one of claims 1 to 10, in which the electrophoresis chambers comprise channeling means opening onto the inlets and / or onto the outlets, preferably etched in the X electrophoresis plate.

12. Device according to one of claims 1 to 11, in which each electrophoresis chamber is configured to each contain at least one membrane of selective permeability, preferably selective in size, positioned so as to be crossed by the solution containing the product to be separated or purified during operation of the device, and / or in which the upper face and / or the lower face of each of the electrophoresis chambers comprises protuberances configured not to disturb, during operation of the device, the circulation in the chamber of the product to be purified and / or separated, and configured to improve heat transfer and to maintain the electrophoresis chambers at a selected temperature, in particular in which said protuberances are made of thermally conductive materials, preferably sapphire or 99% a-Al2O3 alumina.

13. Device according to one of claims 1 to 12, wherein said heat transfer system (4) is a pipe network configured to allow the circulation of one or more heat transfer fluids, said network being placed in direct contact with a portion of the plates Y adjacent to Z, said portion being thermally connected to the electrophoresis chambers, in order to allow the temperature in said electrophoresis chambers to be controlled, in particular wherein said pipe network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers, preferably wherein said pipe network of the heat transfer system comprises, for each of the electrophoresis chambers, channels parallel to the edge C1, said channels being able to contain heat transfer fluids of different temperatures in order to generate said temperature gradient.

14. Device according to one of claims 1 to 13, in which the clamping means for ensuring the sealing of said device comprise two external clamping plates enclosing said device, said means being removable, in particular plate by plate.

15. Method for purifying and / or separating by free-flow electrophoresis a product contained in a solution by using an electrophoresis microcell device according to one of claims 1 to 14, and comprising the following steps: - connecting the supply channels of said device to the supply circuits of the liquid cathode, the liquid anode, an initial solution containing a product to be purified and / or separated, and at least one buffer solution, said channels and circuits being controlled by a central unit (UC1) - connecting the cooling systems to a cooling circuit, controlled by a central unit (UC2) - generating an electric field along the edges Al, via the liquid anode and the liquid cathode, - generating a fluidic circulation in each of the electrophoresis chambers, by the central unit (UC1),so as to: • circulate a liquid cathode from the inlet E(l) to the outlet S(l), • circulate a liquid anode from the inlet E(n) to the outlet E(m), • circulate in the electrophoresis chamber, between the liquid cathode and the liquid anode, the initial solution containing the product to be purified and / or separated and said at least one buffer solution, from the inlets E(2) to E(nl) to the outlets S(2) and S(ml), - select and recover in at least one of the outlets S(2) to S(ml), of each of the electrophoresis chambers of said device, the separated or purified product.,

16. Method according to claim 15, wherein said method is carried out under continuous flow of the initial solution containing the product to be purified and / or separated, in particular at a flow rate of 1 to 5 L / hour, in particular said device comprising 100 electrophoresis chambers, preferably in operation 300 days / year.

17. A method according to any one of claims 15 to 16, wherein the cathode liquid and liquid anode are electrolytic solutions of the same composition.

18. Method according to one of claims 15 to 17, wherein said fluid circulation of the liquid cathode in each of the electrophoresis chambers, controlled by the central unit (UC1), is carried out at a flow rate of 10 to 10,000 pL / min, and / or wherein said fluid circulation of the liquid anode in each of the electrophoresis chambers, controlled by the central unit (UC1), is carried out at a flow rate of 10 to 10,000 pL / min, and / or wherein said fluid circulation of the solution to be purified and / or separated in each of the electrophoresis chambers, controlled by the central unit (UC1), is carried out at a flow rate of 10 to 30,000 pL / min, and / or wherein said fluid circulation of the buffer solution in each of the electrophoresis chambers, controlled by the central unit (UC1), is carried out at a flow rate of 10 to 50,000 pL / min.

19. Method according to one of claims 15 to 17, wherein said device comprises at least 100 electrophoresis chambers positioned in parallel, and / or wherein said device comprises in each electrophoresis chamber at least one size-selective membrane, configured to separate the product to be purified and / or separated from the initial solution during the fluid circulation in each of the electrophoresis chambers, and / or wherein during the fluid circulation in each of the electrophoresis chambers, a pH variation along the edge Al in each electrophoresis chamber is generated, using at least two buffers of different pH, and / or wherein during the fluid circulation in each of the electrophoresis chambers, a temperature gradient along the edge Al of the recessed part is applied in each electrophoresis chamber.

20. Method according to one of claims 15 to 19, wherein during the fluid circulation in each of the electrophoresis chambers, a homogeneous temperature, in particular from 10°C to 40°C, is applied in each of the electrophoresis chambers, and / or wherein, during the fluid circulation in each of the electrophoresis chambers, the electric field generated is from 200V to 4000 V.

21. Method according to one of claims 15 to 20, carried out to purify and / or separate a protein or carried out to purify and / or separate isomers, in particular enantiomers.

22. Method for developing an industrial device for purifying and / or separating a solution comprising the product to be purified and / or separated by electrophoresis comprising the following steps: a. a first study step implementing a device according to claim 3 comprising a single electrophoresis chamber, in particular the height h of the hollowed-out part of which is 25 to 200 pm or 1.0 to 2.0 mm, to determine and optimize the fluid circulation of the product to be purified and / or separated, b. a second step of setting up said industrial device according to one of claims 4 or 5 comprising 10 to 100 electrophoresis chambers.

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