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 its uses.

[0002] High-performance liquid chromatography (HPLC) and zone electrophoresis using a support are analytical and / or preparative techniques for separating molecules, particularly 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 a 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 demanding maintenance, operating in flow, designed for easy and modular operation to implement, in order to allow purification and / or separation of the expected products in volumes and quantities on an industrial scale.

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

[0005] Another object of the invention is a process for purifying and / or separating molecules, in particular biomolecules, adaptable to an 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 in 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 an inert, electrically insulating material and thermal conductor, made of sapphire or alumina (Al2O3) with 99% a-Al2O3,

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

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

[0013] each floor (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 plates Z being respectively adjacent to a plate Y,

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

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

[0019] said device being such that each plate X 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 section • in the shape of a rectangular parallelepiped with 4 lateral 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 µm to 20 mm, in particular from 50 µm to 200 µm or from 1.0 mm to 5.0 mm, • the lateral faces (a, b) being parallel to each other, 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, face (c) being delimited by two edges (Cl, C2) of dimension Loe and face (d) being delimited by two edges (Dl, D2) of dimension Loe, • n successive entries E(1), 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 along 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 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 plates Y 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(l) inputs of each electrophoresis chamber to a micro / milli fluidic circuit for supplying a liquid cathode, - supply channels configured to connect the E(n) inputs of each electrophoresis chamber to a micro / milli fluidic circuit for supplying a liquid anode, - supply channels configured to connect at least one of the E(2) to E(nl) inlets of each electrophoresis chamber to a micro / millifluidic circuit supplying an initial solution containing a product to be purified and / or separated, - supply channels configured to connect the remaining inlets of each electrophoresis chamber to micro / millifluidic circuits supplying at least one buffer solution, - recovery channels configured to connect each of the S(l) to S(m) outputs of each electrophoresis chamber to micro / millifluidic recovery circuits,

[0025] said device being configured so that, 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), - to 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, - to 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 that does not employ a stationary phase, i.e., without the use of a solid phase to support the migration of species during electrophoresis.

[0027] The term "plate" refers to a rigid element, generally in the form of a rectangular parallelepiped, in which at least two faces are parallel to each other and these faces represent primarily the total area of ​​this element, namely the area of ​​said two parallel faces 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 stacking 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 to electrophoresis.

[0031] The term "electrophoresis chamber" refers to a portion of the electrophoresis plate X comprising:

[0032] - a hollowed-out portion 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 plate X 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 return channels connected to the inlets and outlets of said electrophoresis chamber. The electrophoresis cell includes the hollowed-out portion closed by the side walls in the X plate and those of the Y plates, the inlets and outlets of the hollowed-out portion, and the supply and return channels.

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

[0037] The term "cooling plate" refers to the plate, designated Z, containing a heat transfer system. The heat transfer system's function is to transport a heat transfer fluid to enable temperature control. The heat transfer system may, for example, consist of a network of channels formed by recesses in plate Z.

[0038] By “sealing plate”, designated Y, we mean a plate intended to ensure the fluidic sealing of the device.

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

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

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

[0042] Plate Y is made of sapphire or alumina A12O3 with 99% a-Al2O3.

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

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

[0045] By way of 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 series of stacked plates held together by clamping means, in which the surfaces of adjacent plates are in direct contact. The adjacent plates are contiguous plates. Each plate in the device has a maximum of 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 the electrophoresis chambers of the plate X, and to ensure 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 occurring being solely thermal in 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 to the first and second stage and to the second and third stage.

[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 arranged in rows.

[0053] The range of "1 to 100" includes the whole numbers in the following ranges: from 1 to 10; from 10 to 20; from 20 to 30; from 30 to 40; from 40 to 50; from 50 to 60; from 60 to 70; from 70 to 80; from 80 to 90; from 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 free-flow electrophoresis is carried out comprises a hollowed-out part of a plate X, closed by the lateral walls in the plate X, said hollowed-out part being in the general shape of a rectangular parallelepiped.

[0055] The term "rectangular parallelepiped-shaped" means that this hollowed-out portion generally fits within a rectangular parallelepiped of length Loe, width Lae, and height h, corresponding to the thickness of plate X ([Fig. 2]). The general shape of the hollowed-out portion is not taken into account when defining its shape. This includes any features present in the electrophoresis chamber, such as the inlet and outlet channels and any protrusions.

[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, cut 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 part of the electrophoresis chamber.

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

[0068] The range from 25 µm to 20 mm includes the following ranges: from 25 to 50 µm; from 50 to 75 µm; from 75 to 100 µm; from 100 to 200 µm; from 200 to 300 µm; from 300 to 400 µm; from 400 to 500 µm; from 500 to 600 µm; from 600 to 700 µm; from 700 to 800 µm; from 800 to 900 µm; from 900 µm to 1.0 mm; from 1.0 to 2.0 mm; from 2.0 to 3.0 mm; from 3.0 to 4.0 mm; from 4.0 to 5.0 mm; from 5.0 to 6.0 mm; from 6.0 to 7.0 mm; from 7.0 to 8.0 mm; from 8.0 to 9.0 mm; from 9.0 to 10.0 mm; 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.

[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 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" is meant a passage allowing the flow of a liquid fluid 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 entries E(l) to E(n) are located between the edges Al and A2 and aligned along 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 face (a) and / or slightly above or below each other. The positioning of the inlets is configured to introduce the different flows into the electrophoresis chamber in such a way as to allow their circulation in the chamber parallel to the edge CL

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

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

[0078] The entries 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 discharge of the liquid cathode after circulation in the electrophoresis chamber.

[0081] The inlet E(n) is configured for the introduction of a liquid anode. The outlet S(m) is configured for the discharge 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 electrophoresis.

[0083] The liquid anode and the liquid cathode are electrolyte solutions, that is to say 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 action of the generator which charges the liquid anode and the liquid cathode and / or with an increase in the concentration of ions.

[0088] One of the inlets 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 outlets S(2) to S(ml) is configured for the recovery of the purified and / or separated solution after the electrophoresis process implemented 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 fluid flows.

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

[0093] The term "millifluidic circuit" means a set of channels with a cross-sectional dimension on 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 / milli fluidic 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 milli-fluidics 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 outlets S(2) to S(ml) of each electrophoresis chamber (Fi) to a micro / milli fluidic circuit for recovering the purified and / or separated product contained in said initial solution to be purified and / or separated;

[0104] - the other outputs have at least one micro / milli fluidic 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 face (c) - circulate the liquid anode from the inlet E(n) to the outlet S(m), along face (d) - to 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, - to 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 using sapphire or alumina (Al2O3) plates with 99% a-Al2O3 as a means of separation between the heat transfer system and the electrophoresis chambers induces excellent temperature control and homogeneity such that it allows: - a thickness h of the electrophoresis chambers constituting the device that can vary from micrometers to millimeters, thus increasing the processing capacity of the solutions to be purified or separated, - purification and / or separation of heat-sensitive molecules, - the introduction of a temperature gradient in the i chambers electrophoresis.

[0107] Furthermore, the use of the sapphire Y plate provides a robust and watertight device that allows for disassembly and reassembly, facilitating the cleaning of the device's components and its maintenance. The modularity of the device allows for the reuse of the Y and Z plates.

[0108] According to a particular embodiment, the number m of outputs is equal to or greater than the number of inputs 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. Multiplying the 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 S(l) output of each electrophoresis chamber to a micro / millifluidic circuit for liquid cathode recovery, - the S(m) output of each electrophoresis chamber to a micro / millifluidic circuit for liquid anode recovery, - 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 outputs of each electrophoresis chamber to at least one micro / millifluidic recovery circuit of at least one buffer solution.

[0113] The recovery channels of the device are configured to recover, after circulation through 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, such that, within 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 at least one buffer solution used.

[0114] By “separation” means the separation from one another of at least two products present in the initial solution that can be recovered separately, said two products being each recoverable at different outlets of the device of the present invention.

[0115] By “purification” we mean 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, in which p is equal to 1 and i is equal to 1, comprising a single electrophoresis plate having a single electrophoresis chamber, in particular of height h of 25 to 200 pm or of 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 of height h from 25 to 200 pm or from 1.0 to 5.0 mm.

[0118] It constitutes a laboratory tool. It advantageously allows the determination of the influence of the various 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 having 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 electrophoresis plates X and each electrophoresis plate X 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 allows to separate and / or purify continuously a solution of up to 1 to 5 Liters per hour of solution to be purified or separated, namely to allow 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, preferably from 650 pm to 2.0 mm.

[0125] The range from 650 pm to 2.0 mm includes the following ranges: from 650 to 700 pm; from 700 to 750 pm; from 750 to 800 pm; from 800 to 850 pm; from 850 to 900 pm; from 900 to 950 pm; from 950 pm to 1.0 mm; 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.

[0126] The range from 650 µm to 5.0 mm comprises the following ranges: from 650 to 2.0 mm;

[0127] 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;

[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 µm 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 from 650 µm to 20.0 mm includes the following ranges: from 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 from "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; 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.

[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 to determine the influence of the different 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 is understood to mean 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 is understood to mean 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 with a height on the order of millimeters is advantageous for achieving industrial quantities for product purification. For example, a device with 10 to 30 chambers with a height 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, preferably from 650 pm to 2.0 mm,

[0146] or wherein the height h of the electrophoresis chamber is from 25 pm to 200 pm or from 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 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), within the same X-ray electrophoresis plate constitute a row of electrophoresis chambers. The row configuration of the chambers optimizes the use of the X-ray plates in terms of surface area and facilitates their manufacture and machining. It also allows for optimization of the arrangement of distribution channels and cooling systems, for example, by sharing the latter.

[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 entrances 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 part is 2 to 15

[0153] The ratio between the length Loe and the width Lae of the hollowed-out portion 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 portion 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: from 1.0 to 2.0 cm; from 2.0 to 3.0 cm; from 3.0 to 4.0 cm; from 4.0 to 5.0 cm; from 5.0 to 6.0 cm; from 6.0 to 7.0 cm; from 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 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: from 5.0 to 6.0 cm; from 6.0 to 7.0 cm; from 7.0 to 8.0 cm; from 8.0 to 9.0 cm; from 9.0 to 10.0 cm; from 10.0 to 11.0 cm; from 11.0 to 12.0 cm; from 12.0 to 13.0 cm; from 13.0 to 14.0 cm; from 14.0 to 15.0 cm; from 15.0 to 16.0 cm; from 16.0 to 17.0 cm; from 17.0 to 18.0 cm; from 18.0 to 19.0 cm; from 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 selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP sheets.

[0159] According to a particular embodiment, the present invention relates to a device as defined above, in which in each electrophoresis plate X a part of said plate is configured to allow space for a fluidic circuit of supply and recovery channels, which is partially or totally engraved, cut or drilled into the plate X.

[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 part,

[0161] and / or wherein 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 hollowed-out 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 selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA) and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP plates,

[0164] and / or wherein in each electrophoresis plate X a portion of said plate is configured to accommodate a fluidic circuit of supply and recovery channels, which is partially or totally etched, cut or drilled into the plate X.

[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 hollowed part of the electrophoresis chamber and the length "Lo" of the plate X is slightly greater than the length "Loe" of the hollowed part 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 part of an electrophoresis chamber and the length "Lo" of the plate X is substantially greater than 10 times the width "Lae" of the hollowed part of an electrophoresis chamber.

[0171] A slightly higher or substantially higher value is, for example, a value that is 1 to 20 mm higher. Advantageously, to increase the sealing of a row of 10 electrophoresis cells, the width of the borders of the electrophoresis plate X 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 Y plates 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: from 0.5 to 1.0 mm; from 1.0 to 1.5 mm; from 1.5 to 2.0 mm; from 2.0 to 2.5 mm; from 2.5 to 3.0 mm; from 3.0 to 3.5 mm; from 3.5 to 4.0 mm; from 4.0 to 4.5 mm; from 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 1.0 to 5.0 mm.

[0176] The range of "1.0 to 10.0 mm" includes the following ranges: from 1.0 to 2.0 mm; from 2.0 to 3.0 mm; from 3.0 to 4.0 mm; from 4.0 to 5.0 mm; from 5.0 to 6.0 mm; from 6.0 to 7.0 mm; from 7.0 to 8.0 mm; from 8.0 to 9.0 mm; from 9.0 to 10.0 mm.

[0177] The range of "1.0 to 5.0 mm" includes the following ranges: from 1.0 to 1.5 mm; from 1.5 to 2.0 mm; from 2.0 to 2.5 mm; from 2.5 to 3.0 mm; from 3.0 to 3.5 mm; from 3.5 to 4.0 mm; from 4.0 to 4.5 mm; from 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 in which the Y plates have a thickness of 0.5 mm to 5.0 mm,

[0180] and / or in which the cooling plates Z have a thickness from 1.0 to 10.0 mm, in particular from 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 include channeling means opening onto the inlets and / or outlets, preferably engraved in the electrophoresis plate X.

[0182] These channeling means are configured to allow the orientation of the flows in the electrophoresis chamber at the level of 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 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, that is, 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 faces (a) and (b) of the rectangular parallelepiped in which the hollowed-out portion 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 contain at least one selective permeability membrane, preferably size-selective, positioned so as to be traversed by the solution containing the product to be separated or purified during the 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 traversed by the initial solution containing the product to be separated or purified during the operation of the device and

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

[0189] The presence of these membranes also allows for selectivity of sizes 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 includes protrusions configured so as not to disturb, during the 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 faces (e) and / or (f) of the hollowed portion towards the interior of said hollowed 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 protrusions are made of materials thermal conductor, preferably in sapphire or 99% a-Al2O3 alumina, preferably in the same material as the Y plate, to ensure thermal conductivity in the chambers and control the temperature in the cell.

[0193] The presence of these protrusions promotes heat exchange between the cooling plates along the path and thus optimizes the separation of the desired molecule while avoiding denaturation of the molecules, 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 the 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 to avoid a perturbation of the streamlines of the laminar flows around the protrusion and in the electrophoresis chamber, said shape and 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 studs, rectangular-based pillars or ovoid-shaped studs.

[0197] According to a particular embodiment, the present invention relates to a device as defined above, in which the plates X and / or Y are engraved 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 contain at least one selectively permeable membrane, preferably size-selective, positioned so as to be traversed by the solution containing the product to be separated or purified during the operation of the device,

[0199] and / or wherein the upper face and / or lower face of each of the electrophoresis chambers includes protrusions 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,

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

[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 network of pipes configured to allow the circulation of one or more heat transfer fluids, said network being in direct contact with a portion of the plates Y adjacent to Z, the aforementioned part being thermally connected to the electrophoresis chambers, in order to allow temperature control in said electrophoresis chambers.

[0202] According to a particular embodiment, the present invention relates to a device as defined above, in which the piping 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 piping 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 piping 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 piping 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 piping network configured to allow the circulation of one or more heat transfer fluids, said network being in direct contact with a portion of the plates Y adjacent to Z, the aforementioned portion being thermally connected to the electrophoresis chambers, in order to allow temperature control in said electrophoresis chambers,

[0208] preferably wherein said piping 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.

[0209] According to a particular embodiment, the present invention relates to a device as defined above, in which said piping 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 piping network of the heat transfer system is perpendicular to the edge Cl 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 face (c) of the hollowed 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 clamping 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 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 process for the purification and / or separation by free-flow electrophoresis of a product contained in a solution comprising the following steps:

[0219] - to connect 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 processing unit (CPU2)

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

[0222] - generate a fluidic circulation in each of the electrophoresis chambers, by the central processing unit (UC1), so that: • 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 fluid circulation by the central unit (UC1) is achieved, for example, using flow meters and pumps present in the supply and recovery circuits. For example, each inlet of the electrophoresis chamber 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 fluidic 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 a 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 process as defined above, in which the liquid cathode and the liquid anode are electrolytic solutions of the same composition.

[0230] By "liquid electrode" is meant a liquid cathode or anode. It should be noted that an electrode is an electronic or ionic conductor that captures or releases electrons.

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

[0232] The liquid electrode may comprise species in ionic form 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 KCl.

[0235] Advantageously, the liquid electrode has the following composition: HEPES at 10mM, HPMC at 0.2%(w / v), Tween 20 at 0.1%(w / 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 0.01 at 250mS / cm.

[0238] Advantageously the liquid electrode is pre-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 KC1 pH=7.5.

[0239] The use of a liquid cathode and anode implies the absence of metal in the device and prevents 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 process 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 process 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 process as defined above, in which said fluidic circulation of the solution to be purified and / or to be 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 process 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; 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; from 8,500 to 9,000 pL / min; from 9,000 to 9,500 pL / min; from 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; from 13,000 to 14,000 pL / min; from 14,000 to 15,000 pL / min; from 15,000 to 16 000 pL / min; from 16,000 to 17,000 pL / min; from 17,000 to 18,000 pL / min; from 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 process 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 wherein said fluidic 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 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.

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

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

[0255] Thus the supply circuit for 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 process as defined above, in which, during fluid circulation in each of the electrophoresis chambers, a pH variation along the Al edge 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 process 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 process as defined above, in which each electrophoresis chamber comprises at least one size-selective membrane, positioned parallel to the edge Cl and adjacent to an inlet of a buffer solution,

[0259] wherein during the fluidic 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 to 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 process as defined above, in which, during fluid circulation in each of the electrophoresis chambers,

[0261] a temperature gradient along the edge Al of the hollowed-out 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 process 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 fluid circulation in each of the electrophoresis chambers, a pH variation along the Al edge in each electrophoresis chamber is generated, using at least two buffers of different pH,

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

[0267] According to a particular embodiment, the present invention relates to the process as defined above, in which, during the fluidic 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 process as defined above, in which, during the fluidic circulation in each of the electrophoresis chambers, the generated electric field is from 200V to 4000V.

[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 process as defined above, in which, during 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 in which, during fluid circulation in each of the electrophoresis chambers, the generated electric field is from 200V to 4000V.

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

[0274] According to a particular embodiment, the present invention relates to the process 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 process 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 the use of a device according to the invention as defined above, comprising a single electrophoresis chamber, in particular having a hollowed-out portion with a height h of 25 to 200 µm or 1.0 to 5.0 mm, for determining and optimizing the fluidic flow of the product to be purified and / or separated in order to implement an industrial device according to the invention as defined above comprising 10 to 100 electrophoresis chambers

[0277] Another object 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 having a height h of 25 to 200 pm or 1.0 to 5.0 mm, to determine and optimize the fluidic 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.1] represents an exploded view diagram of a microcell electrophoresis device comprising 30 electrophoresis cells arranged in rows of 10 cells on 3 stages, without representation of the clamping means of all the plates.

[0280] (1) represents an electrophoresis plate comprising a row of 10 chambers electrophoresis chambers, each comprising a rectangular parallelepiped-shaped hollowed portion (6), each chamber including inlets or outlets (7) and supply or recovery channels (8), two adjacent hollowed portions are separated by a single wall. (2) represents a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchange. (3) represents a cooling plate comprising a cooling system (4) which includes a hollowed-out section 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 sequences YZY power plants are common to two successive floors, respectively the first and second floor and the second and third floor.

[0281] [Fig. 2] represents the hollowed-out portion (6) of a chamber of an electrophoresis plate (1). The hollowed-out portion is inscribed within 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 lateral walls between the hollowed-out portion and the plate X, the faces (a, b) being parallel to each other and the faces (c, d) being parallel to each other. Face (a) is delimited by the edges (A1, A2) of dimension Lae, face (b) by the edges (B1, B2) of dimension Lae. Face (c) is delimited by the edges (C1, C2) of dimension Loe, face (d) by the edges (D1, D2) of dimension Loe. The edges (A1,B1,C1,D1) delimit face (e) and the edges (A2, B2, C2, D2) delimit 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 for all the plates.

[0283] (1) represents an electrophoresis plate comprising a single chamber comprising a hollowed-out portion (6) in the shape of a rectangular parallelepiped, including inlets or outlets (7) and supply or return channels (8). (2) represents a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchange. (3) represents a cooling plate comprising a cooling system (4) which includes 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 the inlets of the electrophoresis chamber. The device consists of a single stage (5) comprising a succession of plates YZYXYZY.

[0284] [Fig.4] represents in part a) a single-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 of electrophoresis, each comprising a hollowed-out section (6) in the shape of a rectangular parallelepiped, each chamber including inlets or outlets (7) and supply or recovery channels (8), two adjacent hollowed-out sections are separated by the same wall. (2) represents a synthetic sapphire plate ensuring the separation of fluids between two plates but allowing heat exchange. (3) represents a cooling plate comprising a cooling system (4) which includes 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.

[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] shows a diagram of a row of four electrophoresis chambers, in which the inlets of each electrophoresis chamber are identically arranged in part a) or the inlets of adjacent chambers are symmetrical with respect to the wall separating them (part b). The inlets and outlets of the electrophoresis chambers include channeling means (9).

[0289] [Fig. 6] shows an exploded view of 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 includes a heat transfer system having 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 particular embodiment, the clamping means consist of two plates (11) which clamp the entire sequence of plates X, Y, and Z, by means of fastening means (12) connecting the two plates (11), the distance between which can be adjusted. The fastening 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 inputs and 7 outputs, said types being numbered from top to bottom from 1 to 7, the central input is for the sample, inputs E(2) and E(4) for the buffer solution (TS) and inputs E(1) and E(5) for the liquid electrodes; part b) represents that of the KPLE-100-009 chip which has 5 inputs and 5 outputs, said outputs being numbered from top to bottom from 1 to 5, the central input is for the sample, inputs E(2) and E(4) for the buffer solution (TS) and inputs E(1) and E(5) for the liquid electrodes.

[0292] The hollowed-out portion of the electrophoresis chambers has a width Lae and a length Loe. The electrophoresis plate X has a width La and a length Lo. The electrophoresis chambers include means (9) for channeling the inlet and outlet flows. The channeling means are, for example, triangular elements (91) with 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 an electrophoresis chamber with a thickness of 100 pm, during the hydrodynamic test. The visualization of the different flows was made possible by coloring the sample flow and electrode flows yellow.

[0294] [Fig. 10] presents a series of photographs taken of a device comprising an electrophoresis chamber having 5 inlets and 7 outlets, with a thickness of 1 mm, 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 having 5 inlets and 7 outlets, of thickness h of 2 mm, during hydrodynamic tests at flow rates in pL / min of the sample / buffer solution / electrode solutions respectively of 400 / 4000 / 1000.

[0296] [Fig. 12] is a photograph taken of the device comprising an electrophoresis chamber having 5 inlets and 5 outlets, with a thickness of 100 pm, 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 sample / buffer solution / electrode respectively of 10 / 80 / 20.

[0297] [Fig. 13] shows 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 sample / buffer solution / electrode solutions respectively of 10 / 80 / 20

[0298] [Fig. 14] is a photograph taken of the device comprising an electrophoresis chamber having 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 sample / buffer solution / electrode solutions respectively of 10 / 100 / 20.

[0299] [Fig. 15] shows 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, 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 respectively of 10 / 100 / 20

[0300] [Fig. 16] shows 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 flow rates in pL / min of 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 the colored compounds.

[0301] [Fig. 17] shows photographs taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 1.0 mm thick, during a test of separation 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 sample / buffer solution / electrode respectively 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] shows photographs taken of the device comprising an electrophoresis chamber with 5 inlets and 7 outlets, 2.0 mm thick, during a test of separation 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 sample / buffer solution / electrode respectively 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(1) to S(7) 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 adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 0 V and at flow rates in pL / min of the sample / buffer solution / electrode solutions respectively of 10 / 100 / 25.

[0304] [Fig.20] represents the HPLC spectra of the output products S(1) to S(7) 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 adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 1000 V and at flow rates in pL / min of the sample / buffer solution / electrode solutions respectively of 10 / 100 / 25.

[0305] [Fig.21] represents the HPLC spectra of the output products S(1) to S(7) of a device comprising an electrophoresis chamber having 5 inlets and 7 outlets, with a thickness of 100 pm, during a test of separation of a mixture of adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMP) carried out at 2000 V and at flow rates in pL / min of the sample / buffer solution / electrode solutions respectively 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, with a thickness of 100 pm, during a test of separation of a mixture of protein and its linker, carried out at 1500 V and at flow rates in pL / min of the sample / buffer solution / electrode solutions respectively of 10 / 80 / 20.

[0307] [Fig.23] represents the HPLC spectra of the products at the output of S(3) of a device comprising an electrophoresis chamber having 5 inlets and 5 outlets, of thickness of 100 pm, during a test of 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 output of S(4) of a device comprising an electrophoresis chamber having 5 inlets and 5 outlets, of thickness of 100 pm, during a test of 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: Materials and methods

[0311] Electrophoresis device

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

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

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

[0315] The KPLE-100-009 chip has a 5-input, 5-output electrophoresis chamber. The inputs and outputs are symmetrical. The inputs are numbered from top to bottom from 1 to 5, i.e., E(1) to E(5) respectively.

[0316] The KPLE-100-008 chip has a 5-input, 7-output electrophoresis chamber. The increased number of outputs improves recovery capabilities. These outputs are also numbered from top to bottom from 1 to 7, respectively S(1) to S(7), i.e., SI 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 inlet flow rate, the recovery of output products, and their analysis, are computer-controlled and automatically recorded.

[0318] [Tables 1] Hollowed out part of the electrophoresis chamber Length Loe x width Lae Electrophoresis plate (separation plate) Length Loe 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 hydrodynamic flow in the device

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

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

[0323] Test 1: Chip KPLE-100-008 of thickness h = 100 pm.

[0324] The KPLE-100-008 chip was connected to flow meters at each inlet 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), HEPES at 10 mM, HPMC at 0.2% (w / v), Tween 20 at 0.1% (w / v), KCl 1.5 M pH at 7.45 adjusted with NaOH Yellow dye 20

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

[0327] Figure 9 is a photograph taken of the KPLE-100-008 device comprising an electrophoresis chamber with a thickness of 100 pm, during the hydrodynamic test. Visualization of the different fluxes was made possible by the yellow coloring of the sample flux and the electrode fluxes.

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

[0329] A yellow band-like area was observed from E(l) to S(l). Another yellow band-like area was observed from E(1) to S(6) and S(7). These two bands represent the paths of the two liquid electrode flows, respectively.

[0330] Tests 2 to 5: Chip with 5 inputs and 7 outputs of thickness h = 1 mm.

[0331] A chip comprising an electrophoresis chamber having 5 inlets, 7 outlets and a thickness h of 1 mm was connected to flow meters at each inlet 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%(w / v) HPMC, 0.1%(w / v) Tween 20, 1.5M KCl pH 7.45 adjusted with NaOH Yellow Dye 1000 800 1000 500 E(2) Buffer Solution Water 10mM HEPES, 1600 3200 4000 2000 HPMC 0.2%(w / v), Tween 20 0.1%(w / v) pH 7.45 Blue dye E(3) Sample Water 160 320 400 400 E(4) Buffer solution Water HEPES 10mM, HPMC 0.2%(w / v), Tween 20 0.1%(w / v) pH 7.45 Blue dye 1600 3200 4000 2000 E(5) Liquid electrode (anode) Water / methanol (60 / 40), HEPES 10mM, HPMC 0.2%(w / v), Tween 20 0.1%(w / v), KCl 1.5M pH 7.45 Adjusted with NaOH Yellow dye 1000 800 1000 500

[0333] Table 3: Operating conditions of test 2

[0334] Fig. 10 is a photograph taken of the device comprising an electrophoresis chamber having 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] Visualization of the path of the different fluxes was made possible by the blue coloring of the buffer solution and the yellow coloring of the electrode fluxes.

[0336] Without the application of an electric field, a nearly straight path for the sample flow from inlet E(3) to outlet S(4), with the outlet facing E(3), was observed. This was due to the blue coloration of the buffer solutions, which contrasts with the absence of coloration in the sample solution. Indeed, the sample flow was bordered on either side by two darker bands representing the paths of the injected buffer solutions from inlet E(2) to outlets S(2) to S(4) and from inlet E(4) to outlets S(5) to S(6).

[0337] A yellow-colored band from E(l) to S(l) and another yellow-colored band from E(l) to S(7) were observed. These two bands represent the paths of the two liquid electrode flows, respectively.

[0338] Test 6: Chip with 5 inputs and 7 outputs of thickness h = 2 mm.

[0339] A chip comprising an electrophoresis chamber, having 5 inlets, 7 outlets, of 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 NaO2 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), HEPES at 10mM, HPMC at 0.2%(w / v), Tween 20 at 0.1%(w / v), 1000 KCL 1.5M pH at 7.45 adjusted with NaO₂ Yellow dye

[0341] Table 4: Operating conditions of test 3

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

[0343] Visualization of the path of the different fluxes is made possible by the blue coloring of the buffer solution and the yellow coloring of the electrode fluxes.

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

[0345] A yellow band-like area was observed from E(1) to outlets S(1) and S(2), and another yellow band-like area was observed from E(5) to outlets S(6) and S(7). These two bands represent the paths of the two liquid electrode flows, 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 apparatus were set up for the separation of a mixture of three fluorescent molecules: fluorescein, rhodamine B, and rhodamine 6G. The chemical structures of the three molecules are shown below.

[0349] [Chem.l] Rhodamine B Rhodamine 6G Mm = 479.01 g / mol (Violet) Mm = 479.01 g / mL (Red) Fluorescein Mm = 332.3 g / mol (Yellow) CîbHsiCÎNîOs CzbHjiCI N 2O3 CioHnOs

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362] These 3 compounds have a similar size, less than a nanometer, and significantly different charges. The zeta potential of each compound was measured beforehand and reported in Table 5. Fluorescein at pH 7.49 exhibited a zeta potential of -23.5 mV, rhodamine B -0.3 mV and rhodamine 6G +36.2 mV. [Table 5] Compound Zeta Potential at pH 7.49 Fluorescein -23.5 mV Rhodamine B -0.3 mV Rhodamine 6G +36.2 mV Table 5: Zeta potential measured at pH 7.49 Test 7 - Chamber with 5 inlets and 5 outlets - h = 100 jim - V = 1500 V The main characteristics of test 7 are as follows: - Room with 5 entrances and 5 exits - h = 100 pm, - V = 1500 V - Flow rate (pl / min): Sample / Buffer / Electrode: 10 / 80 / 20 During this test, the KPLE-100-009 chip, which has 5 inputs and 5 outputs, with an electrophoresis chamber height h of 100 pm, was connected to flow meters at each input. Inputs E(1) and E(5) were each supplied with an electrolytic solution for the anode and cathode respectively. The two electrolytic solutions for the cathode and anode respectively have the same composition. The liquid electrolytic solution has the following composition: HEPES at 100M, HPMC at 0.2% (w / (v), Tween 20 at 0.1% (w / v), 40% methanol, and 1.5M KCl. The flow rate was set at 20 pL / min. These electrolytic solutions had a pH of 7.49 (which was adjusted with a NaOH solution) and a conductivity of 92.33 mS / cm. The electric field is generated by the electrolytic solutions in the electrophoresis chamber, which contain 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 consisted 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 HEPES at 10 mM, HPMC at 0.2%(w / v), Tween 20 at 0.1%(w / v) in water, at a fixed flow rate of 80 pL / min. The buffer solution had a pH of 7.5 (adjusted with a NaOH solution) and a conductivity of 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 exiting the electrophoresis chamber were collected in tubes and analyzed by HPLC to determine 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 products to be separated.

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

[0368] Results

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

[0370] The HPLC spectra of the products exiting at 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 output S(2), that mainly of rhodamine B at output S(3) and that of rhodamine 6G at output S(4).

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

[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 = 2500 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 setup of the inlet solutions was identical to that of test 7, but the separation process of this test differs in the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 10 / 100 / 20 and in 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. Inputs E(1) and E(5) were each supplied with an electrolytic solution for the anode and cathode, respectively. The two electrolytic solutions for the cathode and anode had the same composition. The liquid electrolytic solution had the following composition: HEPES at 10 mM, HPMC at 0.2% (w / v), Tween 20 at 0.1% (w / v), methanol at 40%, and KCl 1.5 M. The flow rate was set at 20 pL / min. These electrolytic solutions had a pH of 7.49 (which was adjusted with a NaOH solution) and a conductivity of 92.33 mS / cm. The electric field is generated by the electrolytic solutions in the electrophoresis chamber, which come 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 HEPES at 10mM, HPMC at 0.2%(w / v), Tween 20 at 0.1%(w / v) in water, at a fixed flow rate of 100 pL / min. The buffer solution had a pH of 7.5 (adjusted with a NaOH solution) and a conductivity of 600 pS / cm.

[0384] The electric field was fixed in this test at 2500 V. After stabilization of the flows, the products exiting the electrophoresis chamber were collected in tubes and analyzed by HPLC to determine the percentage of each compound at each exit.

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

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

[0387] Results

[0388] Figure 14 shows the 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), an outlet facing inlet E(3). Rhodamine 6G migrated towards the liquid anode, with a smaller displacement than fluorescein, and exited at S(5).

[0389] The HPLC spectra of the products exiting at outlets S(2) to S(6) are presented at the [Fig.15]

[0390] The results of the HPLC analysis for test 8 at 2500 V confirm the presence of fluorescein in the S(2) and S(3) outputs, mainly in S(2) output, with no signal from rhodamine 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 recovered at outlets S(2) and S(3). Rhodamine B showed no migration, while rhodamine 6G showed partial migration of approximately 83.49% to outlet S(5).

[0392] Test 9.

[0393] The main features 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, with in particular a height of the electrophoresis chamber h of 1 mm.

[0399] The setup of the input 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] The [Fig. 16] is a photograph taken of the device during electrophoresis.

[0401] Fig. 16 shows in an electrophoresis chamber 1 mm high, an electrophoretic migration of 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 inlet E(3).

[0403] Under the conditions set up in this test, the electrophoretic migrations of fluorescein and rhodamine 6G were less significant compared to previous tests, so that fluorescein arrives between outputs S(3) and S(4) and rhodamine 6G between outputs 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 inputs and 7 outputs, with an electrophoresis chamber height h of 1 mm.

[0411] The setup of the input 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 / 3000 / 50 and by the voltage of the applied electric field, fixed at 3000 V.

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

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

[0414] Under the conditions set up in this test 10, by increasing the voltage of the electric field 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 fluorescein arrives at the outlet S(3) and the main flow of rhodamine 6G arrives at the 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 features of test 11 are as follows: - chamber with 5 inlets and 7 outlets - h = 2 mm V = 2000 V - Flow rate (lp / 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 setup of the inlet solutions was identical to that of test 9, but the separation process of this test 11 differs in the flow rate of the sample, the buffer solution and the electrodes, respectively in pL / min of 20 / 3000 / 50 and in the applied electric field voltage, fixed at 3000 V, conditions identical to test 11.

[0420] The [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 an electrophoresis chamber 2 mm high.

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

[0423] Separation tests of 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 shown below.

[0425] [Chem.2] Adenosine triphosphate Adenosine monophosphate cydiqiie

[0426] Tests 12 to 14 were carried out 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 lists the compositions of the liquid electrodes and the separation buffer solutions and of the sample to be separated that were used.

[0428] [Tableauxô] Compounds Electrode Liquid Separation Buffer Sample HEPES 10 mM 10 mM / HPMC 0.2% 0.2% / Tween 20 0.1% 0.1% / KCl 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 lists the types of solutions introduced at the device inlets 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 entire experimentation after the system had stabilized.

[0435] Three different tests 12, 13 and 14 were carried out. In these tests, the flow rates at the inlets remained unchanged for all 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 outlets S(1) to S(7) were analyzed by HPLC during the tests.

[0438] The HPLC spectra of the outputs S(1) to S(7) at 0V of test 12 are shown 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, there is partial migration of ATP (first peak) towards S(3), while AMP shows no migration towards the different outputs. Approximately 40% of the ATP is diverted towards the S(3) output.

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

[0444] To increase this migration proportion, 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 the sample preparation.

[0445] Example 5: Protein / linker purification

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

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

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

[0449] The applied electric field was 1500 V.

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

[0451] The hydrodynamic monitoring carried out shows 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 the HPLC analysis.

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

[0455] Analyses show that 80% of the linker is diverted to a different output than the majority of the protein, only 10.43% of the protein is diverted to the same output as the linker.

Claims

Demands

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, in which X represents an electrophoresis plate (1) made of inert material, Y represents a sealed plate (2) made of sapphire or 99% alumina (Al2O3), Z represents a cooling plate (3) comprising a heat transfer system (4), and p, an integer from 1 to 100, represents both the number of stages of said device and the number of plates X. Each stage (5) is defined by the following sequence of plates YZYXYZY, in which: - plate X is located between two plates Y, - each of the two plates Z is respectively adjacent to a plate Y, - and each of the two plates Y located at the ends of the sequence YZYXYZY overlaps a plate Z such that each plate Z is located between two plates Y,said device further comprising clamping means for all the plates enabling the sealing of said device, said device being such that each plate X 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 part • in the form of a rectangular parallelepiped with 4 lateral faces (a, b, c, d) and 2 upper and lower faces (e, f), • said hollowed part having length Loe and width Lae, • of height h corresponding to the thickness of the electrophoresis plate X, from 25 µm to 20 mm, in particular from 50 µm to 200 µm or from 1.0 mm to 5.0 mm, • the lateral faces (a, b) being parallel to each other, 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, face (c) being delimited by two edges (Cl, C2) of dimension Loe and 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 along a direction parallel to Al and A2, • m successive exits from S(l), S(2) to S(ml), S(m), m being an integer from 4 to 12, preferably 5 or 7, distributed on face (b) between Bl and B2 and aligned along a direction parallel to Bl and B2, so that S(l) faces E(l) and S(m) faces E(n) along 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 E(n) inputs of each electrophoresis chamber to a micro / millifluidic circuit supplying a liquid anode 1, - supply channels configured to connect at least one of the E(2) to E(nl) inlets 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 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(D), - 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,- to recover, via a recovery circuit, the purified and / or separated product contained in said initial solution at one of the outlets S(2) to S(ml) of each electrophoresis chamber.

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 at least one buffer solution.

3. A device according to any one of claims 1 to 2, wherein p equals 1 and i equals 1, comprising a single electrophoresis plate comprising a single electrophoresis chamber, in particular of height h from 25 to 200 pm or from 1.0 to 5.0 mm.

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

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

6. Device according to any 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. A device according to any 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 hollowed portion and / or wherein the width Lae of the hollowed portion of the electrophoresis chamber is from 1.0 to 8.0 cm, preferably from 1.0 to 5.0 cm, and / or wherein the length Loe of the hollowed portion of the electrophoresis chamber is from 5.0 to 20.0 cm, preferably from 5.0 to 15.0

8. VIII. Device according to any one of claims 1 to 7, wherein the X-ray electrophoresis plates are made of material selected from polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA) and fluoroethylene propylene (FEP), in particular Teflon™, Teflon™-PFA and Teflon™-FEP plates.

9. Device according to any one of claims 1 to 8, wherein in each electrophoresis plate X a portion of said plate is configured to accommodate a fluidic circuit of supply and recovery channels, which is partially or totally etched, cut or drilled into the plate X.

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

11. Device according to any one of claims 1 to 10, wherein the electrophoresis chambers comprise channeling means leading to the inlets and / or outlets, preferably engraved in the electrophoresis plate X.

12. A device according to any one of claims 1 to 11, wherein each electrophoresis chamber is configured to contain at least one selectively permeable membrane, preferably size-selective, positioned so as to be traversed by the solution containing the product to be separated or purified during operation of the device, and / or wherein the upper and / or lower face of each of the electrophoresis chambers comprises protrusions 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 enhance heat transfer and to maintain the electrophoresis chambers at a selected temperature, in particular wherein said protrusions are made of thermally conductive materials, preferably sapphire or 99% a-Al2O3 alumina.

13. A device according to any one of claims 1 to 12, wherein said heat transfer system (4) is a piping network configured to allow the circulation of one or more heat transfer fluids, said network being 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 temperature control in said electrophoresis chambers, in particular wherein said piping network of the heat transfer system is configured to generate a temperature gradient in each of the electrophoresis chambers, preferably wherein said piping network of the heat transfer system comprises, for each of the electrophoresis chambers, parallel channels to the edge Cl, said channels being able to contain heat transfer fluids of different temperatures in order to generate said temperature gradient.

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

15. A method for purifying and / or separating, by free-flow electrophoresis, a product contained in a solution by implementing a microcell electrophoresis device according to any 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) - connect the cooling systems to a cooling circuit, controlled by a central unit (UC2) - generate an electric field along the Al edges, via the liquid anode and liquid cathode, - 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(D, • 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. A 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 operating 300 days / year.

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

18. A method according to any one of claims 15 to 17, wherein 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, 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, and / or wherein said fluidic 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 50,000 pL / min.

19. A method according to any 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 fluid circulation in each of the electrophoresis chambers, and / or wherein during fluid circulation in each of the electrophoresis chambers, a pH variation along the Al edge in each electrophoresis chamber is generated, using at least two buffers of different pH, and / or wherein during fluid circulation in each of the electrophoresis chambers, a temperature gradient along The Al edge of the hollowed-out part is applied in each electrophoresis chamber.

20. A method according to any one of claims 15 to 19, wherein during 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 fluid circulation in each of the electrophoresis chambers, the generated electric field is from 200V to 4000V.

21. A method according to any one of claims 15 to 20, implemented to purify and / or separate a protein or implemented to purify and / or separate isomers, in particular enantiomers.

22. Method of developing an industrial device for the purification and / or separation of 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 having a height h of the vent part of 25 to 200 pm or 1.0 to 2.0 mm, to determine r and optimize the fluidic 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 from 10 to 100 electrophoresis chambers.

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