Flow cell for electrochemically-modified liquid affinity chromatography in BIO-nanotechnology

Abstract

The present invention relates to a flow cell for electrically controllable ad- and desorption of molecules from and into a liquid. Particularly, the present invention relates to a flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising (a)two electrodes comprising a conductive surface, (b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes, (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

Description

[0001] New International patent application

[0002] Applicant: Technische Universitaet Muenchen

[0003] Our ref: TUM18337PCT

[0004] FLOW CELL FOR ELECTROCHEMICALLY-MODIFIED LIQUID AFFINITY CHROMATOGRAPHY IN BIO-NANOTECHNOLOGY

[0005] TECHNICAL FIELD OF THE INVENTION

[0006] [1] The present invention relates to a flow cell for electrically controllable ad- and desorption of molecules from and into a liquid, said flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising (a) two electrodes comprising a conductive surface, (b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes, (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

[0007] BACKGROUND

[0008] [2] Bio-nanotechnology has grown into a rapidly emerging research field. This discipline combines innovative techniques and new materials with phenomena occurring in nature and proves to be useful in multiple applications. For example, interaction between biomolecules and inorganic nanoparticles can be utilized in such applications as magnetic resonance imaging, drug delivery, nanomedicine for implant coating as well as energy storage. Also, the transfer of electrons between the nanomaterials and the biomolecules can be used for biomolecule detection for e.g. antibodies or pathogens. However, it is very difficult to specifically address processes in nanoscale. In this regard, it is very much of interest to control and manipulate the bio-nano interface in order to improve applications in all these fields.

[0009] [3] For example, it is known to extract directly from crude cell lysates a target biomolecule material by magnetic separation. The method includes modifying the target biomolecule material so as to enhance its affinity to magnetic nanoparticles and then combining the same with the magnetic nanoparticles so that the target biomolecule material may be magnetically manipulated. The target biomolecule material may thereafter be released from the magnetic nanoparticles in an elution buffer with certain pH value. Magnetic one-step purification of His-tagged Proteins by bare iron oxide Nanoparticles has been described in the art (Schwaminger et al. 2019, ACS Omega, 4, 3790-3799). Flow cells involving magnetic separation techniques have been described in the art. However, conventional magnetic separation of biomolecules described in the above non-patent literature requires the use of buffer / pH switch, which adds further costs. Furthermore, it could be timeconsuming as the diffusion takes some time and the elution is not completed before an equilibrium is reached. The buffer-switch approach may also cause aggregation or degeneration of biomolecules.

[0010] [4] Hence, in view of the great potential, there is a constant need for developing improved means for separating, isolating or purifying various types of (bio)molecules in the bio-nanotechnology context.

[0011] [5] The technical problem therefore is to comply with this need.

[0012] SUMMARY OF THE INVENTION

[0013] [6] The technical problem is solved by the subject-matter as defined in the claims. The present invention provides an innovative approach for biomolecule recognition, separation and purification that can be applied for bio-nanotechnology. In particular, the present invention is based on revised, electrically controllable affinity chromatography for purifying various types of (bio)molecules, especially by providing means with greater functionality and / or free from known problems and limitations, e.g., as outlined above, and / or decreasing the costs of such means and methods. In some aspects, the present invention relates to a flow cell for electrically controllable ad- and desorption of molecules from and into a liquid, said flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising

[0014] (a) two electrodes comprising a conductive surface,

[0015] (b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes,

[0016] (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

[0017] [7] It is envisaged that said electrodes of said flow cell form a capacitor. It is further envisaged that said stationary phase acts as a dielectric or insulator between the electrodes. It is also envisaged that each of said electrodes comprises an insulating layer between said electrode and said stationary phase. It is further envisaged that said electrodes are configured to allow flow of a liquid. It is also envisaged that the fluid path is in transverse direction and / or in longitudinal direction in relation to said stationary phase being located between said electrodes. Said voltage may be a DC voltage. Said voltage may be from a constant voltage source, preferably an adjustable constant voltage source. It is also envisaged that said controller is a voltage source which does not react on changing current. It is further envisaged that said electrode is not formed by an electrically conductive coating and / or metallic net which is directly formed and / or disposed on said stationary phase.

[0018] [8] It is particularly envisaged for said flow cell that said stationary phase is porous. Said stationary phase may be a membrane, a polymer, a molecularly imprinted polymer, a non-metal material, a metal-organic framework, a molecular sieve, a chromatography resin, a fluidized bed system, foam or felt. Said stationary phase may be composed of zeolites, monoliths or capillaries and wires. It is further envisaged that said stationary phase comprises at least two stationary phases. It is also envisaged that said at least two stationary phases are stacked, wrapped or tortuous. Further, said at least two stationary phases may be located between two electrodes. It is further envisaged that each stationary phase is located between a separate set of two electrodes. It is also envisaged that said stationary phase is capable of adsorbing said molecules. It is also envisaged that said stationary phase is capable of adsorbing said molecules when the voltage is changed between said electrodes. It is further envisaged that said stationary phase is capable of adsorbing said molecules through a ligand immobilized on said stationary phase.

[0019] [9] For said flow cell it is also envisaged that said ligand is capable of non-covalently binding said molecules. Said ligand may be capable of non-covalently binding said molecules when the voltage is changed between said electrodes. It is further envisaged that said stationary phase comprises molecules already adsorbed to said stationary phase or said ligand immobilized on said stationary phase. Said molecules may be biomolecules, chemical molecules, viruses, virus-like particles, extracellular vesicles, cells (prokaryotic or eukaryotic). Said biomolecule may be a protein or a nucleic acid, such as DNA or RNA. Said chemical molecules may be small molecules or micro plastic.

[0020]

[0010] In some aspects the present invention also relates to the use of said flow cell for purifying, separating, selecting an / or isolating molecules or chemical entities from a liquid and / or for electrically controllable affinity chromatography.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

[0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, respectively.

[0023]

[0012] Figure 1 shows a possible schematic set-up of the components of the device in the volume of the chamber comprised by the flow cell of the present invention (left) and exemplarily stationary phases applicable for said flow cell (right).

[0024]

[0013] Figure 2 shows a schematic illustration of a set-up for controlled crystallization.

[0025]

[0014] Figure 3 shows the pH shift due to crystallization of calcium oxalate particles in an electric field (32 mV, 2 mm distance) over 30 minutes.

[0026]

[0015] Figure 4: Schematic representation of the affinity precipitation of IgG antibodies in a tangential flow electromodulable cell. Precipitation can occur on ligands coupled to an interface (A) or freely present in solution (B). The antibodies are resolubilized (desorbed) from the stationary phase by applying a voltage change. Due to the constant flow, they are then washed out of the flow cell.

[0027]

[0016] Figure 5. Illustration of a modular system for potential-controlled adsorption and desorption. The electrodes are connected to the positive pole (+) and negative pole (-) of a voltage source. The contact rings inside the module are connected via a waterproof connection. This prevents liquid from escaping and enables electrical conduction from the outside to the inside. Inserts for different flow patterns (arrows: axial, radial, and tangential), stationary phases, and electrode geometries can be installed in the holder. This design also allows a dielectric to be placed between the electrode and the liquid so that there is no direct contact between the electrode and the mobile phase.

[0028]

[0017] Figure 6: Potential-controlled desorption of IgG after surface precipitation on Protein A membranes. Representation of the LIV / VIS signal (280 nm) and the pH value over the pumped volume. Both runs were performed at a flow rate of 1.5 mL min-1. Pure IgG (Cutaquig®, Octapharma) was applied in excess to a Sartobind Lab A membrane (Sartorius) as the reactive surface. After the washing step, the antibodies were desorbed from the membrane by applying an electrical potential of 2.5 V (A) or using an acidic pH of 3.5 (B).

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030]

[0018] The present invention is described in detail in the following and is also illustrated by the appended examples and figures.

[0031]

[0019] The use of high-quality, pure and intact biomolecules such as proteins and nucleic acids is a crucial prerequisite for their successful application in the medical and scientific context. Methods such as chromatography, electrophoresis, filtration and / or centrifugation are generally applied for isolating, separating and / or purifying such molecules. Each of said methods relies on certain physicochemical properties of the processed biomolecule.

[0032]

[0020] Affinity chromatography (AC) is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner, i.e. it uses a biologically-related agent as a stationary phase to specifically bind the biomolecule of interest. This technique has been used for decades for the isolation and purification of specific targets by taking advantage of the selective and reversible binding which occurs in many biological interactions (Rodriguez et al. 2020, J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 10; 1157: 122332). New alternatives on the market are membrane adsorbers or so-called membrane chromatography (MC). Pore-based mass transfer limitations are also associated with AC-based methods. However, both chromatography materials and membrane adsorbers are consumables in downstream processing and essentially determine the process costs. Depending on the functionalization, the costs differ greatly. Materials with the affinity ligands can dramatically increase the cost of a process. Due to the high selectivity of AC, high yields of 99% and purities above 95% can be achieved. At the same time, however, due to the strong interaction between e.g. protein A and a mAb, desorption / elution must be performed at low pH values, e.g., pH values of < 3.5. Another known problem is the leakage of the protein A ligand, which occurs when the column is loaded by host cell proteases and when the material is regenerated and cleaned with alkali. This circumstance and also the continuous fouling of the material - especially due to the porous structure - leads to a limited lifetime of the already expensive material.

[0033]

[0021] In sum, for the purification of biomolecules such as DNA and RNA molecules, amino acids, oligopeptides, polypeptides, monosaccharides, oligosaccharides, polysaccharides, fats, fatty acids and lipids, but also other types of molecules by affinity chromatography care must be taken to select binding agents, supports, immobilization schemes and elution conditions that allow both effective dissociation of retained targets and good regeneration of the stationary phase without permanently damaging the binding agents / materials.

[0034]

[0022] The present invention takes these necessities into account and relates to novel and improved means for separating biomolecules from a liquid utilizing liquid affinity chromatography. Particularly, the present invention provides for a novel flow cell for electrically controlled ad- and desorption of molecules from and into a liquid that can be used for purifying, separating, selecting and / or isolating molecules (e.g. biomolecules) or chemical entities. In this respect the provided flow cell allows for the purification of molecules in an specific, simplified and cost-reduced manner by utilizing potential- controlled affinity membrane chromatography techniques that can also be used in the nanobiotechnology context.

[0035]

[0023] Specifically, in contrast to classical AC, when using the flow cell of the present invention, desorption or recovery of adsorbed (bio)molecules is achieved by applying an electrical potential, preferably at mild pH conditions. In particular, the present invention relates to the use of local electric fields to suppress specific electrostatic binding and to break the bonds between the stationary phase and bound (bio)molecule so that the (bio)molecule is desorbed again. This has the surprising advantage that e.g. antibodies aggregate to a lesser extent and the yield of purified biologically active antibodies is increased when using the flow cell of the present invention after purification. A further surprising advantage when using the means provided by the present invention lies in that a ligand immobilized on the stationary phase, e.g. protein A, undergoes a slower denaturation than it does under acidic conditions according to standard AC-based methods and the membranes are available to a higher number of work-up cycles than conventional affinity matrices. Accordingly, the flow cell of the present invention allows for a greater functionality of AC, e.g. without the use of specific buffer solutions, pH shifts and / or salts, making it thus possible to release (bio)molecules in an electrically- induced manner and / or concentrate them without extended desorption kinetics.

[0036]

[0024] Further, the flow cell provided by the present invention not only allows for an efficient enrichment and desorption of (bio)molecules, but also for voltage-induced chemical reactions, such as enzymatic reactions or electrochemical polymerization, crystallization or precipitation of molecules on said stationary phase. In addition, based on the applied electrically controlled ad- and desorption of the molecules, several cycles can be run one after the other so that in relation to the active area of the stationary phase a very effective enrichment is achieved

[0037]

[0025] Thus, the flow cell of the present invention that can be applied for the purification, separation, selection and / or isolation of various (bio)molecules is distinguished from established systems by at least the following novel / improved features / functionalities:

[0038] Focused recovery of the (bio)molecules by potential switch;

[0039] Mild conditions with reduced use or no use of buffer solutions, no salt or pH shifts;

[0040] Possible elution of target molecules in very small volumes;

[0041] Increase of dynamic capacity by additional electrical potentials;

[0042] Multiple use of the chromatographic / filter unit, as the ligand / material is less degenerated.

[0043]

[0026] In some aspects, the present invention relates to a flow cell for electrically controllable ad- and desorption of molecules from and into a liquid, said flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising

[0044] (a) two electrodes comprising a conductive surface,

[0045] (b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes,

[0046] (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

[0047]

[0027] As used herein, a “flow cell” is not particularly limited in terms of its shape, dimension or material as long as the chamber, the inlet and the outlet thereof are configured to allow an electrically controllable ad- and desorption of molecules from and into a liquid. For example, the flow cell may be a structure that allows the separation of the molecules continuously or a structure that allows the separation of the molecules batch-wise. The flow cell may also be a structure for microscopic treatment or a structure for a treatment of a much larger scale. In particular, the flow cell of the present invention is configured to allow a electrically controllable ad- and desorption of molecules from an into a liquid by means of liquid affinity chromatography. Further, the flow cell is configured to comprise a device comprising two electrodes comprising a conductive surface, a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes, and a controller functionally associated with a power supply configured to apply and change a voltage between said electrodes. In this respect the flow cell allows a flow of a liquid or solution and to apply a voltage between the two electrodes, wherein changing the voltage between said electrodes changes the affinity of the molecules to said stationary phase and results in a desorption or adsorption of said molecules from or to said stationary phase.

[0048]

[0028] The flow cell of the present invention may have a housing into which the flow cell is placed. Accordingly, the housing can determine the shape of the flow cell as described elsewhere herein. Moreover, the housing may help to safely hold the flow cell in a stable position to allow the intended separation or purification process. Thus, the flow cell may be fixed in said housing e.g. by suspensions or is embedded in this housing. In some embodiments the housing may comprise more than one flow cell, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more flow cells that are hold or fixed inside said housing and are used in parallel or consecutively for the intended separation or purification process.

[0049]

[0029] The flow cell of the present invention may comprise additional functional units In some embodiments, the flow cell may further comprise or is connected with a supply unit that is in fluid communication with the inlet of the chamber for supplying the liquid comprising the molecule(s) to be separated, purified and / or isolated from said fluid to the chamber. Pre-treatments of the (bio)molecules in the liquid may be carried out in the supply unit, if this is deemed necessary in some embodiments. In some embodiments the flow cell may further comprise a collecting unit in fluid communication with the outlet of the chamber for collecting the purified or separated (bio)molecules, allowing the (bio)molecules to be further treated.

[0050]

[0030] Thus, the flow cell of the present invention is preferably a modular flow cell, i.e. comprises more than one component (see Figure 5).

[0051]

[0031] In some embodiments the flow cell of the present invention is a microfluidic chip, which allows a precise control and manipulation of fluid in a small scale (e.g. sub-millimeter to millimeter). Preferably, a volume of the chamber of the microfluidic chip ranges from 1 pm3to 1 ,000 mm3, more preferably 1 pm3to 100 mm3, and even more preferably 1 pm3to 10 mm3.

[0052]

[0032] In some embodiments the volume of the chamber of the flow cell is much larger and may range from 1 ,000 mm3to 10 m3, more preferably 0.01 to 100 L, and even more preferably 0.1 to 10 L.

[0053]

[0033] The chamber of the flow cell that comprises an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid along said fluid path may be configured in a manner that the liquid is at least temporarily held within the volume of the chamber to allow adsorption of the molecules to the stationary phase. It is thus preferred that the volume for containing the liquid along said fluid path covers a full cross section of the inner volume of the chamber along substantially the length of the stationary phase. Preferably, the volume for containing the liquid medium along said fluid path coincides with the inner volume of the chamber. For avoidance of doubt, the electrodes are partially, preferably fully, arranged within the fluid path, that is, the electrodes are partially, preferably fully, immersed in the liquid flowing through the flow path. However, the volume for containing the liquid medium along said fluid path may also be smaller than and encompassed by the inner volume of the chamber. It is preferred that the volume for containing the liquid along said fluid path covers a full cross section of the inner volume of the chamber along substantially the length of the collecting area / stationary phase. Before the electrically controlled ad- and desorption of the molecules from an into the liquid starts, i.e. the affinity chromatographic process begins, the liquid comprising the molecules to be separated / purified is introduced into the chamber via the inlet followed by an outflow of the liquid via the outlet. Thus, the chamber of the flow cell may be constructed to allow a flow of the liquid along said fluid path. Preferably, said flow is a continuous flow, i.e. the liquid flows continuously trough said flow cell.

[0054]

[0034] Thus, the flow cell may allow that the liquid travels through the chamber, i.e. the molecules or components comprised in the liquid flow constantly through the chamber, thereby ad- and desorbing from and into the liquid in an electrically controlled manner. Thus, in the context of the present invention it is envisaged that the electrodes are configured to allow flow of a liquid. The liquid may also be referred to as mobile phase that passes the fluid path between the inlet and the outlet of the chamber. The term “mobile phase” as used herein may refer to a phase that retains the analyte (e.g. the molecules to be analyzed or the mixture that needs to be separated) during the separation process performed by the flow cell of the present invention. The mobile phase thereby moves in a definite direction in the chromatographic system, i.e. the inlet comprised by the chamber of the flow cell of the present invention is for introducing the fluid and the outlet is for extracting the fluid. Furthermore, it is ensured that any fluid flow from the inlet to the outlet necessitates passing through and / or over the stationary phase described elsewhere herein.

[0055]

[0035] With the means of the present invention, e.g. for the purification of monoclonal antibodies, even at low flow rates in the range of a few mL / (bar*min*cm2filter area) a much faster loading in the order to 100 mg per mL bed volume is possible. Suitable adjustments of the flow rate may be made in view of the respective molecules comprised in said liquid and the desired result of the processes.

[0056]

[0036] In some embodiments, the fluid path is in transverse direction and / or in longitudinal direction in relation to said stationary phase being located between said electrodes. The term “transverse direction” (including radial direction and tangential direction) as used herein may refer to a perpendicular orientation or movement relative to a specified reference point or axis, in particular in relation to said stationary phase being located between said electrodes. The term “longitudinal direction” as used herein may refer to the primary or main orientation or movement along a specified reference point or axis (axial direction), in particular in relation to said stationary phase being located between said electrodes.

[0057]

[0037] The fluid is preferably selected to retain the (bio)molecules or substances in their native state. Preferably, the fluid may be a suitable aqueous solution or buffer, such as a wash solution or a recovery solution as defined elsewhere herein. In some embodiments, the liquid may comprise human / bodily fluids, cell lysates or other supernatant of a cell culture medium. Thus, in some embodiments the liquid may be a “crude cell culture medium” referring to a basic or unrefined liquid mixture that contains essential nutrients, growth factors, and other components necessary to support the growth and maintenance of cells in culture. In come other embodiments the liquid may be a “clarified cell culture medium” referring to a cell culture medium that has undergone a process to remove particulate matter, debris, cells, or other insoluble components, resulting in a clear and transparent solution. This clarification step is often performed to improve the quality of the medium and create a homogeneous environment for cell growth and experimentation. The process typically involves techniques such as centrifugation, filtration, and / or sedimentation, which help separate solid or insoluble materials from the liquid medium. In this respect the “clarified cell culture medium” may be the “supernatant of the cell culture medium”, i.e. refers to the clear liquid portion that remains on top after a mixture of cells and medium has been subjected to a centrifugation or settling process.

[0058]

[0038] In the context of the present invention, the liquid or mobile phase may also be a liquid media or buffer in which biomolecules such as proteins and nucleic acids are stored or stabled. Typical buffers that may be used as liquids in the context of the flow cell of the present invention comprise phosphate-buffered saline (PBS) or Tris-buffered saline (TBS).

[0059]

[0039] The term “stationary phase” as used herein indicates a physical entity which may be brought into contact with the liquid comprising the biomolecules and on which the biomolecules adsorb based on voltage changes as described elsewhere herein. Thus, the “stationary phase” may refers to the “immobile phase” or substance that retains the analyte (e.g., the substance being analyzed or the mixture that needs to be separated) during the separation process. As mixture passes through and / or over this stationary phase, different compounds or molecules (e.g., chemical entities) interact differently with the stationary phase, leading to their separation based on their respective affinities or retention times. In the context of the present invention, the stationary phase may also be referred to as “collecting area”, i.e. is a space in the chamber in which the biomolecules will be collected and from which the biomolecules will be subsequently released when the flow cell is used for desorbing the biomolecules from said stationary phase. Thus, the term “collecting area” indicates a physical entity which may be brought into contact with the liquid or liquid medium comprising the biomolecules and on which the biomolecules bind with the application of voltage changes. When applying a further voltage change, the biomolecules can again be released from the collecting area.

[0060]

[0040] The present invention may be implemented in various systems with different environments. Accordingly, the stationary phase is not limited to any particular size, shape or material as long as the functionalities described herein may be satisfied. As described elsewhere herein, the stationary phase is disposed in the chamber. The term “disposed in the chamber” covers both a configuration where the stationary phase is a component separate and independent from and disposed in the chamber and a configuration where the stationary phase is a part of the chamber.

[0061]

[0041] In the context of the present invention the stationary phase is not in direct contact with the electrodes. The term “not in direct contact” as used herein means that that the stationary phase and the two electrodes do not touch each other. Rather there is a certain space or distance between the stationary phase and the two electrodes. In particular, it is envisaged within the context of the present invention that said electrodes are not formed by an electrically conductive coating and / or metallic net which is directly formed and / or disposed on said stationary phase.

[0062]

[0042] It is further envisaged that the stationary phase can act as a dielectric or insulator between the electrodes. The term “dielectric” as used herein may refer to a material that can store and transmit electrical energy in the form of an electric field without allowing significant electric current to flow through it. Dielectrics are insulating materials that possess the ability to polarize when subjected to an external electric field. This polarization involves the displacement of electric charges within the material, resulting in the buildup of positive and negative charges on opposite sides. Dielectrics are characterized by their electrical properties, including their ability to resist the flow of electric current (high electrical resistance) while still allowing the transmission of electric fields. The term “insulator” as used herein may refer to a material that does not allow the easy flow of electric current through it. It possesses high electrical resistance, meaning that it restricts the movement of electric charges, such as electrons. Insulators are characterized by their ability to resist the passage of electricity and prevent the transfer of electrical energy. Common examples of insulating materials include rubber, plastic, glass, ceramic, and most non-metallic materials. Thus, in preferred embodiments, the flow cell comprises a dielectric. Specifically, the flow cell may preferably comprise a dielectric that prevents direct contact between one or both electrodes and the mobile phase.

[0063]

[0043] It is further envisaged that the flow cell of the present invention comprises one or more inline sensors. The term “in-line sensor” refers to a sensor placed directly in the process stream or system to perform a measurement or inspection. In-line sensors integrated, e.g. using electrochemical impedance spectroscopy or cyclic voltammetry, in the system can be used for the analytical measurement of potential-controlled precipitation, resolubilization, and crystallization processes described herein in order to simultaneously generate further product- and processrelevant information. The sensors also enable adaptive process control. A feedback mechanism creates an adaptive system that is capable of dynamically adjusting to the actual process behavior. The modular design of the system allows it to be used as a reusable, CIP-capable system or as a disposable module. Scaling from nano to industrial scale is also possible. In some embodiments, the in-line sensor applied in the context of the invention is an optical sensor. In some embodiments, the in-line sensor is a sensor for voltammetric measurements.

[0064]

[0044] The nature and composition of the stationary phase may vary depending on the type of chromatography and the intended application, but it typically remains fixed in its position, contrasting with the mobile phase that moves or flows through and / or over it. Suitable stationary phases are known in the art and could include, but are not limited to, stationary phases made from materials such as gels, resins, and / or glass fibers. Moreover, they can also be derived from any suitable porous substrate(s), such as membranes, specifically polymeric membranes, serving as the foundational material for the stationary phase. Preferably, the stationary phase is characterized by porosity and / or permeability. The terms “Porosity” or “porous” as used herein mean that the stationary phase consists of a solid matrix with defined holes or pores which have diameters ranging from less than 2 nm to more than 20 pm. However, also nonporous systems are applicable as stationary phase in the context of the present invention. The term “permeability” refers to the ability of the stationary phase to transmit fluids. The separation of solutes by porous membranes is mainly a function of molecular size and membrane pore size distribution. In particular, the stationary phase is permeable, in particular to fluids, e.g., water and / or alcohol. Thus, the stationary phase preferably allows the passage of fluids like water and / or alcohol. Specifically, the stationary phase may be a membrane, a polymer, as molecularly imprinting polymer, a non-metal material, a metal-organic framework, a molecular sieve, a chromatography resin, a fluidized bed system, foam or felt. Further, the stationary phase may be composed of zeolites, monoliths or capillaries and wires.

[0065]

[0045] The term “electrically controllable” as used herein may refer the ability of the flow cell to manipulated, adjusted, and / or regulated the ad- and desorption of molecules from and into a liquid and the ad- and desorption of molecules to and from a stationary phase using an electric signal or voltage. Thus, the flow cell is based on “electrically controllable” Affinity Chromatography using “electrochemically-modified" means and methods relating to Affinity Chromatography, in particular Liquid Affinity Chromatography. Thus, the flow cell of the present invention specifically utilizes “digital” (i.e. “electrically controllable” and / or “electrochemically-modified") Liquid Affinity Chromatography for ad- and desorbing molecules from and into a liquid, thereby separating, selecting, purifying and / or isolating said molecules.

[0066]

[0046] The term “affinity chromatography” as used herein may refer to a type of liquid chromatography that for example uses a biologically-related agent (e.g., chemical entity or biomolecule, e.g., immobilized chemical entity or biomolecule) as a stationary phase to specifically bind the molecule of interest. Affinity chromatography exploits the specific interaction between two molecules or chemical entities, for example, an enzyme and its substrate, an antibody and its antigen, or a receptor and its ligand. The term “liquid chromatography” as used herein may refer to a separation technique used, for example, in analytical chemistry and biochemistry, to separate and / or analyze components of a mixture based on their interactions with a liquid mobile phase and a stationary phase.

[0067]

[0047] “Adsorption” means that that molecules initially dissolved in the liquid are adsorbed to the stationary phase. “Desorption” means that said molecules are again desorbed from the stationary phase and returned into a liquid (resolubilization). Specifically, changing the voltage between the electrodes comprised by the device comprised by the flow cell of the present invention can change the affinity of the molecules so that to adsorb said molecules to the stationary phase. Similarly, changing the voltage between the electrodes comprised by the device comprised by the flow cell of the present invention can also changes the affinity of the molecules so that to desorb said molecules from the stationary phase. “Changing the affinity” means that the affinity between the molecule and the stationary phase is modified.

[0068]

[0048] The term “affinity” may describe a natural attraction or force between entities or substances that causes them to combine or interact. When referring to "affinity of chemical entities," especially in a biochemical or molecular context, it may denote the measure of the attraction or binding strength between two molecules or chemical entities, such as a ligand and its receptor, an enzyme and its substrate, or an antibody and its antigen. This affinity is crucial for determining the specificity and efficacy of biological interactions and can be influenced by various factors, including the molecular structure, charge, and environmental conditions. The higher the affinity, the stronger the attraction or binding force between the two entities. Thus, in the context of the present invention, “changing the affinity” means that the binding strength of the molecules to the stationary phase is changed by changing the voltage between the two electrodes so that the molecules adsorb to or desorb from the stationary phase.

[0069]

[0049] For example, a first change in the voltage between said electrodes comprised by the flow cell of the present invention may change the affinity of said molecules to said stationary phase so that the affinity to said stationary phase is increased, i.e. the increased affinity leads to an increased binding of said molecules to said stationary phase. Accordingly, said molecules are desorbed from said liquid and adsorbed to said stationary phase. Then, a further change in the voltage between said electrodes may again decrease the affinity of said molecules to said stationary phase so that the molecules are desorbed from said stationary phase and adsorbed into the liquid. In this respect, the (bio)molecule’s adsorption to and desorption from the stationary phase is controlled by voltage changes. Thus, by applying an electric potential, binding and release of the (bio)molecules to and from the stationary phase can be controlled by electrical charges.

[0070]

[0050] The term “voltage” often denoted as Vas used herein may refer to the measure of electrical potential difference between two points in an electrical circuit. It represents the energy (per unit charge) required to move an electric charge between those two points. The unit of voltage is the volt (V), which is defined in the International System of Units (SI) as one joule per coulomb. In some embodiments the voltage is a “direct current voltage”, often abbreviated as “DC voltage”. “DC voltage" may refer to a type of electrical voltage that remains constant in polarity and magnitude over time. In some embodiments the voltage can be from a constant voltage source, preferably an adjustable constant voltage source. The term “constant voltage source” as used herein may refer to an electrical device or circuit that generates a steady and unchanging voltage output, regardless of changes in the connected load or other external factors. The term “adjustable constant voltage source” as used herein may refer to an electrical device or circuit that provides a stable and unchanging voltage output that can be adjusted (e.g., manually) to different levels. “Changing the voltage” means that the voltage between the two electrodes is alter, e.g. decreased or increased, which leads to a variation in the flow of the electric charge over time in an electrical circuit, specifically a variation in the flow of the electric charge between the two electrodes. Thus, the term “changing the voltage between said electrodes” as used herein may refer to the adjustment or modification of the electrical potential difference between two specific electrodes in a given setup or device comprised by the flow cell of the present invention.

[0051] Preferably, the voltage applied is in the range from -100 V to + 100 V, more preferably from -50 V to +50 V. Stronger potentials may lead to oxidation of the (bio)molecules as well as the formation of hydrogen, which might endanger the system.

[0071]

[0052] In the context of the present invention, the voltage between the two electrodes is changed by a controller functionally associated with a power supply configured for applying and changing the voltage between said electrodes. In some embodiments said controller is a voltage source which does not react on changing current. The term “changing current” as used herein may refer to a fluctuation or variation in the flow of electric charge (current) over time in an electrical circuit. Accordingly, the controller is a voltage source that tolerates or is resistant to said fluctuations or variations in the current.

[0072]

[0053] In further aspects, by elevating the voltage applied to a specific level, repulsive forces can be generated, particularly through the electric field brought forth by the applied voltage. This electric field has the potential to alter the overall interaction force between the biomolecules and the stationary phase, especially the binding sites, transitioning it from attractive to repulsive. These effects are contingent upon minor shifts in the biomolecules' conformations, specifically secondary, tertiary, or quaternary structures (e.g., in proteins). These shifts result in the loss of complementary features between at least one biomolecule and / or binding site due to their interaction with the induced or established electric field from the applied voltage. In further aspects, these subtle conformational changes generally do not lead to denaturation, aggregation, or similar modifications of the biomolecules. Additionally, these conformational changes tend to be transient, existing only temporarily and being easily reversed. This reversal typically occurs automatically as the biomolecules move away from the influence of the electromagnetic or electric field. This can involve actions like removing the eluate from the field and / or the stationary phase.

[0073]

[0054] As described above, utilizing voltage for elution circumvents the necessity for traditional elution methods involving pH adjustments or the use of concentrated salt solutions, both of which impose relatively harsh conditions on the biomolecules that can potentially cause denaturation.

[0074]

[0055] The term “electrodes” as used herein may refer to conductive materials that are used to establish an electrical connection with a nonmetallic part of a circuit. Essentially, they may act as the interface between electrical circuits and ionic conductors (like electrolytes in a solution). There are generally two electrodes in an electrochemical cell: Anode, which is the electrode where oxidation (loss of electrons) occurs. In a galvanic or voltaic cell (like batteries), it is the negative electrode. However, in an electrolytic cell (used for electrolysis), it is the positive electrode. Cathode, which is the electrode where reduction (gain of electrons) occurs. In a galvanic or voltaic cell, it's the positive electrode. In an electrolytic cell, it's the negative electrode. The choice of electrode material may be crucial and can vary based on the application. Some common electrode materials include metals like platinum, gold, and silver, as well as carbon in various forms (e.g., graphite rods or carbon paste).

[0056] According to the flow cell of the present invention, said electrodes are envisaged to be configured to allow flow of a liquid. The term “electrode is configured to allow flow of a liquid” as used herein may indicate that the electrode has been designed or set up in a manner that permits the movement or passage of a liquid solution. In this context, the electrode's structure or arrangement is such that the solution can move across or through it. Further, the electrode is arranged and configured to generate an electric field at the collecting area.

[0075]

[0057] In some embodiments the electrode is positioned proximal to the volume of the chamber. As used herein, the expression “proximal to the volume” indicates that the element concerned, i.e. the electrode, is disposed within a sufficiently close distance to the volume so that a electric field sufficient for using the flow cell of the present invention and adsorbing the (bio)molecules at the collecting area may be established. Therefore, this expression encompasses a configuration where the element concerned is not in direct contact with, but is adjacent to, the volume and also a configuration where the element concerned is in direct contact with the volume. For example, the electrode may be disposed in the chamber. In such a scenario, the electrode is in direct contact with the volume of the chamber.

[0076]

[0058] It is particularly envisaged that said electrodes of said flow cell form a capacitor. The term “capacitor” as used herein may refer to an electronic component designed to store and release electrical energy in an electrical circuit. For example, it may consist of two conductive plates separated by an insulating material called a dielectric as described elsewhere herein. The electrodes or conductive plates are made of conductive material that allows electric current to flow through it. Conductive materials have a high density of electric charge carriers (usually electrons) that are free to move, facilitating the transfer of electric charge from one point to another. Conductive surfaces are characterized by their low electrical resistance. Metals, such as copper and aluminum, are some of the most common conductive materials due to their abundance of free electrons that can move easily within their crystalline structures. However, other materials, like certain types of carbon, conductive polymers, and even some liquids and solutions, can also exhibit conductive properties under specific conditions.

[0077]

[0059] In the context of the present invention one or more of the electrodes, including possibly all of them, may be created using a conductive material, particularly a metal coating. The term “electrically conductive coating” may refer to a thin layer or film of material that is applied onto a surface to enhance its electrical conductivity. This coating is designed to allow the passage of electric current across the surface of the electrodes, enabling efficient transfer of electrical charge. The coatings may contain conductive particles or additives, such as metal powders or carbon-based materials, that create pathways for electric charge to flow.

[0078]

[0060] However, in the context of the present invention it is particularly envisaged that said electrode is not formed by an electrically conductive coating and / or metal net which is directly formed and / or disposed on the stationary phase. That means, in the context of the present invention the electrode is not created or composed of a conductive material that is directly applied to the surface of the stationary phase, or the way that the electrodes are present in the stationary phase. Instead, as described elsewhere herein, the stationary phase is not in direct contact with said electrodes.

[0079]

[0061] In the context of the present invention, at least one of the stationary phases, possibly a membrane, or a non-conductive carrier may function as an insulating barrier between the electrodes. To achieve this, materials like gold or platinum can be used for constructing the electrodes or the conductive coatings. These metals can be used in their pure form, and the coatings consist solely of a single metal. In some aspects, the stationary phase, particularly a membrane, itself lacks conductivity, relying on the electrical conductivity exclusively from the conductive coating. In particular, the approach involves the use of only one specific type of conductive material or metal for both the electrodes and conductive coatings.

[0080]

[0062] In the context of the present invention it is further particularly envisaged that each of said electrodes comprises an insulating layer between said electrode and said stationary phase as shown in Fig. 1. Thus, it is envisaged in the context of the present invention that the stationary phase and the electrodes do not directly interact. The term “insulating layer between said electrode and said stationary phase” as used herein may refer to a non-conductive barrier or material positioned between an electrode and a stationary phase (e.g. in a chromatographic system). The "insulating layer" prevents direct electrical contact between the electrode and the stationary phase, maintaining their separation and preventing unwanted interactions. Non-limiting examples of the insulating layer can be made from materials also described herein for the stationary phase when acting as “insulator”, i.e. materials possessing high electrical resistance that do not allow the easy flow of electric current through them. In particular, the "insulating layer" may be made of non-metallic materials like rubber, plastic, glass, ceramic, or coatings with high electrical resistance.

[0081]

[0063] As explained herein above, utilizing voltage changes for elution as described for the flow cell of the present invention circumvents the necessity for traditional elution methods involving pH adjustments or the use of concentrated salt solutions, both of which impose relatively harsh conditions on the biomolecules that can potentially cause denaturation. In fact, when utilizing traditional AC, the binding of proteins such as antibodies can take place at neutral pH. This is followed by washing steps with the wash buffer / binding buffer preferably at a slightly acid pH to elute non-specifically bound contaminants. A pH shift to approximately pH 3 is then used for elution, as the action between the (protein) binding domain, i.e. the ligand (e.g. protein A) and a portion of the bound entity (e.g. the Fc part of an antibody) is largely based on electrostatic interactions, which can thus be suppressed. The flow cell of the present invention instead makes use of local electric fields to suppress specific electrostatic binding and to break the ligand-entity bonds, such as antibodyantigen bonds. When breaking the bonds between the ligand and the bond entity, this results in a change of affinity of the entity to the ligand finally leading to a break of affinity and desorption of the entity into the liquid.

[0082]

[0064] Hence, instead of being eluted by a pH-dependent elution (e.g., at pH < 3.5), when using the flow cell of the present invention the molecules bond to the stationary phase are eluted by the means of a potential switch, which further preferably allows for a more efficient purification of said molecules, e.g. higher quality antibody production when compared to pH-dependent elution (e.g. as described elsewhere herein). Moreover, the means of the present invention also help decreasing the formation of e.g. antibody aggregates as compared to standard AC- or MC- based means and / or methods. If fact, as explained elsewhere herein, it is known that aggregates (e.g., Ab aggregates) often occur during classical elution due to the low pH. However, when using the means of the present invention, such low pH values can be circumvented which helps to reduce the fraction of antibody agglomerates that are unusable for subsequent application. Accordingly, these aggregates in turn are translated into yield losses and additional cleaning effort for their removal.

[0083]

[0065] According, the use of flow cell of the present invention avoids the necessity for elution by altering the pH value overall, particularly through the introduction of fluids. Additionally, any fluids introduced both during and before the elution step, including optional second fluids and rinsing / washing fluids, are preferably characterized by a pH value that equals that of the first fluid or differs by less than 1.0 pH unit, specifically less than 0.5 pH units, and even more specifically less than 0.2 or 0.1 pH units. The goal of the flow cell of the present invention is to ensure that all fluid(s) used maintain a pH value that creates mild conditions for the molecules being purified, separated, selected, and / or isolated. In this context, pH values commonly regarded as "neutral," such as those falling between pH 6 and pH 8, are the preferred range, particularly between pH 7 and 7.8, and most preferably around pH 7.4. Within this framework, the liquids introduced into the flow cell during the process, which may encompass all liquids used, might comprise buffer solutions employed to maintain consistent pH levels and / or minimal amounts of ions and salts, preferably below 150 mM. These buffer solutions contain low levels of salts, contrasting with the higher concentrations typically employed in standard salt elution methods. For example, phosphate-buffered saline (PBS), is likely to fall within this category. PBS or similar solutions used for rinsing, as second fluids, or at any stage following binding and preceding elution, may exhibit salt concentrations approximating or below 150 mM. However, it is preferable to use fluids (rinsing fluids, second fluids, or any fluid) with even lower salt concentrations, such as those typically below 10 mM, particularly below 5 mM, and more specifically below 2 mM, 1 mM, 0.5 mM, and preferably even below 0.25 mM, with an approximate value of 0.15 mM being most favored. As a result, the addition of salt solutions with concentrations exceeding the common buffer concentrations described above is generally avoided, especially before and / or during the elution step.

[0084]

[0066] Thus, in the context of the present invention preference is given to using fluids that have notably lower salt concentrations. In this context, a "free of salts" solution refers to liquids with negligible salt ion concentrations, such as buffer solutions matching the criteria already outlined. Phosphate-buffered saline (PBS), particularly when its salt concentrations fall below the specified thresholds, can be applied as a buffer solution, particularly as a second fluid, to maintain pH levels within the range of pH 6 to pH 8, and notably at pH 7.4, meeting the criteria of a "free of salt" solution within the context of this disclosure. Commonly used PBS concentrations typically provide isotonic conditions, characterized by osmolarity and ion concentrations that mirror those found in the human body.

[0085]

[0067] In some embodiments, as described herein above, using the flow cell of the present invention may also comprise washing steps, i.e. the stationary phase on which the molecules are adsorbed may be washed or treated with a wash solution at least once. Said wash solution preferably comprises a buffer. It is further envisaged that said wash solution has a pH of not less than 5.0. It is also envisaged that said wash solution has an ion concentration of not more than 500 mM. The term “wash solution” as used herein may refer to a solvent or mixture of solvents used to clean or rinse a sample, column, or other chromatographic components, with the purpose of removing unwanted or interfering substances. The wash solution's composition is typically designed to effectively remove impurities without eluting or displacing the compounds of interest from the stationary phase.

[0086]

[0068] The term “buffer” as used herein may refer to a solution that resists significant changes in pH when small amounts of an acid or a base are added to it. A buffer system generally contains a weak acid and its conjugate base or a weak base and its conjugate acid. For example, a common buffer system is made of acetic acid (a weak acid) and its conjugate base, the acetate ion. The pH range over which a buffer effectively resists changes in pH is typically close to the pKa of the weak acid or base used in the buffer system. The Henderson-Hasselbalch equation is often used to calculate the pH of a buffer solution, given the concentrations of the weak acid and its conjugate base (or vice versa).

[0087]

[0069] The term “ion concentration” as used herein may refer to the amount of ions present in a given volume of solution. It is a measure of how many ions of a particular type are in the solution, often expressed in terms such as moles per liter (molarity, denoted as M) or equivalents per liter. For example, in an aqueous solution, when a salt (like sodium chloride, NaCI) dissolves, it dissociates into its constituent ions: Na+and Cl’. The ion concentration can refer to the concentration of any of these ions in the solution.

[0088]

[0070] With the means provided by the present invention, it is possible that the same type of liquid (i.e. having the same salt concentration and the same pH) can be used throughout the whole electrically controlled separation process described herein, i.e. for the adsorption of the molecules to the stationary phase and the desorption of the molecules from the stationary phase (i.e. the elution step).

[0089]

[0071] Nonetheless, within the means provided by the present invention it is also possible to use another liquid for the desorption of molecules from the stationary phase, e.g. a buffer in which the elution step may then be carried out. Specifically, by changing the voltage between the electrodes as described elsewhere herein, the affinity of the molecules bond to the stationary phase is changed so that to desorb the molecules from said stationary phase, thereby collecting the biomolecules desorbed or released from the stationary phase in said liquid. Thus, a specific liquid may be used for eluting and collecting the (bio)molecules desorbed from the stationary phase. This liquid may also be named “recovery solution” and refers to a specific solution or set of conditions applied to the chromatographic system to release and / or recover the biomolecules that have been bound to a stationary phase. Specifically, the flow cell of the present invention may be configured to allow a recovery solution to be applied to said stationary phase in transverse direction and / or in longitudinal direction in relation to said stationary phase being located between at least said two electrodes. Preferably, said recovery solution comprises a buffer and / or salts or is essentially free of salts. The term “essentially free” as used herein with reference to a certain molecule or salt ion may mean that the amount of said molecule or salt ion, if present at all, does not exceed trace amounts and preferably is less than 0.01 pM.

[0090]

[0072] However, the flow cell provided by the present invention is also configured to allow for the use of fluids comprising higher salt concentrations, such liquids (e.g. a recovery solution) that are isotonic with blood. In some embodiments it is envisaged that said salts have a concentration of more than 0.15 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM or 150 mM.

[0091]

[0073] The molecules contained in the liquid described herein, sometimes also named “chemical entities” within the context of the present invention, that can adsorb to and desorb from the stationary phase when using the flow cell of the present invention are preferably biomolecules, chemical molecules, organic compounds, inorganic ions, viruses, virus-like particles, extracellular vesicles or cells (prokaryotic or eukaryotic). Thus, said molecules may relate to different types of molecules or substances or mixtures thereof that are subject to separation and analysis using the affinity chromatographic techniques described herein. Different components or compounds comprised in the liquid interact differently with the stationary phase, leading to their separation from each other as described elsewhere herein. Each of said molecules or chemical entities possesses distinct chemical properties and can be characterized based on their unique chromatographic behaviors.

[0092]

[0074] The term “contained in a liquid” or “contained in a mobile phase” as used herein may refer to the solute (e.g., molecules or chemical entity described herein) or mixture of solutes being dissolved or suspended in the liquid that is passed through or flows over the stationary phase of the flow cell of the present invention. Accordingly, when a molecule or chemical entity is contained in a liquid, this may essentially mean that said molecule is part of the mixture being transported by the liquid and is subject to separation process using the flow cell of the present invention.

[0093]

[0075] The term “biomolecule” as used herein may refer to a biological molecule, which is any molecule that is involved in the structure, function, and / or regulation of living organisms. Biomolecules comprise a wide range of molecules found within cells and organisms, including viruses and macromolecules such as nucleic acids (DNA and RNA), proteins, antibodies, carbohydrates, and lipids, as well as smaller molecules like metabolites, cofactors, vitamins, hormones and signaling molecules. In the context of the present invention, preferred biomolecules are proteins or nucleic acids, such as DNA and RNA. In the context of the present invention, these biomolecules, as mentioned above, are each capable of binding to the stationary phase described elsewhere herein. “Capable of binding” means that each of said (bio)molecules is capable of interacting with the surface of said stationary phase, thereby at least temporarily being immobilized on the stationary phase.

[0094]

[0076] The term “protein” is equally used herein with the term "polypeptide". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins wherein the modification is effected e.g. by post- translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art. In the context of the present invention, the purification, separation and / or isolation of proteins with the flow cell of the present invention may be realized by combining peptide tags to theses proteins as described elsewhere herein. “Peptide tags” means short peptide sequences consisting of up to 30 amino acids, which sometimes are also called binding or recognition tags. These tags facilitate a specific binding to the surface of the stationary phase and thus allow controlled immobilization of proteins fused to such peptide sequences. Typical examples for the peptide tags comprise glutamic acid-based tag, aspartic acid-based tags or Histidine-based tags, and in particular the (His6)-tag, FLAG-tag, Glu6-tag or Strep-tag. Various methods for attaching such peptide tags to proteins are well known in the field and thus are not detailed herein.

[0095]

[0077] The term “antibody” as used herein may refer to a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. In particular, an “antibody” when used herein, is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2, with IgG being preferred in the context of the present invention. An antibody of the present invention is also envisaged which has an IgE constant domain or portion thereof that is bound by the Fc epsilon receptor I. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The constant domains are not involved directly in binding an antibody to an antigen, but can exhibit various effector functions, such as participation of the antibody dependent cellular cytotoxicity (ADCC). If an antibody should exert ADCC, it is preferably of the I gG 1 subtype, while the lgG4 subtype would not have the capability to exert ADCC.

[0096]

[0078] The term “antibody" also includes, but is not limited to, but encompasses monoclonal, monospecific, poly- or multi-specific antibodies such as bispecific antibodies, humanized, camelized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with chimeric or humanized antibodies being preferred. The term "humanized antibody" is commonly defined for an antibody in which the specificity encoding CDRs of HC and LC have been transferred to an appropriate human variable frameworks ("CDR grafting"). The term “antibody” also includes scFvs, single chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs) and nanobodies. In terms of the present invention, the term “antibody” shall also comprise bi-, tri- or multimeric or bi-, tri- or multifunctional antibodies having several antigen binding sites.

[0097]

[0079] Furthermore, the term "antibody" as employed in the context of the present invention also relates to derivatives of the antibodies (including fragments) described herein. A "derivative" of an antibody comprises an amino acid sequence which has been altered by the introduction of amino acid residue substitutions, deletions or additions. Additionally, a derivative encompasses antibodies which have been modified by a covalent attachment of a molecule of any type to the antibody or protein. Examples of such molecules include sugars, PEG, hydroxyl-, ethoxy-, carboxy- or aminegroups but are not limited to these. In effect the covalent modifications of the antibodies lead to the glycosylation, pegylation, acetylation, phosphorylation, amidation, without being limited to these.

[0098]

[0080] As used herein the term "antigen binding portion" refers to a fragment of immunoglobulin (or intact antibody), and encompasses any polypeptide comprising an antigen-binding fragment or an antigen-binding domain. Preferably, the fragment such as Fab, F(ab'), F(ab')2, Fv, scFv, Fd, disulfide- linked Fvs (sdFv), and other antibody fragments that retain antigen-binding function as described herein. Typically, such fragments would comprise an antigen-binding domain and have the same properties as the antibodies described herein.

[0099]

[0081] The terms "antigen-binding domain", “antigen binding portion”, "antigen-binding fragment" and “antibody binding region” when used herein may refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. The part of the antigen that is specifically recognized and bound by the antibody is referred to as the "epitope" as described herein. As mentioned above, an antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain.

[0100]

[0082] The term "antigen" as used herein may refer to a molecule or substance which induces an immune response (preferably an antibody response) in an animal, preferably a non-human animal immunized therewith (i.e. the antigen is "immunogenic" in the animal).

[0101]

[0083] In the context of the present invention, the term “protein” may also comprise fusion proteins. The term “fusion protein” may refer to a genetically engineered protein that is created by joining two or more individual protein coding sequences.

[0102]

[0084] As used herein, the terms “nucleic acids” or “nucleotide sequences” may refer to DNA molecules (e.g. cDNA or genomic DNA), RNA (mRNA), combinations thereof or hybrid molecules comprised of DNA and RNA. The nucleic acids can be double- or single-stranded and may contain double- and single-stranded fragments at the same time.

[0103]

[0085] The term “small molecule” as used herein refers to any organic material that is not a polymer. Small molecules may include repeat units in some circumstances. For example, using a long chain alkyl group as a substituent does not remove a molecule from the “small molecule” class. Small molecules may also be incorporated into polymers, for example as a pendent group on a polymer backbone or as a part of the backbone. In general, a small molecule has a well-defined chemical formula with a single molecular weight, whereas a polymer has a chemical formula and a molecular weight that may vary from molecule to molecule.

[0104]

[0086] The term “micro plastic” refers to fragments of any type of plastic less than 5 mm (0.20 in) in length and are described as contaminants of increasing ecotoxicological concern in aquatic environments, as well as for human health (Akdogan and Guven 2019, Environmental Pollution Volume 254, Part A, 113011).

[0105]

[0087] As described herein above, the molecules comprised in the liquid that are to be purified, separated, selected and / or isolated from said liquid using the flow cell of the present invention are each capable of adsorbing to and desorption from the stationary phase defined elsewhere herein. Preferably, the adsorption and desorption are realized by changing the voltage between the electrodes leading to changes in the affinity, so that the molecules can adsorb to or desorb from the stationary phase.

[0106]

[0088] In the context of the flow cell of the present invention, it envisaged that the molecule is adsorbed to and desorbed from the stationary phase in a electrically controllable manner. Accordingly, the molecule, when desorbed from the liquid, is at least temporarily immobilized on the stationary phase. The terms “immobilized” or “immobilization" as used herein may refer to the process of fixing or attaching a molecule, substance, or entity in a stable and relatively fixed position, onto a surface or within a matrix. This immobilization is typically achieved through chemical or physical means, and it results in the molecule or substance becoming effectively stationary or restricted in its movement.

[0089] Within the context of the present invention it is particularly envisaged that said stationary phase is capable of adsorbing said molecules through a ligand immobilized on said stationary phase. The term “immobilized on a stationary phase” as used herein may mean that the ligand is firmly attached or affixed to the solid or semi-solid medium (i.e., the stationary phase) through which the mobile phase passes. The stationary phase can be a solid or a viscous liquid, depending on the type of chromatography being used. Immobilization can be achieved through various mechanisms, for example, including but not limited to: (1) Physical adsorption, where molecules or chemical entities adhere to the surface of the stationary phase because of weak forces like van derWaals interactions; (2) Covalent bonding, where molecules or chemical entities are chemically bound to the surface, often through linker molecules or specific functional groups on the surface; (3) Entrapment, where molecules or chemical entities are trapped in a network or matrix, but not directly bound to it; (4) Ionic interactions, often used in ion-exchange chromatography, where charged species or chemical entities are retained on a stationary phase with an opposite.

[0107]

[0090] Said ligands immobilized on the stationary phase may also be referred to as “immobilization sites” of the molecules or chemical entities comprised in the liquid. The immobilization sites / ligands are preferably affixed to either the inner or outer surface of a (exemplary porous) stationary phase. In some aspects, the inner surface refers to a surface within the open pores of the stationary phase. These inner surfaces are preferably accessible to external fluids. In some embodiments, the stationary phase is predominantly constructed from a porous substrate, e.g., a membrane, preferably composed of polymer / s. The immobilization sites of the molecules or chemical entities can be on either the inner or outer surface of this porous substrate. In some aspects, immobilization of chemical entities comprise, among others, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC) or Dicyclohexylcarbodiimid (DCC) coupling and / or radical coupling methods. Specifically, these also include methods like electronic beam treatment.

[0108]

[0091] In some aspects, utilizing EDC or DCC couplling for immobilization of e.g. ligands that create the binding sites for the molecules to be purified from the liquid generally results in a reasonably uniform orientation of these binding sites on the surface(s) of said stationary phase. However, the yield of binding is often relatively low. Notably, employing EDC or DCC requires an initial grafting step to prepare the surface(s) of the stationary phase(s) for immobilization. This preparation involves generating amine- or carboxy- moieties on these surfaces, which then serve as sites for subsequent immobilization of chemical entities using processes like amination or similar methods. Conversely, the use of radical coupling techniques presents a less extensive approach for immobilizing binding sites onto the stationary phase(s). Typically, stationary phases that incorporate binding sites generated through radical coupling methods exhibit greater quantities of binding sites. Nonetheless, these binding sites might exhibit a less uniform arrangement or orientation.

[0109]

[0092] Within the setup of the flow cell of the present invention, it is envisaged that the ligand immobilized on the stationary phase and representing the binding sites for the molecules in the liquid is capable of non-covalently binding to said molecules, i.e. the binding between the ligand and the molecules is preferably a non-covalent binding. The terms “non-covalent binding” or “non-covalently bind” or “non-covalently bound” as used herein can be used interchangeably and do not involve the permanent sharing of electrons by way of a chemical bond. It involves variations of electromagnetic interactions between molecules. Examples of electromagnetic interactions are ionic interactions, hydrogen bonding, halogen bonding, van der Waals interaction, dipole-dipole interactions, dipole- induced dipole interaction, London dispersion interaction. In the context of the present invention it is envisaged that the molecules are known to non-covalently bind to the ligand immobilized on the stationary phase. In particular, it is envisaged that said ligand is capable of non-covalently binding said molecules when the voltage is changed between said electrodes as described elsewhere herein.

[0110]

[0093] Exemplarily ligands that may be immobilized to the stationary phase and can be used for non-covalently binding biomolecules, such as proteins, are protein affinity tags, such as the E2 epitope, FLAG epitope, EE-tag, HA tag, HSV epitope, KT3 epitope, Myc epitope, S1-tag, T7 epitope, VSV-G, V5-tag. Further, protein A (e.g., having Uniprot Accession Number: P02976), protein G (e.g., having Uniprot Accession Number: P06654) and / or protein L (e.g., having Uniprot Accession Number: Q53291) can be used as ligands to e.g. non-covalently binding antibodies. The term “protein A” as used herein may refer to a surface protein derived from the bacterium Staphylococcus aureus. It has the unique ability to bind to the constant region (Fc region) of immunoglobulins, particularly immunoglobulin G (IgG), from various species. Protein A (e.g., having Uniprot Accession Number: P02976) is commonly used in affinity chromatography to purify antibodies. The interaction between protein A and antibodies can also be exploited for antibody detection, immunoprecipitation, and / or other applications. Additionally, recombinant forms of protein A are engineered to optimize binding efficiency and specificity, enhancing its utility in various processes. The term “protein G” as used herein may refer to bacterial surface protein originally isolated from certain strains of bacteria, primarily of the species Streptococcus. Similar to protein A, protein G possesses the ability to bind to the Fc region of immunoglobulins (IgG antibodies), making it a valuable tool for purification. Protein G (e.g., having Uniprot Accession Number: P06654) can be used in affinity chromatography to purify antibodies from complex samples. When immobilized on a solid support matrix, protein G columns can selectively capture and purify IgG antibodies. This process takes advantage of the specific interaction between protein G and the Fc region of antibodies. While protein A has a higher affinity for IgG antibodies from certain species (such as human, rabbit, and pig), protein G offers broader species reactivity, making it particularly useful for purifying antibodies from a wider range of sources, including mouse, rat, and bovine. Recombinant forms of protein G have also been engineered to optimize its binding properties and enhance its effectiveness in various processes. The term “protein L” as used herein may refer to a bacterial surface protein derived from certain strains of bacteria, such as Peptostreptococcus magnus. Protein L is unique in its ability to bind to the light chain of immunoglobulins (Ig) from various species, regardless of the species' heavy-chain subclass. This characteristic sets protein L (e.g., having Uniprot Accession Number: Q53291) apart from other antibody-binding proteins like protein A and protein G, which primarily bind to the Fc region of IgG antibodies. Due to its ability to interact with the variable region of immunoglobulin light chains, protein L is particularly useful for the purification and detection of antibodies that might not bind well to protein A or protein G, such as camelid antibodies (e.g., llama, alpaca) that lack a traditional Fc region. Recombinant forms of protein L have also been engineered to optimize its binding properties and enhance its effectiveness in various processes. In this respect the provided means can be particularly utilized for a potential-controlled affinity membrane chromatography for antibody purification.

[0111]

[0094] In the context of the present invention said ligands immobilized to the stationary phase may also comprise peptide and nuclei acid aptamers. The term “aptamer” as used herein may refer to short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. Aptamers can be selected (e.g., through SELEX (Systematic Evolution of Ligands by Exponential Enrichment)) for their ability to bind to a specific target molecule with high affinity and specificity. Typically aptamers can selectively recognize and bind to a wide range of target molecules, including proteins, nucleic acids, small molecules, and even cells.

[0112]

[0095] The term “peptide aptamer” as used herein may refer to a short peptide sequence that binds a specific target molecule, or family of target molecules. Peptide aptamers can be selected (e.g., through SELEX (Systematic Evolution of Ligands by Exponential Enrichment)) for their ability to bind to a specific target molecule with high affinity and specificity. Peptide aptamer is preferably based on a scaffold, e.g. thioredoxin, adnectin, anticalin, avimer, knottin, fynomer, atrimer, darpin, affibody, affilin, armadillo repeat, Obody (e.g., Reyerdatto et al. (2015), doi : 10.2174 / 1568026615666150413153143).

[0113]

[0096] The term “DNA aptamer” as used herein may refer to short single-stranded DNA molecule that binds a specific target molecule, or family of target molecules. DNA aptamers can be selected (e.g., through SELEX (Systematic Evolution of Ligands by Exponential Enrichment)) for their ability to bind to a specific target molecule with high affinity and specificity.

[0114]

[0097] The term “RNA aptamer” as used herein may refer to a short single-stranded RNA molecule that binds a specific target molecule, or family of target molecules. RNA aptamers can be selected (e.g., through SELEX (Systematic Evolution of Ligands by Exponential Enrichment)) for their ability to bind to a specific target molecule with high affinity and specificity.

[0115]

[0098] However, in the context of the present inventions also ligands based on carbohydrate structures which depict an epitope for recognition of a specific target molecule, such as the biomolecules described herein, may be immobilized to the stationary phase. Carbohydrates applicable in this respect are e.g. monosaccharides and polysaccharides, such as glycans. Alternatively, the ligand recognized by the molecules may be glycosylized. In the context of the present invention proteins may bind to these carbohydrates, but also distinct carbohydrate epitopes can be immobilized to the stationary phase.

[0099] Further, in the context of the present inventions also ligands based on lipid structures which depict an epitope for recognition of a specific target molecule, such as the biomolecules described herein, may be immobilized to the stationary phase. For example, lipids can act as epitopes for scavenger receptors. Further, there are also specific antibodies binding to distinct lipids and lipoproteins. Thus, in the context of the present invention these lipid regions can be immobilized to the stationary phase to remove or purify these antibodies.

[0116]

[0100] Exemplarily pairs of a ligand immobilized to the stationary phase and the corresponding molecules in the liquid binding to said ligand are selected from, e.g. an antigen and its corresponding antibody (preferably a monoclonal antibody), albumin (e.g., having Uniprot Accession Number: P02768) and albumin-binding protein (ABP), avidin (e.g., having Uniprot Accession Number: P02701) and biotin-carboxy carrier protein (BCCP) (e.g., having Uniprot Accession Number: P0ABD8), streptavidin (e.g., having Uniprot Accession Number: P22629) and biotin-carboxy carrier protein (BCCP) (e.g., having Uniprot Accession Number: P0ABD8), calmodulin (e.g., having Uniprot Accession Number: P62152) and calmodulin binding peptide (CBP), chloramphenicol (e.g., CAS Registry Number: 56-75-7) and chloramphenicol acetyl transferase (CAT) (e.g., having Uniprot Accession Number: P11504), cellulose and cellulose binding domain (CBP), chitin and chitin binding domain (CBD), choline and choline-binding domain (CBD), galactose and galactose-binding protein (GBP) (e.g., having Uniprot Accession Number: P0AEE5), glutathione and glutathione S-transferase (GST) (e.g., having Uniprot Accession Number: P08515), divalent metal ion, such as Ni2+, Co2+, Cu2+ or Zn2+ and histidine affinity tag (HAT), divalent metal ion, such as Ni2+, Co2+, Cu2+ or Zn2+ and poly-histidine (His-tag), cross-linked amylose or maltose and maltose-binding protein (MBP) (e.g., having Uniprot Accession Number: P0AEX9), streptavidin (e.g., having Uniprot Accession Number: P22629) and streptavidin binding peptide (SBP), Strep-Tactin and Strep-tag, protein A and antibody (in particular lgG1 , lgG2 or lgG4), protein G and antibody (in particular lgG1 , lgG2, lgG3 or lgG4), protein L and antibody (in particular, IgG, IgA, IgM, IgD, IgE), strep-tactin and strep-tag (e.g., Kimple et al. (2013), doi: 10.1002 / 0471140864. ps0909s73), but the invention is not limited thereto.

[0117]

[0101] In the context of the present invention it is also envisaged that said stationary phase comprises molecules already adsorbed to said stationary phase or molecules already adsorbed to said ligands immobilized on said stationary phase. Thus, the stationary phase between the two electrodes comprised by the flow cell of the present invention may already comprise the molecules or chemical entities that shall be desorbed from said stationary phase by changing the voltage between the electrodes as described elsewhere herein. Accordingly, in some embodiments the flow cell comprises a stationary phase already comprising (bio)molecules immobilized on the chosen stationary phase or bond to a ligand immobilized on the stationary phase which is placed between the two electrodes inside the flow cell of the present invention. Then, applying a voltage change between the two electrodes, thereby changing the affinity between said (bio)molecules and said stationary phase or said ligand immobilized to said stationary phase leads to a decreased binding affinity of said (bio)molecules to said stationary phase or said ligand immobilized to said stationary phase, thereby desorbing the (bio)molecules from the stationary phase and adsorbing the (bio)molecule into the liquid. Then, the (bio)molecules can be collected as described elsewhere herein.

[0118]

[0102] In the context of the flow cell of the present invention it is also envisaged that the stationary phase may be separated from a (first) liquid used to adsorb said (bio)molecules to said stationary phase and is then brought into contact with another liquid. “Separated” means in this respect that the liquid is partly or completely removed from the chamber and a second (or further) liquid is introduced into the chamber. This second or further liquid may then be used when desorbing the (bio)molecules from the stationary phase, i.e. for adsorbing the molecules into the liquid. Alternatively, the stationary phase comprising the molecules already adsorbed to said stationary phase may be removed from the chamber containing the (first) liquid and disposed into another chamber which contains another liquid in which the elution step may then be carried out.

[0119]

[0103] The Flow cell of the present invention may be configured to comprise a stationary phase (or more than one stationary phase) varying regarding size, shape or material as long as the aforementioned functionalities may be satisfied. In particular, in some embodiments the flow cell of the present invention is configure the way that the stationary phase comprises at least two stationary phases. In further embodiments, the at least two stationary phases (e.g., distinct), including all of them if applicable, along with the affinity substrates, particularly membranes, and the specific affinity substrates, also membranes, or the respective non-conductive carriers that constitute these stationary phases, can be structured in various arrangements. These arrangements might include stacked, wrapped or tortuous arrangement, or being positioned separately in a disjunct pattern. Thus, in some embodiments the at least two stationary phases are stacked. In some embodiments the at least two stationary phases are wrapped. In some embodiments the at least two stationary phases are tortuous. In some embodiments the at least two stationary phases are located between two electrodes. In some embodiments, each of the two stationary phases is located between a separate set of two electrodes.

[0120]

[0104] Specifically, the distinct stationary phases may be positioned apart from one another along the direction of fluid flow containing the (bio)molecules. This separation could involve the fluid passing sequentially over each specific stationary phase. Particularly, each segment of the initial fluid is directed across each distinct specific stationary phase. In scenarios involving numerous identical specific stationary phases, one has the option to guide each segment of the initial fluid over every single stationary phase or selectively over certain stationary phase(s).

[0121]

[0105] In some aspects, in a stacked configuration, the at least two distinct, specifically different specific stationary phases, along with the affinity substrates, particularly membranes, and the specific affinity substrates, also membranes, or the respective non-conductive carriers, are organized as successive layers, forming a cohesive stack. This stack is ideally composed of at least two porous substrates, e.g., permeable membranes, that are aligned as stacked layers. More specifically, the stack is structured such that one of the electrodes, potentially formed by a conductive material like a metallic coating or metallic net and placed on a first affinity substrate, or membrane, and / or first non-conductive carrier, is positioned at one end of the stack. Correspondingly, the second electrode, also potentially formed by a conductive material and located on a second affinity substrate, or membrane, and / or second non-conductive carrier, is positioned at the opposite end of the stack. The stack encompasses an affinity substrate, particularly a membrane, core positioned between these first and second electrodes. This core comprises at least one affinity substrate, especially a membrane, and / or specific affinity substrates, also membranes, serving as the insulating barrier between the two electrodes.

[0122]

[0106] In some aspects, electrodes can be positioned between each distinct stationary phase and / or amidst the layers, particularly affinity substrates or membranes, forming the stationary phases. Similarly, electrodes can also be positioned between layers of specific affinity substrates, or membranes, within the distinct stationary phases. Additionally, electrodes can be placed between layers, stationary phases, or substrates, especially membranes, forming sub-cores. These sub-cores could include their respective ligands and / or receptors.

[0123]

[0107] In some aspects, the at least two distinct stationary phases, preferably specific ones, are ideally positioned sequentially along the direction of fluid flow, particularly the flow of the first fluid. This arrangement involves aligning these stationary phases in a consecutive manner or one after the other. Alternatively, the first fluid can be directed in sequence across these at least two specific stationary phases. In this context, it is preferred that the two stationary phases represent different specific configurations.

[0124]

[0108] In further aspects, a configuration involving the wrapped or tortuous of stationary phases, specifically constructed from substrates like membranes, particularly layers of stacked membranes incorporating at least two or all electrodes, is contemplated. In this arrangement, a stack of stationary phases and electrodes is preferably combined through wrapping or twisting, winding, or curving in a complex or convoluted manner. This kind of arrangement holds significant utility within chromatographic methods, including but not limited to the methods described herein. Such a configuration could effectively function as an integral component of various chromatographic apparatuses suitable for these purposes.

[0125]

[0109] In some aspects, the creation of the wrapped arrangement involves folding a stationary phase or, alternatively, a stack of stationary phases (e.g., membranes), preferably encompassing at least two or all electrodes, into multiple wraps. In the case of the tortuous arrangement, the stationary phase or stack of stationary phases is preferably arranged in a series of twisted, wound, or curved in a complex or convoluted manner layers. This wrapped or tortuous configuration is subsequently positioned within a housing.

[0126]

[0110] In further aspects, the chosen housing of a device can accommodate the wrapped or tortuous configuration in a manner that permits fluids to pass over and / or through the stationary phase(s) (e.g., membranes). The housing is designed to enable a specified flow direction, typically facilitated by at least one inlet for introducing fluids and at least one outlet for extracting them as described elsewhere herein. Within this housing, the wrapped or tortuous arrangement is positioned to obstruct direct passage from the inlet to the outlet. Furthermore, it ensures that any fluid flow from the inlet to the outlet necessitates passing through and / or over the stationary phase(s). This interaction typically occurs at least once, preferably multiple times, and can span various sections or regions of the stack and / or substrates. This encompasses traversal over and / or through all distinct specific phases within the arrangement. The wrapped or tortuous configuration is usually positioned within the housing in such a way that its main extension is oriented parallel, perpendicular, or oblique to the aforementioned designated flow direction.

[0127]

[0111] In some aspects, cylindrical structures resembling tubes can serve as enclosures for configurations of the present invention. These tube-like structures may exhibit various base shapes, which can range from rectangular to circular, and include other forms. Additionally, suitable enclosures can take on a box-like structure, equipped with inlets and outlets placed between which the stationary phase(s) are positioned. These stationary phase(s) placed between the inlets and outlets can receive lateral fluid inputs. In this arrangement, the stationary phase(s) are positioned to obstruct direct passage from inlet to outlet. Fluid being channeled through the outlets is therefore compelled to traverse through and, preferably, over the stationary phase(s) at least once. Consequently, the inlets need to be situated on one side of the stationary phase(s), while the outlets are positioned on the opposite side, ideally diagonally opposite, of the stationary phase(s). This arrangement promotes the desired fluid flow dynamics.

[0128]

[0112] In further aspects, circular enclosures characterized by an inner core and an outer cage, separated by a gap, can be used as housing. The gap, defined by the shortest distance between the core and the cage, serves as a crucial element in this context. Within this gap, the wrapped or tortuous surface is positioned. Both the inner core and the outer cage, in an ideal scenario, exhibit permeability to fluids and might also include inlets and outlets for fluid supply to the surface(s) or for the removal of fluids / eluate. The fluid, for optimal flow, is expected to pass through the arrangement radially within the gap, facilitating its flow from the inlet to the outlet. Typically, only either the inner core or the outer cage is equipped with inlets, while the other component has outlets. An inlet could be any form of fluid-permeable passage facilitating supply, and an outlet refers to a passage for removal. For example, the volume within the core can be utilized for either the introduction of fluids or the removal of fluids.

[0129]

[0113] In some aspects, the wrapped or tortuous configuration comprises a minimum of two porous substrates, preferably made from polymeric membranes. On one side of each respective membrane, a conductive coating or layer of metal is applied, forming a stacked assembly. Additionally, non- conductive porous substrates, ideally made from polymeric membranes, are interposed between the coated membranes. This arrangement establishes at least one stationary phase within the system.

[0114] In some aspects, an insulator (e.g., made from one of the porous substrates, e.g., polymeric membranes), is positioned amidst the electrically conductive coatings or layers. This configuration can be harnessed to intentionally administer release voltages exclusively to chosen substrates.

[0130]

[0115] In some aspects, the ratio of surface area to volume can be influenced by the choice of stationary phase(s) arrangement, particularly concerning the housing structure. In this context, the wraps are comprised of flat sections positioned between local folding points. Each of these flat areas has a length, and the preferred form for the pleats is an M-shape. For every wrap, a first length is selected to exceed at least one dimension such as width, diameter, or similar attributes of the housing. Specifically, for circular housings with an outer cage and inner core, this pertains to the gap distance. Considering that the core has a smaller circumference than the cage, wraps with uniform, nearly identical lengths of surface areas would lead to suboptimal surface area to volume ratios. Hence, wraps with irregular M-shapes are more desirable as they yield higher, optimally optimized surface area to volume ratios. These irregular M-shapes consist of flat areas with at least one second length that differs from the first length. Preferably, sets of wraps encompassing more than one wrap are used. These wrap sets can include wraps characterized by at least two distinct irregular M- shapes. These sets of wraps are preferably repeated in a regular manner within the housing. Particularly in the context of circular housing, the wraps or wraps sets can create a complete circle within the housing. Regarding tortuous arrangements, circular housings can also offer advantages. The winding can be achieved by enveloping the surface(s) or stack of surface(s) around the inner core of the housing, with the inner core playing a crucial role in supporting the wrapped structure.

[0131]

[0116] In some further aspects, the present invention relates to a flow cell comprising fewer membranes stacked on top of each other (e.g., compared to Sartobind Protein A75) enabling a much faster loading (e.g., adsorption) of the membranes with antibodies. The potential-controlled rapid discharge (e.g. desorption) of the membrane and / or immediate rechargeability according to the present invention results in a better purification performance (e.g., compared to standard AC-based purification).

[0132]

[0117] Further, the flow cell provided by the present invention may also be used for voltage-induced chemical reactions, such as enzymatic reactions on said stationary phase. Thus, the means provided by the present invention also allow for catalysis. In this respect the molecules that undergo the chemical reaction (i.e. in case of an enzymatic reaction the respective substrate and respective enzyme) directly bind to the surface of the selected stationary phase, in particular to the enzymatic center comprised by or placed on the surface of said stationary phase. Then, a voltage change as described elsewhere herein leads to the chemical reaction between the molecules and an enrichment of the product on said stationary phase. Then, the product of said chemical reaction can be desorbed from said stationary phase by a further voltage change.

[0133]

[0118] Moreover, the flow cell provided by the present invention may also be applied for electrochemical polymerization, crystallization or precipitation of molecules on said stationary phase (see Fig. 1 and 4). The term “polymerization” refers to a process of chemically binding monomers to form larger molecules. Commercial polymer molecules are usually thousands of repeat units long. The two most common polymerization methods are addition and condensation polymerization. The term “crystallization” refers to a process by which solids form, wherein the atoms or molecules are highly organized into a structure known as a crystal. “Precipitation” occurs when cations and anions in aqueous solution combine to form an insoluble ionic solid called a precipitate. In the context of the present invention, a voltage change as described elsewhere herein leads to the polymerization, crystallization or precipitation and an enrichment of the polymer, crystal or precipitate on said stationary phase. Then, the polymer, crystal or precipitate can be desorbed from said stationary phase by a further voltage change.

[0134]

[0119] It is noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0135]

[0120] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0136]

[0121] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0137]

[0122] The term “less than” or in turn “more than” does not include the concrete number.

[0138]

[0123] For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, e.g., more than 80 % means more than or greater than the indicated number of 80 %.

[0139]

[0124] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”. When used herein “consisting of" excludes any element, step, or ingredient not specified.

[0140]

[0125] The term “including” means “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0141]

[0126] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0142]

[0127] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0143]

[0128] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. All documents, including patent applications and scientific publications, referred to herein are incorporated herein by reference for all purposes.

[0144]

[0129] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0145]

[0130] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0146]

[0131] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0147]

[0132] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[0148]

[0133] The present invention is further characterized by the following items:

[0149] 1. A flow cell for electrically controllable ad- and desorption of molecules from and into a liquid, said flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising

[0150] (a) two electrodes comprising a conductive surface,

[0151] (b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes,

[0152] (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

[0153] 2. The flow cell of item 1 , wherein said electrodes form a capacitor.

[0154] 3. The flow cell of any one of the preceding items, wherein said stationary phase acts as a dielectric or insulator between the electrodes.

[0155] 4. The flow cell of any one of the preceding items, wherein each of said electrodes comprises an insulating layer between said electrode and said stationary phase.

[0156] 5. The flow cell of any one of the preceding items, wherein said electrodes are configured to allow flow of a liquid.

[0157] 6. The flow cell of item 5, wherein the fluid path is in transverse direction and / or in longitudinal direction in relation to said stationary phase being located between said electrodes.

[0158] 7. The flow cell of any one of the preceding items, wherein said voltage is DC voltage.

[0159] 8. The flow cell of any one of the preceding items, wherein said voltage is from a constant voltage source, preferably an adjustable constant voltage source.

[0160] 9. The flow cell of any one of the preceding items, wherein said controller is a voltage source which does not react on changing current. The flow cell of any one of the preceding items, wherein said electrode is not formed by an electrically conductive coating and / or metallic net which is directly formed and / or disposed on said stationary phase. The flow cell of any one of the preceding items, wherein said stationary phase is porous. The flow cell of item 11 , wherein said stationary phase is a membrane, a polymer, a molecularly imprinted polymer, a non-metal material, a metal-organic framework, a molecular sieve, a chromatography resin, a fluidized bed system, foam or felt. The flow cell of item 11 , wherein said stationary phase is composed of zeolites, monoliths or capillaries and wires. The flow cell of any one of the preceding items, wherein said stationary phase comprises at least two stationary phases. The flow cell of item 14, wherein said at least two stationary phases are stacked, wrapped or tortuous. The flow cell of item 14 or 15, wherein said at least two stationary phases are located between two electrodes. The flow cell of item 14 or 15, wherein each stationary phase is located between a separate set of two electrodes. The flow cell of any one of the preceding items, wherein said stationary phase is capable of adsorbing said molecules. The flow cell of any one of the preceding items, wherein said stationary phase is capable of adsorbing said molecules when the voltage is changed between said electrodes. The flow cell of any one of the preceding items, wherein said stationary phase is capable of adsorbing said molecules through a ligand immobilized on said stationary phase. The flow cell of item 20, wherein said ligand is capable of non-covalently binding said molecules. The flow cell of item 20 or 21 , wherein said ligand is capable of non-covalently binding said molecules when the voltage is changed between said electrodes. The flow cell of any one of the preceding items, wherein said stationary phase comprises molecules already adsorbed to said stationary phase or said ligand immobilized on said stationary phase. The flow cell of any one of the preceding items, wherein said flow cell comprises one or more inline sensors. The flow cell of any one of the preceding items, wherein said molecules are biomolecules, chemical molecules, viruses, virus-like particles, extracellular vesicles, or cells (prokaryotic or eukaryotic). The flow cell of item 25, wherein said biomolecule is a protein or a nucleic acid, such as DNA or RNA. The flow cell of item 25, wherein said chemical molecules are small molecules or micro plastic.

[0161] EXAMPLES

[0162]

[0134] An even better understanding of the present invention and of its advantages will be evident from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0163]

[0135] Example 1 - Flow cell setup for crystallization

[0164] For crystallization experiments, 3 mL sodium oxalate (5 mmol / L) have been added to 3 mL calcium chloride (10 mmol / L). The concentrations are similar to previously published experiments (Werner et al. 2021 , In ACS omega 6 (40), pp. 26566-26574. DOI: 10.1021 / acsomega.1c03938.). The experiments have been conducted at 25 °C (room temperature). All experiments have been conducted in the same set-up containing two electrodes (Figure 2). In this set-up two large circular electrodes which are protected by insulating layers made of thin polymers (rubber) have been used. A glass slide from a microscopy cover slip has been used as stationary phase. Only for the “potential” experiments a potential of 32 V has been applied. The experiments have been conducted over 30 minutes and the pH has been monitored at the end of the experiments. The experiments have been conducted in duplicates.

[0165] The results indicate an immediate crystallization of calcium oxalate at the investigated concentration which is according to the solubility product. However, Figure 3 shows a significant change of pH for the sample in the applied potential and therefore a significantly higher amount of crystallized calcium oxalate.

[0166]

[0136] Example 2 - Affinity precipitation of IgG antibodies using a modular flow cell

[0167] The mild and electrically controllable desorption of biomolecules such as IgG antibodies that have previously precipitated on a stationary phase of flow cell according to the invention could be illustrated. Specifically, desorption was achieved by means of a controlled electric field, which affects the surface charge conditions and dipoles of the precipitated biomolecules and ligands (see Figure 4). The electric field was generated by oppositely charged electrodes that are aligned with each other in a capacitor-like arrangement. Furthermore, the system for potential-controlled desorption consisted of modular components that can be combined in a detachable manner (see Figure 5). This allows individual components such as the stationary phase(s), the electrodes, or the housing of the flow cell to be exchanged or combined in an application-specific manner. This means that various processes on the stationary phase, such as the interaction of target molecules with protein A, G, L, aptamers, or synthetic binding motifs, can be influenced within the system by the electric field. In addition, the various inserts allow the technology to be used for axial, tangential, and radial flow. The modular design also allows in-line sensors to be integrated. For example, using electrochemical impedance spectroscopy or cyclic voltammetry, the system can be used for the analytical measurement of potential-controlled precipitation, resolubilization, and crystallization processes in order to simultaneously generate further product and process-relevant information. The sensors also enable adaptive process control. A feedback mechanism creates an adaptive system that is able to dynamically adapt to the actual process behavior. The modular design of the system allows it to be used as a reusable, CIP-compatible system or as a disposable module. Scaling from nano to industrial scale is also possible.

[0168]

[0137] Example 3 - Potential-controlled desorption of IgG after surface precipitation on Protein A membranes

[0169] The functionality of electrically-modulated desorption could be demonstrated using the modular system shown in Figure 5 (axial) for a pure IgG antibody solution (Cutaquig®, Octapharma). Axial flow was achieved using a single Sartobind Lab A affinity membrane (Sartorius). The electrodes consisted of V4A stainless steel grids with a mesh size of 25 pm. IgG was pumped into the flow cell at a flow rate of 1.5 mL min-1. A voltage of 2.5 V was then applied for 75 s. An increase in the LIVA / IS signal was observed (see Figure 6 (A)). This indicates that the antibodies can be desorbed (resolubilized) by the electric field. As a comparison experiment, the standard procedure, pH-based elution, was performed using the same experimental setup (see Figure 6 (B)).

Claims

CLAIMS1. A flow cell for electrically controllable ad- and desorption of molecules from and into a liquid, said flow cell comprising a chamber comprising an inlet and an outlet which define therebetween a fluid path for the liquid and a volume for containing the liquid provided along said fluid path, the volume comprising a device comprising(a) two electrodes comprising a conductive surface,(b) a stationary phase between said two electrodes, wherein said stationary phase is not in direct contact with said electrodes,(c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of molecules so as to adsorb said molecules to said stationary phase or desorb molecules from said stationary phase when said molecules are adsorbed to said stationary phase.

2. The flow cell of claim 1 , wherein said electrodes form a capacitor.

3. The flow cell of any one of the preceding claims, wherein said stationary phase acts as a dielectric or insulator between the electrodes.

4. The flow cell of any one of the preceding claims, wherein each of said electrodes comprises an insulating layer between said electrode and said stationary phase.

5. The flow cell of any one of the preceding claims, wherein said electrodes are configured to allow flow of a liquid.

6. The flow cell of claim 5, wherein the fluid path is in transverse direction and / or in longitudinal direction in relation to said stationary phase being located between said electrodes.

7. The flow cell of any one of the preceding claims, wherein said voltage is DC voltage.

8. The flow cell of any one of the preceding claims, wherein said voltage is from a constant voltage source, preferably an adjustable constant voltage source.

9. The flow cell of any one of the preceding claims, wherein said controller is a voltage source which does not react on changing current.

10. The flow cell of any one of the preceding claims, wherein said electrode is not formed by an electrically conductive coating and / or metallic net which is directly formed and / or disposed on said stationary phase.11 . The flow cell of any one of the preceding claims, wherein said stationary phase is porous.

12. The flow cell of claim 11, wherein said stationary phase is a membrane, a polymer, a molecularly imprinted polymer, a non-metal material, a metal-organic framework, a molecular sieve, a chromatography resin, a fluidized bed system, foam or felt.

13. The flow cell of claim 11, wherein said stationary phase is composed of zeolites, monoliths or capillaries and wires.

14. The flow cell of any one of the preceding claims, wherein said stationary phase comprises at least two stationary phases.

15. The flow cell of claim 14, wherein said at least two stationary phases are stacked, wrapped or tortuous.

16. The flow cell of claim 14 or 15, wherein said at least two stationary phases are located between two electrodes.

17. The flow cell of claim 14 or 15, wherein each stationary phase is located between a separate set of two electrodes.

18. The flow cell of any one of the preceding claims, wherein said stationary phase is capable of adsorbing said molecules.

19. The flow cell of any one of the preceding claims, wherein said stationary phase is capable of adsorbing said molecules when the voltage is changed between said electrodes.

20. The flow cell of any one of the preceding claims, wherein said stationary phase is capable of adsorbing said molecules through a ligand immobilized on said stationary phase.

21. The flow cell of claim 20, wherein said ligand is capable of non-covalently binding said molecules.

22. The flow cell of claim 20 or 21 , wherein said ligand is capable of non-covalently binding said molecules when the voltage is changed between said electrodes.

23. The flow cell of any one of the preceding claims, wherein said stationary phase comprises molecules already adsorbed to said stationary phase or said ligand immobilized on said stationary phase.

24. The flow cell of any one of the preceding claims, wherein said flow cell comprises one or more in-line sensors.

25. The flow cell of any one of the preceding claims, wherein said molecules are biomolecules, chemical molecules, viruses, virus-like particles, extracellular vesicles, or cells (prokaryotic or eukaryotic).

26. The flow cell of claim 25, wherein said biomolecule is a protein or a nucleic acid, such as DNA or RNA.

27. The flow cell of claim 25, wherein said chemical molecules are small molecules or micro plastic.

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