WO2025036567A1

WO2025036567A1 - Electrochemically-modified liquid affinity chromatography

Info

Publication number: WO2025036567A1
Application number: PCT/EP2023/072725
Authority: WO
Inventors: Sonja Berensmeier; Sebastian SCHWAMINGER; Tobias STEEGMUELLER
Original assignee: Technische Universität München, in Vertretung des Freistaates Bayern
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2025-02-20
Also published as: WO2025036999A1

Abstract

The present invention relates to means and methods utilizing "digital" ("electrochemically-modified" and/or "electrically controllable") affinity membrane chromatography (dAMC) for purifying, separating and/or isolating chemical entities (e.g., antibodies). Particularly, the present invention relates to means and methods utilizing a stationary phase chromatography of a pair of chemical entities, wherein a first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between at least two electrodes, wherein such method and device comprise applying a voltage between said electrodes and changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities, wherein a stationary phase that is eluted in a specific manner, wherein a recovery solution is applied to the stationary phase in transverse direction and/or in longitudinal direction in relation to said stationary phase being located between said at least two electrodes.

Description

ELECTROCHEMICALLY-MODIFIED LIQUID AFFINITY CHROMATOGRAPHY

TECHNICAL FIELD OF THE INVENTION

[1] The present invention relates to means and methods utilizing “digital” (i.e., “electrochemically-modified” and/or “electrically controllable”) liquid affinity membrane chromatography (dAMC) for purifying, separating and/or isolating chemical entities (e.g., biomolecules such as antibodies and/or proteins). Accordingly, in some aspects, the present invention relates to means and methods comprising/utilizing a potential-controlled affinity membrane chromatography for antibody purification (i.e., so-called “digital” antibody purification). In further aspects, the present invention relates to means and methods comprising/utilizing a stationary phase chromatography of a pair of chemical entities, wherein a first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non- covalently bind to each other, wherein said stationary phase is located between at least two electrodes, wherein such method and device comprise applying a voltage between said electrodes and changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities. Furthermore, the present invention relates to means and methods comprising a stationary phase that is eluted in a specific manner, e.g., where a recovery solution is applied to the stationary phase in transverse direction and/or in longitudinal direction in relation to said stationary phase being located between said at least two electrodes.

BACKGROUND

[2] Monoclonal antibodies (mAbs) are homogeneous binding partners specific for a particular antigen epitope originating in a single B cell clone. Their generally high binding affinity and specificity are exploited in many ways in research and clinics. For example, they are suitable for application in detection, therapeutics and/or diagnostics. Of particular note are immunoassays and immunohistology for specific detection of certain infectious diseases, autoimmune diseases, and cancers. Particularly groundbreaking is an application of antibodies as therapeutics. Since the approval of the first antibody drug in the mid-1990s, more than 65 additional antibodies have now been approved. Most of these antibodies are used particularly serious diseases in the field of oncology and hematology. However, it is precisely these medications for which treatment with antibody therapeutics is particularly expensive (can reach up to and over $100000 per patient per year). Against the backdrop of rising populations worldwide, this circumstance is leading to the exclusion of numerous patients from necessary treatment with mAb drugs, which often have few alternatives.

[3] These high costs result in part from the manufacturing process, which is typically particularly demanding in terms of purity for protein-based (e.g., antibody-based) therapeutics. The downstream process accounts for the majority of total production costs, with affinity chromatography (AC) costs clearly dominating.

[4] Affinity chromatography is a type of liquid chromatography that uses a biologically- related agent as a stationary phase to specifically bind the molecule of interest. Affinity chromatography exploits the specific interaction between two molecules, for example, an enzyme and its substrate, an antibody and its antigen, or a receptor and its ligand. Affinity membrane chromatography combines the principles of affinity chromatography with the use of membranes as the stationary phase instead of traditional bead-based matrices. Accordingly, in standard AC bead-based matrices are used for the stationary phase, whereas in affinity membrane chromatography, the stationary phase is a membrane, which offers benefits like faster flow rates and shorter binding/elution times due to the thinner structure and higher surface area.

[5] In the downstream process of AC, cell debris, host proteins, host DNA and other contaminants are removed. First, cell components are separated by centrifugation and depth filtration. Next, protein A chromatography is typically the central capture step, where the volume of the mAb fraction is drastically reduced. 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 protein A and mAb, desorption/elution must be performed, e.g., at pH values < 3.5.

[6] The extremely low pH enables combined chemical inactivation of viruses, but at the same time it is responsible for two main disadvantages: (1) The fraction of antibody agglomerates that is unusable for subsequent application increases due to lower pH values; (2) The functionality of proteinogenic affinity ligands steadily decreases at low pH, so that protein A- based materials are not as durable or can be reused frequently. In addition to the known problem of low pH during elution, AC as a capture step achieves only low binding capacities due to its very large particles and also mass transport-limited pore accessibility. Secondly, with AC, the particle-based fixed bed limits procedural scale-up. Following AC and virus inactivation, classical cation and anion exchange chromatography are typically combined with filtration steps to separate the remaining host cell proteins, as well as protein A components and mAb aggregates.

[7] Therefore, there is an ongoing need for improving affinity chromatography-related means and methods, especially by providing improved means and methods 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.

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

SUMMARY OF THE INVENTION

[9] The technical problem is solved by the subject-matter as defined in the claims.

[10] In some aspects, the present invention relates to a method of changing the affinity of a pair of chemical entities, comprising: (a) contacting a first member of said pair of chemical entities with a second member of said pair of chemical entities, wherein said first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between at least two electrodes; (b) applying a voltage between said electrodes; and (c) changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities.

[11] In some aspects, the present invention relates to a method for electrically controllable affinity chromatography, comprising: (a) placing a stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities between two electrodes; (b) applying a voltage between said electrodes; and (c) changing the voltage between said electrodes, thereby changing the affinity between said first and second member of said pair leading to decreased binding of said second member to said first member of said pair of chemical entities.

[12] In other aspects, the present invention relates to a use of an electrical field for eluting a second member of a pair of chemical entities, which is bound non-covalently to a first member of said pair of chemical entities, said first member being immobilized on a stationary phase located between two electrodes.

[13] In further aspects, the present invention relates to a device for changing the affinity of a pair of chemical entities, comprising: (a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution; (b) a stationary phase between said two electrodes, wherein a first member of said pair of chemical entities is immobilized on said stationary phase, said stationary phase allowing flow of a solution; (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes; wherein said device being configured to allow flow of a mobile phase comprising a second member of said pair of chemical entities such that said first member of said pair is contacted with s aid second member of said pair, thereby allowing said first and second member to non-covalently bind to each other and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to increase binding of said second member to said first member of said pair or decrease binding of said second member to said first member of said pair.

[14] In yet other aspects, the present invention relates to a device for electrically controllable affinity chromatography, comprising: (a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution; (b) a stationary phase between two electrodes, said stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities; (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes; wherein said device being configured to allow flow of a solution and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to decrease binding of said second member to said first member of said pair.

[15] In some aspects of the present invention, means or methods of the present invention have a surprising advantage that the antibodies aggregate to a lesser extent and the yield of biologically active antibodies is increased. A further surprising advantage of the means and methods of the present invention lies in that the protein A ligand in said means and methods of the present invention 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, means and methods of the present invention allowing for a greater functionality without the use of buffer solutions, pH shifts or salting, making it thus possible to release antibodies electrically-induced and thus concentrated without extended desorption kinetics.

BRIEF DESCRIPTION OF THE DRAWINGS

[16] 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. The Figures show:

[17] Figure 1 shows: (A) Schematic diagram and (B) photograph of the set-up for voltage- controlled elution.

[18] Figure 2: Voltage-based elution of an antibody pursuant to the teaching of the present invention. The illustrative antibody is Trastuzumab.

[19] Figure 3: Absorbance of released protein (mAB) after a potential switch to 2400 mV.

DETAILED DESCRIPTION OF THE INVENTION

[20] The present invention is described in detail in the following and is also illustrated by the appended examples and figures. [21] In the biopharmaceutical industry, the production of monoclonal antibodies (i.e., upstream process), established in the 1970s, is the area with the highest growth rate. Before these can be used in research, diagnostics and/or therapy, they must be purified to almost 100% purity by means of elaborate multi-stage separation processes (i.e., downstream process).

[22] A downstream process for the purification of IgG from animal cell cultures is known in the art. The core process is usually based on protein A-based affinity chromatography (AC), as this can efficiently bind many different target antibodies. The established platform process can be easily and quickly transferred to different antibody products. However, there are numerous known disadvantages of protein A-based methods, such as for example the acid-induced aggregation of antibodies as well as the degeneration of protein A and the associated high costs, which are however accepted due to the lack of real alternatives. In order not to forego the advantage of protein A (selectivity leading to high purity), a wide variety of strategies are being pursued. Hence, new matrices such as membranes are used as alternative to particle-based chromatography. Accordingly, known AC-based antibody purification means and methods achieve dynamic adsorption capacities of 40 to 80 mg/ml bed volume, with a relatively long antibody residence time of about 5 minutes. 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. There are no real alternatives to the protein A ligand as required for the antibodies. Antibody titers in production could also be improved from 0.1 g/L to 10 g/L by a factor of 100. Binding capacities of the materials, on the other hand, could only be doubled, so that enormously more separation matrices have to be used as product titers increase and market demand rises.

[23] The present invention relates to novel and improved means and methods utilizing affinity chromatography for purifying, separating and/or isolating chemical entities. Particularity, the present invention relates to novel and improved means and methods utilizing “digital” (i.e., “electrochemically-modified” and/or “electrically controllable”) affinity membrane chromatography (dAMC) for purifying, separating and/or isolating chemical entities (e.g., biomolecules such as antibodies, e.g., mAbs). Accordingly, in some aspects, the present invention relates to means and methods comprising/utilizing a potential-controlled affinity membrane chromatography for antibody purification (i.e., so-called “digital” antibody purification).

[24] Therefore, in contrast to classical alkaline desorption at < pH 3.5, desorption or recovery of adsorbed antibodies according to the present invention is achieved by applying an electrical potential, preferably at a mild pH conditions. This has a surprising advantage that the antibodies aggregate to a lesser extent and the yield of biologically active antibodies is increased. A further surprising advantage of the means and methods of the present invention lies in that the protein A ligand in said means and methods of the present invention 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, means and methods of the present invention allow for a greater functionality, e.g., without the use of buffer solutions, pH shifts and/or salts, making it thus possible to release antibodies in an electrically-induced manner and/or concentrate them without extended desorption kinetics.

[25] In some aspects, the present invention relates to utilizing a potential-controlled desorption of bound antibodies according to means and methods of the present invention. Typically, the binding of antibodies to protein A ligands can take place at a neutral pH. This is followed by washing steps with the wash buffer/binding buffer preferably at a slightly acidic pH to elute non-specifically bound contaminants. A pH shift to approximately 3 is used for elution, as the interaction between protein A binding domain and Fc portion of the antibody is largely based on electrostatic interactions, which can thus be suppressed. Accordingly, in some aspects, the present invention relates to the use of local electric fields to suppress specific electrostatic binding and to break antibody-antigen bonds. In further aspects, the present invention relates to breaking of affinity binding domain from protein A to the Fc part of the antibody. In yet further aspects, the present invention relates to modifying (e.g., breaking) an antibody-antigen binding and/or protein A-Fc part binding at different pHs and/or potentials (e.g., as described elsewhere herein).

[26] In some aspects the present invention relates to means and methods utilizing a stationary phase chromatography of a pair of chemical entities, wherein a first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between at least two electrodes, wherein such method and device comprise applying a voltage between said electrodes and changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities. Furthermore, the present invention relates to means and methods comprising a stationary phase that is eluted in a specific manner, e.g., where a recovery solution is applied to the stationary phase in transverse direction and/or in longitudinal direction in relation to said stationary phase being located between said at least two electrodes.

[27] In other aspects, the present invention relates to a method of changing the affinity of a pair of chemical entities, comprising: (a) contacting a first member of said pair of chemical entities with a second member of said pair of chemical entities, wherein said first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between at least two electrodes; (b) applying a voltage between said electrodes; and (c) changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities.

[28] In some aspects, the present invention relates to a method for electrically controllable affinity chromatography, comprising: (a) placing a stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities between two electrodes; (b) applying a voltage between said electrodes; and (c) changing the voltage between said electrodes, thereby changing the affinity between said first and second member of said pair leading to decreased binding of said second member to said first member of said pair of chemical entities.

[29] In other aspects, the present invention relates to a use of an electrical field for eluting a second member of a pair of chemical entities, which is bound non-covalently to a first member of said pair of chemical entities, said first member being immobilized on a stationary phase located between two electrodes.

[30] In further aspects, the present invention relates to a device for changing the affinity of a pair of chemical entities, comprising: (a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution; (b) a stationary phase between said two electrodes, wherein a first member of said pair of chemical entities is immobilized on said stationary phase, said stationary phase allowing flow of a solution; (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes; wherein said device being configured to allow flow of a mobile phase comprising a second member of said pair of chemical entities such that said first member of said pair is contacted with s aid second member of said pair, thereby allowing said first and second member to non-covalently bind to each other and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to increase binding of said second member to said first member of said pair or decrease binding of said second member to said first member of said pair.

[31] In yet other aspects, the present invention relates to a device for electrically controllable affinity chromatography, comprising: (a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution; (b) a stationary phase between two electrodes, said stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities; (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes; wherein said device being configured to allow flow of a solution and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to decrease binding of said second member to said first member of said pair.

[32] In some further aspects, the present invention relates to means and methods comprising/utilizing a potential-controlled affinity membrane chromatography for antibody purification.

[33] In yet other aspects, the present invention relates to means and methods comprising/utilizing an immobilization of a selective protein ligand on Au-polymer membrane surfaces for reversible binding and potential-controlled elution of monoclonal antibodies.

[34] In further aspects, the present invention relates to means and methods comprising/utilizing monoclonal antibodies that are selectively bound to a membrane modified with protein A, preferably wherein said antibodies instead of being eluted by a pH-dependent elution (e.g., at pH < 3.5) are eluted by the means of a potential switch, which further preferably allows for a more efficient purification of said antibodies and/or higher quality antibody production, e.g., compared to pH-dependent elution (e.g., as described elsewhere herein).

[35] In further aspects, the metallization, which can be used for potential control, can be achieved with, for example gold, which beyond its chemical inactivity can facilitate the immobilization of protein A domains on the membrane. In further aspects, the coupling of cysteine-tagged protein A to gold allows directional binding via only one amino acid of the protein A-based ligand and a resulting high effective binding capacity for monoclonal antibodies.

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

[37] In some further aspects, the present invention relates to means and methods of the present invention (e.g., “digital” ion exchange membranes) showing that even at low flow rates in the range of a few mL/(bar*min*cm2 flilter area) a much faster loading in the order of 100 mg per mL bed volume is possible.

[38] The contemplated dAMC of the present invention (e.g., for the purification of monoclonal antibodies) is distinguished from the established membrane chromatography and its products by at least the following novel/improved features/functionalities:

[39] - Focused recovery of the antibodies by potential switch;

[40] - Reduced use or no use of buffer solutions, no pH shifts;

[41] - Increase of dynamic capacity by additional electrical potentials; [42] Multiple use of the filter unit, as protein A is less degenerated.

[43] In other aspects, the present invention relates to the following parameters for characterizing the purification performance of antibodies according to the present invention (e.g., according to means and methods of the present invention), all of which have been improved compared to standard AC-based and MC-based purification methods:

[44] 1. Adsorption capacity of antibodies in mg mAb per ml bed volume of the membrane is up to 10% breakthrough;

[45] 2. Duration of loading in minutes up to 10% breakthrough;

[46] 3. Duration of mAb recovery (e.g., desorption) up to 90% or 95%;

[47] 4. Auxiliary media required (e.g., salts and/or buffers, etc.) for one purification cycle

[48] 5. Quality of antibodies (e.g., balance aggregates and/or monomers)

[49] 6. Total duration of a purification cycle

[50] 7. Number of possible purification cycles until adsorption capacity falls below 80%.

[51] In other aspects, the present invention relates to decreasing the formation of antibody aggremates, e.g., as compared to standard AC- or MC- based means and/or methods. It is known that aggregates (e.g., Ab aggregates) often occur during classical elution due to the low pH.

[52] In other aspects, the present invention relates to an improved target value achieved by the means and methods of the present invention as compared to standard AC- or MC-based means and methods, which is the integral time in which which quantity of functional antibodies can be purified. From this, it can also be derived, among other things, which performance can be achieved by upscaling (e.g., by using filter modules with filter areas of > 100 cm² effective filter area).

[53] For the most part, membrane adsorbers are not used for the direct recovery and concentration of IgG, but rather in subsequent polishing steps of the purification cascade. However, it is known in the field of protein A-based membrane adsorbers that, for example, the equilibrium binding capacity of one of the first commercialized protein A membranes could be increased 7-fold by increasing the ligand density and the specific surface area. In contrast, the latest generation of commercial protein A chromatography materials have only about a 5% increase in ligand density compared to the previous generation. Accordingly, membrane adsorbers have the ability to run a process in less time while using less buffer, which is further improved by the means and methods of the present invention, e.g., such effects are further enhanced by potential-controlled elution according to the present invention (e.g., according to means and methods of the present invention).

[54] Further, depending on the pH of the eluate, antibodies, being quite flexible, can lose hydrodynamic size at increasingly acidic pH values and thus have a greater tendency to form aggregates. These aggregates in turn are translated into yield losses and additional cleaning effort for their removal. Another knonwn 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. Means and methods according to the present invention overcome the aforementioned problems and thus improve known means and methods significantly.

[55] In some aspects, the present invention relates to means and methods comprising/utilizing a stationary phase that is used to purify, separate, select, and/or isolate biomolecules (preferably antibodies and/or proteins) using affinity chromatography, e.g., using a bind-elute mode. In some aspects, such stationary phase is provided according to “step (a)” of the method/means of the present invention (e.g., a method/device of changing the affinity of a pair of chemical entities or method/device for electrically controllable affinity chromatography).

[56] In some aspects, the stationary phase is characterized by porosity and/or by permeability, allowing the passage of fluids like water and/or alcohol. The immobilization sites of of chemical entities are preferably affixed to either the inner or outer surface of such 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 further aspects, the stationary phase is predominantly constructed from a porous substrate, e.g., a membrane, preferably composed of polymer/s. The immobilization sites of chemical entities can be on either the inner or outer surface of this porous substrate. In some aspects, methods for immobilization of chemical entities comprise, among others, EDC or DCC coupling and/or radical coupling methods. Specifically, these also include methods like electronic beam treatment.

[57] In some aspects, utilizing EDC or DCC coupling for immobilization of chemical entities that create the binding sites generally results in a reasonably uniform orientation of these binding sites on the surface(s). 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.

[58] The chemical entities (e.g., biomolecules) intended for purification, separation, selection, and/or isolation include but are not limited to proteins, particularly immunoglobulins or antibodies (e.g., as described elsewhere herein), including natural or monoclonal antibodies, as well as enzymes or nucleic acids such as RNA or DNA. Notably, the term "biomolecules" also encompasses the inclusion of viruses.

[59] 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.

[60] In some aspects, 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.

[61] In further aspects, it is preferred that the method according to the 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 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 is to ensure that all fluids used maintain a pH value that creates mild conditions for the biomolecules 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 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 fluids) 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. Instead, the 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.

[62] In some aspects, one or more of the electrodes, including possibly all of them, are created using a conductive material, particularly a metal coating. This conductive coating could also involve a metallic net that is positioned on a non-conductive substrate. This non-conductive substrate might consist of polymeric membranes or porous substrates. Alternatively, these conductive coatings might be placed directly onto the affinity substrate, especially a membrane.

[63] Preferably, at least one of the affinity substrates, possibly a membrane, or the non- conductive carrier could 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 affinity substrate, 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. Notably, the conductive coating does not involve a metallic net coated with gold or platinum, and more specifically, it is not formed by a metallic net.

[64] In further aspects, the stationary phase might include at least one electrode (e.g., metal or carbon electrode or an electrode made from other suitable material), particularly formed through metal (or carbon) coatings or metallic (or carbon) nets positioned on the affinity substrate, such as a membrane, or the non-conductive carrier. Within this setup, the ligands and/or receptors responsible for the binding sites could be immobilized on the electrode present in the stationary phase. This immobilization could involve methods such as covalent or associative binding of the receptor and/or ligand to the metal. Notably, the use of sulfur-gold bonding might be employed to bind the ligands and/or receptors to the electrode. [65] In further aspects, 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. Specifically, the distinct stationary phases could be positioned apart from one another along the direction of fluid flow containing the biomolecules. 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).

[66] 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.

[67] 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 subcores. These sub-cores could include their respective ligands and/or receptors.

[68] 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. [69] 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.

[70] 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.

[71] 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. 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.

[72] 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. [73] 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.

[74] 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.

[75] 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.

[76] 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.

[77] The present invention further relates to the following items:

1. A method of changing the affinity of a pair of chemical entities (e.g., biomolecules, preferably proteins and/or antibodies), comprising:

(a) contacting a first member of said pair of chemical entities with a second member of said pair of chemical entities, wherein said first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between at least two electrodes;

(b) applying a voltage between said electrodes; and

(c) changing the voltage between said electrodes, thereby changing the affinity of said pair of chemical entities.

2. The method of any one of the preceding items, wherein said change of said affinity is an increase in affinity leading to increased binding of said second member to said first member of said pair.

3. The method of any one of the preceding items, wherein steps (a) to (c) are contemporaneously performed.

4. The method of any one of the preceding items, further comprising step (o’) further changing the voltage between said electrodes, thereby decreasing binding of said second member to said first member of said pair.

5. The method of any one of the preceding items, further comprising step (a’) contacting said stationary phase comprising said pair of chemical entities with a wash solution at least once.

6. The method of any one of the preceding items, wherein said wash solution comprises a buffer.

7. The method of any one of the preceding items, wherein said wash solution has a pH of not less than 5.0. 8. The method of any one of the preceding items, wherein said wash solution has an ion concentration of not more than 500 mM.

9. A method for electrically controllable affinity chromatography, comprising:

(a) placing a stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities between two electrodes,

(b) applying a voltage between said electrodes; and

(c) changing the voltage between said electrodes, thereby changing the affinity between said first and second member of said pair leading to decreased binding of said second member to said first member of said pair of chemical entities.

10. The method of of any one of the preceding items, wherein step (a) is preceded by the following step (a⁰) contacting a first member of a pair of chemical entities with a second member of said pair of chemical entities, wherein said first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between two electrodes.

11. The method of any one of the preceding items, wherein said change of said affinity is a decrease in affinity leading to decreased binding of said second member to said first member of said pair.

12. The method of any one of the preceding items, wherein the decrease in affinity results in detachment of said second member from said first member of said pair.

13. The method of any one of the preceding items, further comprising step (d) contacting said stationary phase comprising said pair of chemical entities with a recovery solution.

14. The method of any one of the preceding items, wherein step (c) or and step (d) or step (o’) and step (d) are contemporaneously performed.

15. The method of any one of the preceding items, wherein the recovery solution applied in step (d) is applied to the stationary phase in transverse direction and/or in longitudinal direction in relation to said stationary phase being located between said at least two electrodes. 16. The method of any one of the preceding items, wherein said recovery solution comprises a buffer and/or salts or is essentially free of salts.

17. The method of any one of the preceding items, wherein 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.

18. The method of any one of the preceding items, wherein said mobile phase is a solution comprising said second member of said pair, preferably an aqueous solution comprising said second member of said pair.

19. The method of any one of the preceding items, wherein said aqueous solution is crude cell culture medium, clarified cell culture medium or supernatant of cell culture medium.

20. The method of any one of the preceding items, wherein said aqueous solution further comprises contaminants selected from host cell proteins, host cell metabolites, host cell constitutive proteins, nucleic acids, endotoxins, viruses, product related contaminants, lipids, media additives and media derivatives.

21. The method of any one of the preceding items, wherein said first member of said pair is known to non-covalently bind to said second member of said pair; preferably wherein said non-covalent binding does not involve the permanent sharing of electrons by way of a chemical bond; further preferably said non-covalent binding involves variations of electromagnetic interactions between molecules (e.g., 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).

22. The method of any one of the preceding items, wherein said second member of said pair is known to non-covalently bind to said first member of said pair.

23. The method of any one of the preceding items, wherein said pair of chemical entities comprises a first member and a second member selected from antigen and 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).

24. The method of any one of the preceding items, wherein said antigen is a protein affinity tag.

25. The method of any one of the preceding items, wherein said protein tag is the E2 epitope, FLAG epitope, EE-tag, HA tag, HSV epitope, KT3 epitope, Myc epitope, S1-tag, T7 epitope, VSV-G, V5-tag.

26. The method of any one of the preceding items, wherein said first member of said pair is 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).

27. The method of any one of the preceding items, wherein said first member is an aptamer, e.g. a peptide aptamer, DNA aptamer or RNA aptamer, preferably said peptide aptamer is 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).

28. The method of any one of the preceding items, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule, e.g. as a fusion protein.

29. The method of any one of the preceding items, wherein said biomolecule is a protein, preferably an antibody or a nucleic acid, such as DNA or RNA.

30. The method of any one of the preceding items, wherein the voltage in step (c) or step (o’) is such that the second member of said pair is eluted in the form of a single peak. 31. The method of any one of the preceding items, wherein the voltage applied is in the range from -100 V to + 100 V, preferably -10 V to +10 V.

32. The method of any one of the preceding items, wherein said voltage is DC voltage.

33. The method of any one of the preceding items, wherein said voltage is from a constant voltage source, preferably an adjustable constant voltage source.

34. The method of any one of the preceding items, wherein said voltage is from a voltage source which does not react on changing current.

35. The method of any one of the preceding items, wherein the electrodes form a capacitor.

36. The method of any one of the preceding items, wherein the electrode comprises a conductive surface.

37. The method of any one of the preceding items, wherein the electrode comprises an insulating layer between said electrode and said stationary phase.

38. The method of any one of the preceding items, wherein the electrode is configured to allow flow of a solution.

39. The method of any one of the preceding items, wherein said electrode is not in contact with said mobile phase, e.g. not in direct contact.

40. The method 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.

41. The method of any one of the preceding items, wherein said stationary phase acts as a dielectric or insulator between the electrodes.

42. The method of any one of the preceding items, wherein said stationary phase is a carrier allowing immobilization of said first member of said pair of chemical entities.

43. The method of of any one of the preceding items, wherein said carrier is porous.

44. The method of any one of the preceding items, wherein said stationary phase is a liquid, a membrane, a polymer, a non-metal material, felt or foam.

45. The method of any one of the preceding items, wherein the stationary phase comprises at least two stationary phases. 46. The method of any one of the preceding items, wherein said at least two stationary phases are stacked, wrapped or tortuous.

47. The method of any one of the preceding items, wherein said at least two stationary phases are located between two electrodes.

48. The method of any one of the preceding items, wherein each stationary phase is located between a separate set of two electrodes.

49. The method of any one of the preceding items, wherein said stationary phase is in contact with said electrodes is in contact with said electrodes.

50. The method of any one of the preceding items, wherein said contact allows flow of a solution.

51. The method of any one of the preceding items, wherein said stationary is not in contact with said electrodes.

52. The method of any one of the preceding items, wherein said stationary is not in direct contact with said electrodes.

53. The method of any one of the preceding items, wherein said first member of said pair of chemical entities is not directly immobilized on the surface of an electrode.

54. The method of any one of the preceding items, wherein said method is for use in performing electrically controllable affinity chromatography.

55. The method of any one of the preceding items, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities.

56. The method of any one of the preceding items, wherein said method is an antibody purification method, preferably a monoclonal antibody purification method.

57. The method of any one of the preceding items, wherein said method is an antibody purification method, wherein eluted antibody having less dimers and/or fragments compared to that from pH-based elution.

58. Use of an electrical field for eluting a second member of a pair of chemical entities which is bound non-covalently to a first member of said pair of chemical entities, said first member being immobilized on a stationary phase located between two electrodes.

59. A device for changing the affinity of a pair of chemical entities, comprising: (a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution,

(b) a stationary phase between said two electrodes, wherein a first member of said pair of chemical entities is immobilized on said stationary phase, said stationary phase allowing flow of a solution,

(c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, said device being configured to allow flow of a mobile phase comprising a second member of said pair of chemical entities such that said first member of said pair is contacted with s aid second member of said pair, thereby allowing said first and second member to non-covalently bind to each other and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to increase binding of said second member to said first member of said pair or decrease binding of said second member to said first member of said pair. A device for electrically controllable affinity chromatography, comprising:

(a) two electrodes comprising a conductive surface and which are configured to allow flow of a solution,

(b) a stationary phase between two electrodes, said stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non- covalently bound to said first member of said pair of chemical entities,

(c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, said device being configured to allow flow of a solution and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to decrease binding of said second member to said first member of said pair. The device of any one of the preceding items, where said mobile phase is a solution comprising said second member of said pair, preferably an aqueous solution comprising said second member of said pair. 62. The device of any one of the preceding items, wherein said aqueous solution is crude cell culture medium, clarified cell culture medium or supernatant of cell culture medium.

63. The device of any one of the preceding items, wherein said aqueous solution further comprises contaminants selected from host cell proteins, host cell metabolites, host cell constitutive proteins, nucleic acids, endotoxins, viruses, product related contaminants, lipids, media additives and/or media derivatives.

64. The device of any one of the preceding items, wherein said solution is a wash solution and/or recovery solution.

65. The device of any one of the preceding items, wherein said wash solution comprises a buffer.

66. The device of any one of the preceding items, wherein said wash solution has a pH of not less than 5.0.

67. The device of any one of the preceding items, wherein said wash solution has an ion concentration of not more than 500 mM.

68. The device of any one of the preceding items, wherein said recovery solution comprises a buffer and/or salts or is essentially free of salts.

69. The device of any one of the preceding items, wherein 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.

70. The device of any one of the preceding items, wherein the decrease in affinity results in detachment of said second member from said first member of said pair.

71. The device of any one of the preceding items, wherein said device is 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.

72. The device of any one of the preceding items, wherein said first member of said pair is known to non-covalently bind to said second member of said pair; preferably wherein said non-covalent binding does not involve the permanent sharing of electrons by way of a chemical bond; further preferably said non-covalent binding involves variations of electromagnetic interactions between molecules (e.g., 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). 73. The device of any one of the preceding items, wherein said second member of said pair is known to non-covalently bind to said first member of said pair.

74. The device of any one of the preceding items, wherein said pair of chemical entities comprises a first member and a second member selected from antigen and 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).

75. The device of any one of the preceding items, wherein said antigen is a protein affinity tag.

76. The device of any one of the preceding items, wherein said protein tag is the E2 epitope, FLAG epitope, EE-tag, HA tag, HSV epitope, KT3 epitope, Myc epitope, S1-tag, T7 epitope, VSV-G, V5-tag.

77. The device of any one of the preceding items, wherein said first member of said pair is 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).

78. The device of any one of the preceding items, wherein said first member is an aptamer, e.g. a peptide aptamer, DNA aptamer or RNA aptamer, preferably said peptide aptamer is 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).

79. The device of any one of the preceding items, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule, e.g. as a fusion protein.

80. The device of any one of the preceding items, wherein said biomolecule is protein, preferably an antibody, or a nucleic acid, such as DNA or RNA.

81. The device of any one of the preceding items, wherein the voltage is such that the second member of said pair is eluted in the form of a single peak.

82. The device of any one of the preceding items, wherein the voltage applied is in the range from -100 V to + 100 V, preferably -10 V to +10 V.

83. The device of any one of the preceding items, wherein said voltage is DC voltage.

84. The device of any one of the preceding items, wherein said voltage is from a constant voltage source, preferably an adjustable constant voltage source.

85. The device of any one of the preceding items, wherein said controller is a voltage source which does not react on changing current.

86. The device of any one of the preceding items, wherein the electrodes form a capacitor.

87. The device of any one of the preceding items, wherein the electrode comprises a conductive surface.

88. The device of any one of the preceding items, wherein the electrode comprises an insulating layer between said electrode and said stationary phase.

89. The device of any one of the preceding items, wherein the electrode is configured to allow flow of a solution.

90. The device of any one of the preceding items, wherein said electrode is not in contact with said mobile phase, e.g. not in direct contact.

91. The device 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. 92. The device of any one of the preceding items, wherein said stationary phase acts as a dielectric or insulator between the electrodes.

93. The device of any one of the preceding items, wherein said stationary phase is a carrier allowing immobilization of said first member of said pair of chemical entities.

94. The device of any one of the preceding items, wherein said carrier is porous.

95. The device of any one of the preceding items, wherein said stationary phase is a liquid, a membrane, a polymer, a non-metal material, felt or foam.

96. The device of any one of the preceding items, wherein the stationary phase comprises at least two stationary phases.

97. The device of any one of the preceding items, wherein said at least two stationary phases are stacked, wrapped or tortuous.

98. The device of any one of the preceding items, wherein said at least two stationary phases are located between two electrodes.

99. The device of any one of the preceding items, wherein each stationary phase is located between a separate set of two electrodes.

100. The device of any one of the preceding items, wherein said stationary phase is in contact with said electrodes.

101. The device of any one of the preceding items, wherein said contact allows flow of a solution.

102. The device of any one of the preceding items, wherein said stationary is not in contact with said electrodes.

103. The device of any one of the preceding items, wherein said stationary is not in direct contact with said electrodes.

104. The device of any one of the preceding items, wherein said first member of said pair of chemical entities is not directly immobilized on the surface of an electrode.

105. The device of any one of the preceding items, wherein said device is for use in performing electrically controllable affinity chromatography.

106. The device of any one of the preceding items, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

107. Use of a device of any one of the preceding items for performing electrically controllable affinity chromatography, preferably said electrically controllable affinity chromatography is for antibody (e.g., mAb) purification.

108. The use of any one of the preceding items, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

109. Use of a device of any one of the preceding items, for purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

110. The use of any one of the preceding items, wherein said biomolecule is a protein, preferably an antibody or a nucleic acid, such as DNA or RNA.

111. The method, device or use of any one of the preceding items, carried out and/or configured as shown in Example 1 and/or in Example 2 herein (e.g., as shown Figures 1 and/or 2 and/or 3 herein), preferably wherein an eluted antibody having less dimers and/or fragments and/or aggregates compared to that derived from a pH-based elution (e.g., as in AC- or MC-based elution).

112. The method, device or use of any one of the preceding items, wherein said method, device or use is electrochemically-modified and/or electrically controllable.

****

[78] Definitions:

[79] 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.

[80] 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.

[81] 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".

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

[83] 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 %.

[84] 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.

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

[86] The term “chemical entities” as used herein may relate to different types of molecules or substances that are subject to separation and analysis using chromatographic techniques. These entities can encompass a wide range of compounds, including small molecules, biomolecules, and other substances, each possessing distinct chemical properties. The term "chemical entities" within chromatography may also refer to the diverse substances subjected to separation, such as organic compounds, inorganic ions, biomolecules, proteins, nucleic acids, and other molecules, each of which can be characterized based on their unique chromatographic behaviors.

[87] The term “stationary phase” as used herein may have a meaning of one of the components in chromatography (e.g., according to means and methods depicted herein). It 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 phase, different compounds (e.g., chemical entities) interact differently with the stationary phase, leading to their separation based on their respective affinities or retention times. The nature and composition of the stationary phase may vary depending on the type of chromatography, but it typically remains fixed in its position, contrasting with the mobile phase that moves through and/or over it. Suitable stationary phases are known in the art and could include, but are not limited to, stationary phase made from materials such as gels, resins, and/or glass fibers. Moreover, they can also be derived from any sutable porous substrate(s), such as membranes, specifically polymeric membranes, serving as the foundational material for the stationary phase. Preferably, the stationary phase is porous, in particular permeable, in particular to fluids, e.g., water and/or alcohol.

[88] The term “affinity” as used herein 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.

[89] The term “immobilized on a stationary phase” as used herein may mean that the substance or chemical entity 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 der Waals 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 charge.

[90] The term “contained in a mobile phase” as used herein may refer to the solute (e.g., chemical entity as used herein) or mixture of solutes being dissolved or suspended in the liquid or gas that is passed through the stationary phase. For example, before the chromatographic process begins, the sample, or analyte as used herein, is introduced into the mobile phase. As the mobile phase travels through the stationary phase (e.g., like a column packed with some material), different components or compounds of the analyte interact differently with the stationary phase, leading to their separation from each other. Accordingly, the term “second member of said pair is contained in a mobile phase” may essentially mean that said second member of said pair is part of the mixture being transported by the mobile phase and is subject to separation based on its interactions with the stationary phase. [91] The term “mobile phase” as used herein may refer to a phase that moves in a definite direction in a chromatographic system. It can be a liquid, a gas, or a supercritical fluid. The mobile phase carries the analyte (the mixture that needs to be separated) through the system.

[92] 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).

[93] The terms “non-covalent binding” or “non-covalently bind” or “non-covalently bound” as used herein can be used interchangeably and does 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.

[94] The term “voltage” often denoted as V as 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.

[95] The terms “direct current voltage”, often abbreviated as "DC voltage," as used herein may refer to a type of electrical voltage that remains constant in polarity and magnitude over time.

[96] 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.

[97] 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. [98] 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.

[99] 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.

[100] The term “conductive surface” as used herein may refer to a material or area 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.

[101] 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 like plastics, ceramics, or coatings with high electrical resistance.

[102] The term “electrode is configured to allow flow of a solution” 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.

[103] The term “electrically conductive coating” as used herein 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, 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.

[104] 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.

[105] 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.

[106] The term “immobilization" as used herein may refers 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.

[107] The term “electrically controllable” as used herein may refer the ability of a device, system, method or component to be manipulated, adjusted, and/or regulated using an electric signal or voltage.

[108] The term “electrode is not formed by an electrically conductive coating” as used herein may refer to an electrode that is not created or composed of a layer of material applied to the surface to make it conductive. In other words, the electrode is not coated with a substance that enhances its electrical conductivity.

[109] 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 or method.

[110] The term “contemporaneously” as used herein may refer to something happening at or around the same time as something else. It can be used to describe events, actions, or situations that occur simultaneously or in a close time frame (e.g., within time frame of 1 , 2 or 3 seconds).

[111] 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. [112] 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).

[113] 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.

[114] 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.

[115] 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.

[116] The term “digital” as used herein may have a meaning of “electrochemically-modified” and/or “electrically controllable”, which in the context of the present invention relating to means and methods relating to Affinity Chromatography (e.g., Liquid Affinity Chromatography) can accordingly mean that said means and methods relating to the Affinity Chromatography (e.g., Liquid Affinity Chromatography) are electrochemically-modified and/or electrically controllable.

[117] The term “recovery solution” as used herein may refer to to a specific solution or set of conditions applied to the chromatographic system to release and/or recover the target biomolecule that has been bound to a stationary phase.

[118] The term “transverse direction” as used herein may refer to a perpendicular orientation or movement relative to a specified reference point or axis. [119] The term “longitudinal direction” as used herein may refer to to the primary or main orientation or movement along a specified reference point or axis.

[120] 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.

[121] The term “crude cell culture medium” as used herein may refer 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.

[122] The term “clarified cell culture medium” as used herein may refer 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.

[123] The term “supernatant of cell culture medium” as used herein may refer to a clear liquid portion that remains on top after a mixture of cells and medium has been subjected to a centrifugation or settling process.

[124] The term “polypeptide” is equally used herein with the term "protein". 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.

[125] 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, lgG-4, 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 lgG1 subtype, while the lgG4 subtype would not have the capability to exert ADCC.

[126] 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.

[127] Furthermore, the term "antibody" as employed in the 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 amine-groups 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.

[128] 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.

[129] 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.

[130] 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).

[131] The term “protein tag” as used herein may refer to a short amino acid sequence that is genetically fused to a target protein. A purpose of adding a protein tag to a target protein may be to facilitate its detection, purification, localization, and/or manipulation within cells and/or in experimental settings.

[132] The term “protein affinity tag” as used herein may refer to a short amino acid sequence that is genetically fused to a target protein to enable its easy purification, detection, and/or manipulation. Affinity tags may have specific properties that allow them to interact with complementary molecules, such as antibodies or affinity resins, facilitating the isolation and/or study of the tagged protein. These tags are designed to possess an affinity for certain binding partners. [133] As described herein references can be made to UniProtKB Accession Numbers (http://www.uniprot.org/, e.g., as available in UniProt release 2023_03 published 28 Jun 2023).

[134] 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.

[135] 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.

[136] 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. [137] 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.

[138] 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.

[139] 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.

[140] 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.

[141] The term “biomolecule” as used herein may refer to 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, and signaling molecules.

[142] The term “fusion protein” as used herein may refer to a genetically engineered protein that is created by joining two or more individual protein coding sequences.

[143] 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.

[144] 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. [145] 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.

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

EXAMPLES

[147] 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.

[148] Example 1 :

[149] Membranes (e.g. Sartorius Sartobind Rapid A) are punched out (d=22 mm). The Sartobind membrane which has Protein A immobilized was incubated overnight in a 0.3 mg/mL IgG (Trastuzumab)//PBS solution at 4 °C (saturation of the affinity membrane). The membranes are sandwiched together as shown in Figure 1A and installed in a syringe filter (see Figure 1 B). The "sandwich" is then washed with Millipore water for 15 min at 0.5 mL/min. Once equilibrium is reached start the potential gradient, increasing from 0 to 900 mV in 50 mV/min steps. The corresponding fraction is collected (see Figure 2).

[150] An antibody, e.g. Trastuzumab eluted pursuant to the teaching of the present elutes in the form of a single peak. A thus eluted antibody shows significantly less dimers and fragments than antibodies eluted traditionally via, e.g. pH shift.

[151] Of course, this example is only illustrative for the principle of eluting a second member of a pair of chemical entities comprising a first and second member as described herein. Indeed, as explained herein and pursuant to the teaching of the present invention affinity between a first and second member of a pair of chemical entities, e.g. through non-covalent binding between a first and second member of a pair of chemical entities can be changed by voltage to, e.g. increase or decrease the affinity.

Example 2:

Membranes (e.g. Sartorius Sartobind Rapid A) are punched out (d=22 mm). The Sartobind membrane which has Protein A immobilized was loaded with 700 pg IgG. The membranes are sandwiched together as shown in Figure 1A and installed in a syringe filter (see Figure 1 B). The "sandwich" is then washed with buffer (10 mM salt containing phosphate buffer at pH 7.4) for 15 min at 0.5 mL/min. Once equilibrium is reached, the potential is switched to 2400 mV. 89.95% of the protein is recovered via potential switch. The remaining protein was eluted via pH switch to pH 2.8; see Figure 3.

[152] 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.

[153] 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.

[154] 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.

[155] 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.

Claims

1. A method of changing the affinity of a pair of chemical entities, comprising:

(b) applying a voltage between said electrodes; and

2. The method of claim 1 , wherein said change of said affinity is an increase in affinity leading to increased binding of said second member to said first member of said pair.

3. The method of claim 2, wherein steps (a) to (c) are contemporaneously performed.

4. The method of any one of claims 1 to 3, further comprising step (o’) further changing the voltage between said electrodes, thereby decreasing binding of said second member to said first member of said pair.

5. The method of any one of the preceding claims, further comprising step (a’) contacting said stationary phase comprising said pair of chemical entities with a wash solution at least once.

6. The method of claim 5, wherein said wash solution comprises a buffer.

7. The method of claim 5 or 6, wherein said wash solution has a pH of not less than 5.0.

8. The method of any one of claims 5 to 7, wherein said wash solution has an ion concentration of not more than 500 mM.

9. A method for electrically controllable affinity chromatography, comprising:

(a) placing a stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities between two electrodes, (b) applying a voltage between said electrodes; and

10. The method of claim 9, wherein step (a) is preceded by the following step (a⁰) contacting a first member of a pair of chemical entities with a second member of said pair of chemical entities, wherein said first member of said pair is immobilized on a stationary phase and said second member of said pair is contained in a mobile phase, thereby allowing said first and second member to non-covalently bind to each other, wherein said stationary phase is located between two electrodes.

11. The method of any one of claims 1 and 5 to 10, wherein said change of said affinity is a decrease in affinity leading to decreased binding of said second member to said first member of said pair.

12. The method of claim 4 or 11 , wherein the decrease in affinity results in detachment of said second member from said first member of said pair.

13. The method of any one of the preceding claims, further comprising step (d) contacting said stationary phase comprising said pair of chemical entities with a recovery solution.

14. The method of any one of the preceding claims, wherein step (c) or and step (d) or step (o’) and step (d) are contemporaneously performed.

15. The method of any one of the preceding claims, wherein the recovery solution applied in step (d) is applied to the stationary phase in transverse direction and/or in longitudinal direction in relation to said stationary phase being located between said at least two electrodes.

16. The method of any one of claims 13 to 15, wherein said recovery solution comprises a buffer and/or salts or is essentially free of salts.

17. The method of claim 16, wherein 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.

18. The method of any one of the preceding claims, wherein said mobile phase is a solution comprising said second member of said pair, preferably an aqueous solution comprising said second member of said pair.

19. The method of claim 18, wherein said aqueous solution is crude cell culture medium, clarified cell culture medium or supernatant of cell culture medium.

20. The method of claim 18 or 19, wherein said aqueous solution further comprises contaminants selected from host cell proteins, host cell metabolites, host cell constitutive proteins, nucleic acids, endotoxins, viruses, product related contaminants, lipids, media additives and media derivatives.

21. The method of any one of the preceding claims, wherein said first member of said pair is known to non-covalently bind to said second member of said pair; preferably wherein said non-covalent binding does not involve the permanent sharing of electrons by way of a chemical bond; further preferably said non-covalent binding involves variations of electromagnetic interactions between molecules (e.g., 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).

22. The method of any one of the preceding claims, wherein said second member of said pair is known to non-covalently bind to said first member of said pair.

23. The method of any one of the preceding claims, wherein said pair of chemical entities comprises a first member and a second member selected from antigen and corresponding antibody, albumin and albumin-binding protein (ABP), avidin and biotin-carboxy carrier protein (BCCP), streptavidin and biotin-carboxy carrier protein (BCCP), calmodulin and calmodulin binding peptide (CBP), chloramphenicol and chloramphenicol acetyl transferase (CAT), cellulose and cellulose binding domain (CBP), chitin and chitin binding domain (CBD), choline and choline-binding domain (CBD), galactose and galactose-binding protein (GBP), glutathione and glutathione S-transferase (GST), 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), streptavidin 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)).

24. The method of claim 23, wherein said antigen is a protein affinity tag.

25. The method of claim 24, wherein said protein tag is the E2 epitope, FLAG epitope, EE-tag, HA tag, HSV epitope, KT3 epitope, Myc epitope, S1-tag, T7 epitope, VSV-G, V5-tag.

26. The method of any one of the preceding claims, wherein said first member of said pair is protein A, protein G or protein L.

27. The method of any one of the preceding claims, wherein said first member is an aptamer, e.g. a peptide aptamer, DNA aptamer or RNA aptamer, preferably said peptide aptamer 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)).

28. The method of any one of the preceding claims, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule, e.g. as a fusion protein.

29. The method of claim 28, wherein said biomolecule is a protein, preferably an antibody or a nucleic acid, such as DNA or RNA.

30. The method of any one of the preceding claims, wherein the voltage in step (c) or step (o’) is such that the second member of said pair is eluted in the form of a single peak.

31. The method of any one of the preceding claims, wherein the voltage applied is in the range from -100 V to + 100 V, preferably -10 V to +10 V.

32. The method of any one of the preceding claims, wherein said voltage is DC voltage.

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

34. The method of any one of the preceding claims, wherein said voltage is from a voltage source which does not react on changing current.

35. The method of any one of the preceding claims, wherein the electrodes form a capacitor.

36. The method of any one of the preceding claims, wherein the electrode comprises a conductive surface.

37. The method of any one of the preceding claims, wherein the electrode comprises an insulating layer between said electrode and said stationary phase.

38. The method of any one of the preceding claims, wherein the electrode is configured to allow flow of a solution.

39. The method of any one of the preceding claims, wherein said electrode is not in contact with said mobile phase, e.g. not in direct contact.

40. The method 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.

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

42. The method of any one of the preceding claims, wherein said stationary phase is a carrier allowing immobilization of said first member of said pair of chemical entities.

43. The method of claim 42, wherein said carrier is porous.

44. The method of any one of claims 41 to 43, wherein said stationary phase is a liquid, a membrane, a polymer, a non-metal material, felt or foam.

45. The method of any one of the preceding claims, wherein the stationary phase comprises at least two stationary phases.

46. The method of claim 45, wherein said at least two stationary phases are stacked, wrapped or tortuous.

47. The method of claim 45 or 46, wherein said at least two stationary phases are located between two electrodes.

48. The method of claim 45 or 46, wherein each stationary phase is located between a separate set of two electrodes.

49. The method of any one of the preceding claims, wherein said stationary phase is in contact with said electrodes is in contact with said electrodes.

50. The method of claim 49, wherein said contact allows flow of a solution.

51. The method of any one of the preceding claims, wherein said stationary is not in contact with said electrodes.

52. The method of any one of the preceding claims, wherein said stationary is not in direct contact with said electrodes.

53. The method of any one of the preceding claims, wherein said first member of said pair of chemical entities is not directly immobilized on the surface of an electrode.

54. The method of any one of the preceding claims, wherein said method is for use in performing electrically controllable affinity chromatography.

55. The method of claim 54, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities.

56. Use of an electrical field for eluting a second member of a pair of chemical entities which is bound non-covalently to a first member of said pair of chemical entities, said first member being immobilized on a stationary phase located between two electrodes.

57. A device for changing the affinity of a pair of chemical entities, comprising:

(c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, said device being configured to allow flow of a mobile phase comprising a second member of said pair of chemical entities such that said first member of said pair is contacted with s aid second member of said pair, thereby allowing said first and second member to non- covalently bind to each other and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to increase binding of said second member to said first member of said pair or decrease binding of said second member to said first member of said pair.

58. A device for electrically controllable affinity chromatography, comprising:

(b) a stationary phase between two electrodes, said stationary phase comprising a first member of a pair of chemical entities which is immobilized on said stationary phase and a second member of said pair of chemical entities which is non-covalently bound to said first member of said pair of chemical entities, (c) a controller functionally associated with a power supply configured for applying and changing a voltage between said electrodes, said device being configured to allow flow of a solution and further configured to apply a voltage between said electrodes, wherein changing the voltage between said electrodes changes the affinity of said pair of chemical entities so as to decrease binding of said second member to said first member of said pair.

59. The device of claim 57, where said mobile phase is a solution comprising said second member of said pair, preferably an aqueous solution comprising said second member of said pair.

60. The device of claim 59, wherein said aqueous solution is crude cell culture medium, clarified cell culture medium or supernatant of cell culture medium.

61. The device of claim 59 or 60, wherein said aqueous solution further comprises contaminants selected from host cell proteins, host cell metabolites, host cell constitutive proteins, nucleic acids, endotoxins, viruses, product related contaminants, lipids, media additives and/or media derivatives.

62. The device of any one of claims 57 to 61, wherein said solution is a wash solution and/or recovery solution.

63. The device of claim 62, wherein said wash solution comprises a buffer.

64. The device of claim 62 or 63, wherein said wash solution has a pH of not less than 5.0.

65. The device of any one of claims 62 to 64, wherein said wash solution has an ion concentration of not more than 500 mM.

66. The device of any one of claims 62 to 65, wherein said recovery solution comprises a buffer and/or salts or is essentially free of salts.

67. The device of claim 66, wherein 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.

68. The device of any one of claims 57 to 67, wherein the decrease in affinity results in detachment of said second member from said first member of said pair.

69. The device of any one of claims 57 to 68, wherein said device is 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.

70. The device of any one of claims 55 to 69, wherein said first member of said pair is known to non-covalently bind to said second member of said pair; preferably wherein said non- covalent binding does not involve the permanent sharing of electrons by way of a chemical bond; further preferably said non-covalent binding involves variations of electromagnetic interactions between molecules (e.g., 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).

71. The device of any one of claims 57 to 70, wherein said second member of said pair is known to non-covalently bind to said first member of said pair.

72. The device of any one of claims 57 to 71 , wherein said pair of chemical entities comprises a first member and a second member selected from antigen and corresponding antibody, albumin and albumin-binding protein (ABP), avidin and biotin-carboxy carrier protein (BCCP), streptavidin and biotin-carboxy carrier protein (BCCP), calmodulin and calmodulin binding peptide (CBP), chloramphenicol and chloramphenicol acetyl transferase (CAT), cellulose and cellulose binding domain (CBP), chitin and chitin binding domain (CBD), choline and choline- binding domain (CBD), galactose and galactose-binding protein (GBP), glutathione and glutathione S-transferase (GST), 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 polyhistidine (His tag), cross-linked amylose or maltose and maltose-binding protein (MBP), streptavidin and streptavidin binding peptide (SBP), 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)).

73. The device of claim 72, wherein said antigen is a protein affinity tag.

74. The device of claim 73, wherein said protein tag is the E2 epitope, FLAG epitope, EE-tag, HA tag, HSV epitope, KT3 epitope, Myc epitope, S1-tag, T7 epitope, VSV-G, V5-tag.

75. The device of any one of claims 57 to 74, wherein said first member of said pair is protein A, protein G or protein L.

76. The device of any one of claims 57 to 74, wherein said first member is an aptamer, e.g. a peptide aptamer, DNA aptamer or RNA aptamer, preferably said peptide aptamer 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)).

77. The device of any one of the claims 57 to 76, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule, e.g. as a fusion protein.

78. The device of claim 77, wherein said biomolecule is protein, preferably an antibody, or a nucleic acid, such as DNA or RNA.

79. The device of any one of claims 57 to 78, wherein the voltage is such that the second member of said pair is eluted in the form of a single peak.

80. The device of any one of claims 57 to 79, wherein the voltage applied is in the range from - 100 V to + 100 V, preferably -10 V to +10 V.

81. The device of any one of claims 57 to 80, wherein said voltage is DC voltage.

82. The device of any one of claims 57 to 81, wherein said voltage is from a constant voltage source, preferably an adjustable constant voltage source.

83. The device of any one of claims 57 to 82, wherein said controller is a voltage source which does not react on changing current.

84. The device of any one of claims 57 to 83, wherein the electrodes form a capacitor.

85. The device of any one claims 57 to 84, wherein the electrode comprises a conductive surface.

86. The device of any one of claims 57 to 85, wherein the electrode comprises an insulating layer between said electrode and said stationary phase.

87. The device of any one of claims 57 to 86, wherein the electrode is configured to allow flow of a solution.

88. The device of any one of claims 57 to 87, wherein said electrode is not in contact with said mobile phase, e.g. not in direct contact.

89. The device of any one of claims 57 to 88, 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.

90. The device of any one of claims 57 to 89, wherein said stationary phase acts as a dielectric or insulator between the electrodes.

91. The device of any one of claims 57 to 90, wherein said stationary phase is a carrier allowing immobilization of said first member of said pair of chemical entities.

92. The device of claim 91 , wherein said carrier is porous.

93. The device of any one of claims 90 to 92, wherein said stationary phase is a liquid, a membrane, a polymer, a non-metal material, felt or foam.

94. The device of any one of claims 57 to 93, wherein the stationary phase comprises at least two stationary phases.

95. The device of claim 94, wherein said at least two stationary phases are stacked, wrapped or tortuous.

96. The device of claim 94 or 95, wherein said at least two stationary phases are located between two electrodes.

97. The device of claim 94 or 95, wherein each stationary phase is located between a separate set of two electrodes.

98. The device of any one of claims 57 to 97, wherein said stationary phase is in contact with said electrodes.

99. The device of claim 98, wherein said contact allows flow of a solution.

100. The device of any one of claims 57 to 99, wherein said stationary is not in contact with said electrodes.

101. The device of any one of claims 57 to 100, wherein said stationary is not in direct contact with said electrodes.

102. The device of any one of claims 57 to 101, wherein said first member of said pair of chemical entities is not directly immobilized on the surface of an electrode.

103. The device of any one of claims 57 to 102, wherein said device is for use in performing electrically controllable affinity chromatography.

104. The device of claim 103, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

105. Use of a device of any one of claims 57 to 102 for performing electrically controllable affinity chromatography.

106. The use of claim 105, wherein affinity chromatography aids in purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

107. Use of a device of any one of claims 57 to 102 for purifying, separating or isolating said second member of said pair of chemical entities, wherein said second member of said pair is a biomolecule or is comprised by a biomolecule.

108. The use of claim 107, wherein said biomolecule is a protein, preferably an antibody or a nucleic acid, such as DNA or RNA.