Selective isolation method for amphisexual species

Abstract

The present invention relates to a method for selectively separating amphoteric species, particularly biomolecules or biomolecular complexes, e.g., vectors containing molecules, based on their isoelectric point. For selective separation, a charged porous surface is provided, which is electrically conductive and / or includes an electrically conductive coating or layer. A fluid containing amphoteric species is contacted with the surface, resulting in the capture of the species on the surface through attractive interaction forces. At least a portion of the adsorbed species is then released by applying a release voltage selected to change the net interaction force between the surface and the adsorbed species from attractive to repulsive. Selectivity can be achieved, for example, by selective adsorption, in which at least one species is not captured, and / or by selective desorption, in which some pre-adsorbed species are selectively released while others remain bound to the surface, preferably only one pre-adsorbed species.

Description

[Technical Field]

[0001] The present invention relates to a method for the selective separation of amphoteric species, in particular (biomolecules), such as proteins, amino acids or nucleic acids, or molecular complexes, such as vectors, in particular capsids, plasmids or vesicles, with respect to their isoelectric points and molecules, such as genomes contained within molecular complexes, in particular vectors, by selective (electro)adsorption and electrodesorption, in particular to prepare highly pure samples of desired amphoteric species, in particular vectors and / or biomolecules. [Background technology]

[0002] Biomolecules are typically species in high demand for medical applications, such as proteins used as drugs. Therefore, desired biomolecules must be produced with high purity and therefore separated from impurities resulting from their formation. Such separations are often achieved by a bind-elute process. The desired molecules are bound to, for example, an exchange resin, gel, or membrane, and then eluted from the resin, gel, or membrane by a change in pH, e.g., acidic elution using an acetate buffer at pH 3, and / or by using a salt solution that displaces the desired biomolecule with salt ions. Both of these elution methods can result in harsh conditions that can be harmful to any biomolecule, e.g., proteins, and can cause aggregation or denaturation of the desired species. Examples of such methods are isoelectric focusing or ion-exchange chromatography.

[0003] A particularly interesting species in the field of biomedicine is vectors. Such vectors are commonly used as tools for delivering molecules, especially biologically active substances, into cells. As such, vectors play an important role in the fields of gene therapy and vaccines or other treatments, especially in personalized cancer therapy, for example. For these purposes, vectors are prepared that contain molecules, especially in the form of biologically active substances, for example, vectors that contain genomes in the form of deoxyribonucleic acid (DNA), single-stranded deoxyribonucleic acid (ssDNA), ribonucleic acid (RNA) or single-stranded ribonucleic acid (ssRNA).

[0004] Commonly used viral vectors include those based on retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses. In all of these, capsid proteins form a shell in which molecules, particularly biologically active substances such as pharmaceuticals and / or genomes in the form of DNA, RNA, ssDNA, or ssRNA, can be placed. As a result, capsids can be used, for example, as a means of delivering biologically active substances, particularly as a protective shell for the molecules to be delivered, and can be used as a vehicle for artificially transporting biologically active substances, such as nucleic acid sequences, usually DNA or RNA, into cells, for example, for expression or replication. Plasmids, which are primarily found in bacteria and are often small, circular, and primarily double-stranded DNA or RNA molecules, are also commonly used as vectors. For gene therapy, plasmids are primarily used by incorporating the desired genomic sequence into the plasmid itself. The plasmid can then move into cells and participate in the replication of the integrated genomic sequence, among other things.

[0005] For gene therapy and other medical applications, it is desirable to use highly purified vectors. However, when preparing vectors containing molecules, the vectors may generally differ in the molecules they contain, particularly in terms of molecular weight and / or the molecules they contain. For example, they may contain only a specific molecule or a portion of such a molecule, or some vectors may not contain any molecule at all. For example, when preparing genome-containing capsids, the resulting vectors usually contain the entire genome, a partial genome, or no genome at all. In order to use them in therapy, it is desirable to selectively separate vectors containing the correct, especially the entire genome or molecule in the correct amount, from vectors that do not meet these conditions, especially those that do not completely meet them.

[0006] Several techniques are known for separating vectors containing the entire genome from vectors containing partial or no genomes, such as anion exchange high performance liquid chromatography (AEX HPLC), capillary isoelectric focusing (cIF), ultracentrifugation (UCF) or gel isoelectric focusing (gIF). Of these, AEX HPLC, cIF and gIF are used as preliminary techniques in the preparation of vectors for gene therapy etc., while UCF is mostly common in analytics. Notably, these techniques are not limited to vectors but can be applied to amphiphilic species in general, especially biomolecules or biomolecular complexes.

[0007] Both the AEX HPLC and IF methods are based on the overall charge of the capsid, which is a function of the amount of genome contained in the capsid relative to the pH value of the surrounding environment. However, the differences in isoelectric points between full capsids, partial capsids, and empty capsids are small, e.g., in the 0.1 regime.

[0008] In the AEX HPLC method, capsids with a cationic overall charge, and therefore a positive net overall charge, are adsorbed onto a chemically negatively charged surface, such as a resin, containing anionic binding sites. To elute vectors bound to the anionic binding sites, it is common to change the pH value of the environment to change the net overall charge of the vector from negative to neutral or positive, or to use salt to displace the bound vectors by removing them from the binding sites, effectively blocking binding due to their high affinity and releasing the vectors. For example, MgCl and / or NaCl solutions can be used as eluents. However, vectors released using such salt solutions must be subsequently separated from the salt, as high concentrations of salt can be harmful in medical applications. Furthermore, elution by changing the pH value of the environment can harm the vectors themselves, as aggregation of the capsid protein can occur, especially if the capsid protein has a neutral charge.

[0009] Similarly, isoelectric focusing techniques such as gel isoelectric focusing or capillary isoelectric focusing suffer from the same problems and limitations as AEX HPLC. In this isoelectric focusing technique, for example, a gel consisting of several regions with different pH values ​​is prepared. A solution containing amphoteric biomolecules with different isoelectric points is contacted with the gel. The different pH value regions are separated and increase in a gradient along one axis. An electric field is applied within the axis. The amphoteric biomolecules then migrate along the electric field relative to their charge. When the molecules reach a region with a pH value equal to their isoelectric point, the amphoteric biomolecules become charge-neutral and stop migrating along the axis. This separates the biomolecules based on their isoelectric points. However, because the charge of the biomolecules becomes neutral, they are prone to aggregation, which may damage the biomolecules.

[0010] Furthermore, due to the small difference in isoelectric point, these techniques are often limited in terms of purity. Typically, both AEX HPLC and IF methods produce samples that are, for example, 70% full-genome-containing capsids, with the remainder being primarily capsids with partial genomes or empty capsids. As a result, it is desirable to achieve a high level of separation between these vectors to obtain highly pure capsid samples.

[0011] Other methods for separating biomolecules generally involve filtration by electrosorption. Among these, European Patent No. 3115099 describes a method for enhancing biomolecule retention by using a metal-coated polymer membrane and applying a voltage to the membrane. Furthermore, International Publication No. 2021 / 084080 describes a method for electrodesorption of molecules adsorbed to a charged membrane having a metal coating by applying a voltage of opposite polarity to the charge of the membrane to the coating. However, such methods cannot achieve the selectivity required for separating biomolecules with only slight differences, particularly vectors containing whole genomes, partial genomes, or no genomes. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] European Patent No. 3115099 [Patent Document 2] International Publication No. 2021 / 084080 Summary of the Invention [Problem to be solved by the invention]

[0013] Based on this, the problem is to provide a suitable method for separating amphoteric species, especially biomolecules or biomolecular complexes, especially vectors. The differences may be very slight, but still provide a high-purity separation. Desorption due to changes in pH and / or application of salt solutions is avoided, thereby eliminating potential harm from degradation of the separated biomolecules and biomolecular complexes. If the separated biomolecules and biomolecular complexes are degraded in any way, this could pose a health risk, especially in medical applications. [Means for solving the problem]

[0014] The problem of the present invention is solved by a method according to claim 1. The subsequent claims 2 to 25 provide advantageous embodiments of the method according to the invention.

[0015] The present invention provides a method for selectively separating amphoteric species, particularly based on their isoelectric points. The species to be separated consist of at least two specific species, each of which possesses a specific isoelectric point. This allows each specific species to have a specific net overall charge at a predetermined pH value, particularly according to its specific isoelectric point. The predetermined pH value may particularly be a defined pH value.

[0016] The predetermined pH value is preferably in the range of pH 6 to pH 9, preferably in the range of pH 6.5 to pH 8.5, and more preferably in the range of pH 7 to pH 8, and is the least harmful to species commonly used for treatment or naturally occurring in the human body.

[0017] In this context, the term "species" may refer to any (bio)molecule or (bio)molecule complex. (Bio)molecules that can be separated using the present invention include, but are not limited to, amino acids, proteins, nucleic acids such as RNA or DNA.

[0018] (Bio)molecular complexes can include, but are not limited to, vectors containing molecules, in particular biomolecules.

[0019] The term "vector containing a molecule" refers to a vector that contains a molecule in at least a portion of the vector, which molecule differs in each portion of the vector, e.g., in amount and / or type, thereby providing at least two populations of vectors with different contained molecules as at least two distinct species that are segregated.

[0020] In this context, the term "having different contained molecules" includes different molecules and / or different amounts of molecules, in particular different amounts of the same molecule and / or different parts of the same molecule and / or different specific molecules. In particular, the term "different amounts of molecules" can include a molecular weight of zero. As a result, some vectors may not contain any molecule at all, and thus "empty" vectors may form or be part of a vector population.

[0021] In particular, a vector may consist of a vector of the same type only and / or may have the same basic structure, but without the particular molecules contained therein. In particular, a vector may consist of a capsid and / or a plasmid. In particular, a vector may be a viral vector, for example based on a retrovirus, lentivirus, adenovirus or adeno-associated virus vector.

[0022] Every vector has a reference isoelectric point and therefore a reference net overall charge, particularly at a given pH value in the surrounding environment. In particular, the reference isoelectric point corresponds to the isoelectric point of an empty vector, i.e., a vector that does not contain any molecules. Similarly, the reference net overall charge is the net overall charge of the empty vector. Among vectors containing molecules, the reference isoelectric point and reference net overall charge of any particular vector are shifted by the molecules contained in the particular vector. As a result, a shifted isoelectric point different from the reference isoelectric point and a shifted net overall charge at a given pH value different from the reference overall charge at the given pH value are caused by the molecules contained in the particular vector. Thus, at least two vector groups are defined based on the molecules contained in each vector group, particularly with respect to different molecules and / or different amounts of molecules contained. In particular, the different molecules can be different types of molecules and / or different parts of the same molecule and / or the same type of molecule.

[0023] In particular, among at least two vector groups, a first vector group has a first isoelectric point and / or a range of first isoelectric points and / or a first overall charge and / or a range of first net overall charges at a given pH value, and a second vector group has a second isoelectric point and / or a range of second isoelectric points and / or a second net overall charge and / or a range of second net overall charges at a given pH value. In particular, groups defined by a range of isoelectric points and / or a range of net overall charges may not be uniform and may in particular be composed of more than one type of vector and / or vectors containing different molecules and / or different amounts of molecules. In particular, the included molecules affect the electronic structure of the vector. This effect may cause a shift in the isoelectric point and / or net overall charge of vectors containing molecules. In particular, the shifted isoelectric points and / or shifted net overall charges may be classified into ranges, and it is desirable to classify the vectors into vector groups falling within these ranges. In particular, for undesired impurities, it may not be necessary to separate them into several groups. As a result, where applicable, a wide range of isoelectric points and / or net overall charges can be defined, consisting of a large number of different impurities, e.g., empty vectors and partially filled vectors.

[0024] In particular, the at least two populations of vectors can be selectively separated from each other by carrying out the method according to the invention.

[0025] In yet another embodiment, the method of the present invention can be used to separate groups of species. Among these groups of species, each group may consist of more than one specific species. In particular, such species may be species of comparable types with minor differences, such as biomolecules with comparable medical applicability. For example, enantiomers may not have enantiomer-specific uses, particularly if separation is not required for medical use, such as protein variants that are part of a protein family. For such groups, the specific isoelectric points and / or specific net overall charges of the species in such groups may be particularly diverse, ranging in isoelectric points and / or net overall charges, e.g., one group having a first range and a second group having a different, particularly diverse range. In particular, such specific groups of species may be defined by such a wide range of isoelectric points and / or net overall charges. DETAILED DESCRIPTION OF THE INVENTION

[0026] The method comprises: a) providing an electrically charged, in particular chemically and / or electrically charged, porous surface, the electrically charged porous surface being electrically conductive and / or comprising an electrically conductive coating or layer, in particular a conductive layer which may preferably be integrated into and / or integrally formed within the porous surface, or may be a separate, in particular non-integral, layer; b) contacting a fluid having a predetermined pH value with a charged porous surface, the fluid containing species, particularly vectors containing molecules, thereby adsorbing at least a first of the at least two species or at least a first group of species, particularly the first vector or first group of vectors, to the charged porous surface, which is due to an interaction force between the net overall charge of the adsorbed species, particularly the vectors, and the charged porous surface, the interaction force being an attractive force; c) preferably passing at least a portion of the fluid past and / or through an electrically charged porous surface; d) applying a release voltage to the electrically conductive, charged porous surface and / or the electrically conductive coating or layer, the release voltage being selected so that the net interaction force between the charged surface and at least a portion of the adsorbed species, in particular vectors, changes from an attractive force to a repulsive force, thereby releasing said portion of the adsorbed species, in particular vectors; Includes:

[0027] Advantageously, the porous surface may be formed by one or more filter membranes, especially polymer membranes, or one or more nonwoven substrates, with a conductive coating or layer, especially a metallic coating or layer, or a coating or layer formed of other conductive materials, especially on at least one portion of one or more filter membranes, especially polymer membranes. In particular, the metallic coating or layer is also porous. Alternatively, the porous surface may be provided by at least one metal membrane. Preferably, gold or platinum is used as the material for the conductive metal coating and / or layer. Preferably, the metal used is pure, and / or the coating or layer consists of only a single metal. In particular, the polymer membrane itself is nonconductive, and only the coating is conductive. In particular, only one conductive material and / or metal is used for the charged porous surface. In particular, the conductive coating or layer is not formed by a metallic net, in particular not by a metallic net coated with gold or platinum. This is particularly advantageous for pleated or wound forms as described later in this disclosure, which use at least one membrane as the porous surface, and the at least one membrane is coated, especially coated with a metal, most preferably a metal coating, using gold or platinum as the metal.

[0028] The term "charged surface" means that a surface has a charge on its surface, particularly a clear polarity. In this context, a chemically charged surface can be achieved, for example, by ionic groups and / or compounds located on the surface, particularly by chemical bonding of quaternary ammonium groups, particularly to achieve a positively charged surface, or by chemical bonding of carboxymethyl groups or compounds containing the same, particularly to achieve a negatively charged surface. For this purpose, anion-exchange or cation-exchange membranes can be used as charged porous surfaces, particularly with an additional conductive, particularly metallic, coating or layer on the surface. In particular, an electrically charged porous surface can be achieved by applying a base voltage, which is applied to the conductive, particularly metallic, coating or layer, or to the membrane itself if the membrane is conductive, for example, a metallic membrane. In particular, electrically charging a surface can be used to adjust the charge of a chemically charged membrane and increase or decrease the overall charge of the charged porous surface. In particular, the porous surface is not formed by a gel or resin. In particular, the charged surface can also be both chemically and electrically charged. In this regard, a chemically charged surface, such as an ion exchange membrane, is provided in step a) with a conductive coating disposed on the surface, particularly the membrane, to which a base voltage is applied. This allows the total charge on the charged surface to then be composed of a combination of the disposed chemical charge and the applied charge. Thus, the charge applied by the base voltage can be used to create, affect, or modify a chemical "pre-charge" on the surface. In this regard, the applied charge can be of the same polarity, thereby enhancing the "pre-charge." Using an opposite polarity can reduce or negate the "pre-charge," or the polarity of the total charge on the charged surface can be changed to the opposite polarity compared to the "pre-charge."

[0029] In particular, to apply a voltage, especially a release voltage and / or a base voltage, a conductive charged porous surface and / or a conductive coating or layer on the charged porous surface can be used as a first electrode and / or the first electrode can be part of the charged porous surface, and a second electrode can be provided as a counter electrode. The second electrode can also be formed by an additional conductive coating or layer. In particular, the second electrode can be preferably provided on a second side of the porous surface opposite to the first side on which the conductive coating or layer forming the first electrode is located. In this regard, the first and second electrodes can be arranged on different porous surfaces, especially membranes. For example, the first electrode can be formed by a conductive coating on a first porous membrane, and the second electrode can be formed by a conductive coating on a second porous membrane.

[0030] In particular, the applied voltage, especially the base voltage and / or release voltage, ranges from 0.1 to 50 volts, preferably from 0.1 to 3 volts, especially up to 2.5 and / or more than 2 volts. In particular, voltages above 3 volts, especially in some embodiments above 2 volts, are applied for only a short time, especially typically less than 2 seconds.

[0031] In particular, during step d), and especially before step d, a further fluid is brought into contact with the charged porous surface. This further fluid may be of the same type or a different type, in particular having a predetermined pH value and / or a pH value that differs from the predetermined pH value by less than 1, in particular 0.5, in particular 0.2, in particular 0.1, and / or does not contain any species. At least a portion of the further fluid can pass over and / or through the porous surface. Furthermore, in step d), in particular the fluid that has passed over and / or through the porous surface is collected. This fluid contains the species released in step d), in particular (biological) molecules or vectors containing (biological) molecules, in particular a single specific species, in particular the same or identical type of (biological) molecules, or vectors of a single vector group, thereby selectively separating the single specific species from the remaining other specific species, in particular the single type of biomolecules from different biomolecules, or the one vector group from other vectors. The single specific species, in particular the single vector group, is particularly homogeneous and / or contains a specific molecule or vector group with a specific isoelectric point. This isoelectric point is particularly a well-defined isoelectric point, particularly not described by a broad isoelectric point and / or not a broad isoelectric point. It therefore particularly comprises vectors containing the same species, particularly identical molecules, or molecules of the same type having the same contained molecules and / or molecules contained in the same amounts. In this context, the term identical does not necessarily mean exact identical, but also includes essentially identical, and includes molecules / vectors that a person skilled in the art would consider identical due to, for example, the same molecular weight, the same functionality and / or functional groups, especially for medical applications without the need for, for example, enantiomeric purity, different enantiomers of the same molecule.

[0032] The pH value different from the predetermined pH is preferably in the range of pH 6 to 9, preferably in the range of pH 6.5 to 8.5, more preferably in the range of pH 7 to 8, which is least harmful to species commonly used for treatment or which occur naturally in the human body.

[0033] In particular, steps b) and c) may be repeated in the form of multiple cycles, especially before performing step d). Performing multiple cycles in this manner can be used to increase the amount of desired species adsorbed to the membrane. Typically, in the preparation of the species, the concentration in the fluid is low. Consequently, the amount of fluid containing the species used, which is usually high to bind the desired amount of species, can be reduced by using less additional fluid in step d) while reducing the amount of fluid that contacts, passes over, and / or passes through the surface in the subsequent release in step d), and / or by ensuring that most of the fluid used in step c) passes over and / or passes through the surface.

[0034] Furthermore, all of steps b) to d) may be used in multiple cycles. This may be beneficial if several different impurities are present in the preparation of the desired species. For example, vectors containing undesirable amounts of molecules, or containing no molecules at all, or containing undesirable molecules, may be considered impurities in this regard. These should be separated from the desired vector containing the molecule. In general, a particularly pure product is required, especially for (biological) molecules intended for pharmaceutical use.

[0035] By performing multiple cycles, for example, in a first cycle, a first impurity can be selectively separated from the desired vector, while a second impurity is not separated. Then, by adjusting the process conditions, for example, in a second cycle having a different predetermined pH value and / or adjusted voltage, it may be possible to separate the desired vector from the second impurity. Furthermore, if the selective separation is incomplete, performing multiple cycles can achieve a higher purity of separation.

[0036] In particular, for vectors containing a molecule, at least two vector populations may contain different amounts of the molecule, such that a first vector population contains the molecule in a first amount and vectors in a second vector population contain the molecule in a second amount.

[0037] In an embodiment particularly advantageous from the viewpoint of gene therapy, the molecule contained in the vector is a genome, in particular RNA or DNA. Advantageously, one of the at least two vector groups can consist of vectors containing the entire genome, while at least another of the at least two vector groups consists of vectors containing partial genomes and / or vectors containing no genome. In particular, the vectors containing partial genomes and vectors containing no genome can form separate vector groups, but may also be considered as a single range group, and therefore the range of isoelectric points and net overall charges must be selected.

[0038] In another embodiment, the molecule involved can be a drug or a protein or another bioactive substance.

[0039] Regarding pH values, acidic or alkaline pH values ​​can generally have a detrimental effect on biomolecules. For example, proteins may aggregate at acidic pH values, e.g., pH values ​​below pH 5, particularly when buffers with these pH values ​​are used. Human blood, for example, has a pH of roughly 7.4, which is close to a neutral pH. Therefore, a neutral pH value is preferably used for all fluids used in the disclosed method, particularly pH 6 to 8, and most preferably pH 7.4. Advantageously, the pH value is constant and / or remains constant throughout all of steps b) to d), particularly for all fluids used in the method and / or on and / or around the charged porous surface and / or the fluid on and / or around the charged porous surface. In this context, pH values ​​with negligible differences, e.g., pH values ​​that are equal to one decimal place, are considered constant. In this regard, any applied voltage may cause local pH changes, e.g., due to locally protonated or deprotonated species. Such effects are negligible relative to the overall pH value of the fluid and are therefore also considered constant. However, during the execution of a cycle, in particular a complete cycle including steps b) to d), the pH value is held constant in one cycle but can be adjusted between cycles, for example, the first cycle at pH 8 and the second cycle at pH 6. In this regard, changes in the pH value are not used to effect any release of previously adsorbed species. However, the pH value can be selected so that it is close to the isoelectric point of any of the adsorbed amphoteric species, in particular vectors, but does not cause release, and can therefore be adjusted between cycles, in particular with respect to different species, in particular vectors, released in any cycle. In this regard, a pH value close to the isoelectric point of any particular species, in particular vectors, to be released but does not cause release (in particular the difference between the pH value and the isoelectric point is less than 1, in particular less than 0.5), can generally be selected to be smaller than the release voltage required to initiate the release of that particular species, in particular vectors, in step d).

[0040] In particular, particularly if the species is a biomolecule, e.g., a vector, no particularly highly concentrated salt solution is used to carry out any release of the previously adsorbed species. In particular, all fluids used are "salt-free." In this context, liquids with negligible salt ion concentrations are considered "salt-free." In particular, common pH buffers between pH 6 and pH 8 are considered "salt-free" in the context of the present disclosure. In this regard, the liquids introduced in the present method, particularly all liquids, may be or contain buffers used to maintain a constant pH value and / or a small amount of ions and / or a small amount of salt, preferably less than 150 mM. Therefore, these buffers contain a small amount of salt, and this amount is small compared to the concentration of salt solutions commonly used for general salt elution. In particular, these buffers, e.g., phosphate-buffered saline (PBS), may be particularly isotonic, thus providing osmolality and ion concentrations consistent with those of the human body. This may be particularly useful for medical applications, particularly with biomolecules, e.g., vectors, as species. PBS, particularly at common concentrations, is usually used in isotonic conditions, in particular with an osmolality and ionic concentration that corresponds to that of the human body. In particular, the release of species in step d) is carried out simply by applying a release voltage. However, for example, after the separation and / or recovery of the desired species, in particular the vector, especially after step d), if any undesired impurities remain bound to the surface, the pH value can be changed for cleaning purposes, especially after step d) of the last cycle if the method is carried out in cycles.

[0041] According to the invention, a particularly selective separation of at least two species can be achieved by: I. in step b), at least a portion of a first species of the at least two species, in particular a first group of vectors of the at least two groups, is selectively adsorbed, while at least one other species of the at least two species, in particular at least one other group of vectors of the at least two groups, in particular another species, in particular another group of the at least two groups, is not adsorbed; and / or II.i) in step b) by adsorbing at least a part of the first and at least a second species of the at least two species, in particular a first and at least a second group of vectors of the at least two vectors, in particular all species of the at least two species, in particular a group of at least two groups of vectors; and ii) In step d), the method is carried out by selectively releasing a first species, in particular a first group of vectors, while at least one more species, in particular a group of vectors, adsorbed in step b) remains adsorbed, and while all other species, in particular all other groups of vectors, adsorbed in step b) remain adsorbed.

[0042] In this regard, the selectivity in the separation based on option I is not limited to only the first species being adsorbed; more than one species can be adsorbed in step b). In this regard, at least one species of the at least two species does not need to be adsorbed in step b) to achieve selectivity based on selectivity concept I. Thus, option I particularly involves adsorbing a group of species consisting of more than one species, particularly a first group of species, while at least one other species, particularly a second group consisting of one or more additional species different from the first group of species, is not adsorbed. In this regard, the first and second groups of species are selectively separated. However, to selectively separate species included in the first group, the method may then further use selective desorption according to concept II in step d) and / or may be carried out in multiple cycles, for example by adjusting the general process conditions to cycles using, for example, different predetermined pH values, etc.

[0043] In particular, for the separation of species, in particular vectors, of at least two species, in particular vectors, a first species, in particular vectors, has a first isoelectric point and / or a range of first isoelectric points and / or a first overall charge and / or a range of first net overall charges at a predetermined pH value, and a second species, in particular vectors, has a second isoelectric point and / or a range of second isoelectric points and / or a second net overall charge and / or a range of second net overall charges at a predetermined pH value.

[0044] In a particularly advantageous embodiment, the predetermined pH is selected so that, at the selected predetermined pH, the polarity of the net overall charge of one of the at least two species or groups of species, in particular vectors, the first and second species or groups of species, in particular vectors, is opposite to the polarity of the charge of the charged porous surface, and the net overall charge of the other of the first and second groups is zero or has the same polarity as the charge of the charged porous surface, thereby, in particular, adsorbing only one of the first and second species or groups of species, in particular vectors, in steps b) and c). In this embodiment, the particularly selective separation occurs according to option I. Advantageously, only vectors containing the desired species, in particular the desired molecule or a desired amount of molecules, are adsorbed, or all but the vectors containing the desired species, in particular the desired molecule or a desired amount of molecules, are adsorbed, so that only vectors containing the desired species, in particular the desired molecule or a desired amount of molecules, are not adsorbed to the surface in step b).

[0045] Advantageously, the predetermined pH value is selected so that it lies between the isoelectric point of the first species, particularly the group of vectors, and the isoelectric point of the second species, particularly the group of vectors, and between the first isoelectric point and the second isoelectric point in a range, and between the first isoelectric point and the second isoelectric point in a range and / or between the second isoelectric point and the first isoelectric point in a range. In particular, at such a selected predetermined pH, the first and second species, particularly the group of species, particularly the vectors, have different polarities of net overall charges. As a result, since one specific species and group of species, particularly the group consisting of vectors, has a polarity opposite to that of the surface charge, and another specific species and group of species, particularly the group consisting of vectors, has a polarity similar to that of the surface charge, only one specific species and group of species, particularly the vectors of a single group of the first and second groups, are adsorbed in step b). Since only species having a polarity opposite to the charge of the surface can be adsorbed, a species, in particular one of the species group, in particular the vector group, is selectively adsorbed to the surface, while the first and second species and the other of the group are not adsorbed to the surface and are removed in step c), in particular together with the fluid contacting, passing by and / or passing through the surface.

[0046] In another particularly advantageous embodiment, both the first and second species and species groups, in particular vectors, in particular all species, in particular species groups, in particular vectors, are adsorbed onto a charged porous surface. This can correspond to selective separation according to option II. Advantageously, the predetermined pH value is selected so that the selected pH value is higher or lower than the isoelectric point and range of isoelectric points of both the first and second species and species groups, in particular all species and species groups, in particular vectors. In particular for both the first and second groups, in particular for all species and groups of vectors, the particularly net overall charge of both the first and second species and groups has the same polarity at the predetermined pH value, which polarity is opposite to the polarity of the charge of the charged porous surface.

[0047] Advantageously, the release voltages of the adsorbed first and second species and groups, in particular the first and second groups of adsorbed vectors, are different in terms of releasing them in step d). By applying a first release voltage to the conductive surface and / or conductive coating or layer, the net interaction force for one of the first and second species and groups, in particular the vector groups, in particular a single group of all species, in particular the vectors, changes to a repulsive force, while the net interaction force for another of the first and second species and groups, in particular all groups, still adsorbed on the surface remains attractive, thereby selectively desorbing only the first or second species and groups, in particular the vector groups, while the other species and groups, in particular all other groups, remain adsorbed on the charged porous surface.

[0048] Subsequently, after applying a first release voltage to desorb one of the previously adsorbed first and second species and groups, particularly vectors and vector groups, advantageously the applied voltage is adjusted to a second release voltage, and by applying the second release voltage the net interaction force with respect to the other of the first and second species and groups, particularly vectors, is changed from an attractive force to a repulsive force, thereby desorbing the other of the first and second species and groups, particularly vectors. In particular, when more than two species and groups, particularly vectors, are adsorbed to the surface, the first and second release voltages are selected so that a third species and group, particularly vectors, remains adsorbed while the first and second vector groups are desorbed.

[0049] In another advantageous embodiment, the porous surface is electrically charged by applying a base voltage. The desorption in step d) can then be initiated by changing, in particular decreasing, the base voltage to a release voltage, in particular by stepwise changing, e.g. decreasing, the voltage to a first release voltage and then to a second release voltage. A "change or decrease in voltage" may therefore include a reversal of polarity, thus in particular stepwise, e.g. from a negative voltage to a positive voltage or vice versa.

[0050] In particular, in the presence of more than two species and species groups, in particular vectors, it may be beneficial to combine options I and II to achieve selective separation, such that specific target species and species groups, in particular vectors, are selectively adsorbed in step b), the remaining species and groups are not selectively adsorbed in step b), and subsequently at least one of the specific target species previously adsorbed in step b) is selectively released in step d), in particular all species are gradually released in step d).

[0051] In yet another advantageous embodiment, at least two charged porous surfaces, particularly formed of polymeric membranes, are provided in step a). These at least two charged porous surfaces comprise at least a first and a second charged porous surface, particularly polymeric membranes. Furthermore, the first charged porous surface is electrically conductive and / or comprises an electrically conductive coating or layer. Optionally, the second charged porous surface is also electrically conductive and / or comprises an electrically conductive coating or layer. For example, having more than one charged porous surface can strengthen the overall porous surface, thus increasing the ability to bind species to the surface, particularly when combined with stacked and especially pleated configurations, particularly according to the present disclosure found further below.

[0052] Preferably, at least two charged porous surfaces, especially when they are formed from polymer films, are arranged so that the stacked layers form a stack. In such an arrangement, the stack is formed so that the first charged porous surface and / or the conductive coating or layer of the first charged porous surface is located at the first end of the stack. The counter electrode is preferably located at the second end of the stack, opposite the first end. Furthermore, particularly, a plurality of second charged porous surfaces are preferably arranged between the first porous surface and the counter electrode. Particularly advantageously, the second charged porous surface is preferably not conductive and does not include a conductive coating or layer, and is particularly chemically charged. Alternatively, the second charged porous layer may be conductive and / or include a conductive coating or layer. The counter electrode is preferably formed by the second charged porous surface itself or a conductive coating or layer of the second charged porous surface. Therefore, in such an arrangement, the second charged porous surface is located at the second end of the stack.

[0053] Advantageously, no further electrodes are arranged between the first and second charged porous surfaces, in particular all charged porous surfaces and / or surfaces forming the stacked layers, and only the surfaces arranged at the first or second end are conductive and / or comprise a conductive layer. In particular, at least one of the polymer films can act as an insulator between the conductive coatings or layers. Alternatively and additionally, a further insulator, which may be formed, for example, by a further polymer film, is arranged between the conductive coatings or layers. Thus, the insulator can be formed in particular by at least one, in particular a number, in particular up to 30, preferably 5 to 15, further polymer films and / or non-conductive surfaces of polymer films comprising conductive coatings, in particular the non-conductive surfaces of the films forming the first and second surfaces.

[0054] In particular, stacked arrangements of charged porous surfaces, particularly polymer membranes, can provide a large surface area in any suitable device used to carry out the methods of the present disclosure in the space required to provide the surfaces, particularly membranes. In particular, such arrangements can also provide fluid-permeable channels between the surfaces, particularly membranes, for example, by spacing the surfaces, particularly membranes, closely together. Such arrangements, in particular, allow fluid to flow past and through the surfaces simultaneously.

[0055] In particular, the stacked arrangement as disclosed may preferably consist of a large number of surfaces, in particular membranes, in particular at least two membranes, in particular up to 50, preferably 5 to 20 membranes.

[0056] In yet another advantageous embodiment, the first porous surface, particularly the polymer membrane, has a first polarity or is charged with a first polarity. Furthermore, the second porous surface has a second polarity or is charged with a second polarity. Preferably, the second polarity is particularly opposite to the first polarity. The polarities of the first and second charges are the polarities of the respective total charges, which may be provided and / or defined by the sum of the chemical charge and the charge applied to the porous surface by the base voltage. In particular, the use of more than one charged porous surface can provide several beneficial effects, particularly different performance, particularly charged porous surfaces with different charges, particularly charges of different polarities.

[0057] A particularly advantageous embodiment is based on the use of two charged surfaces, where both the first and second charged porous surfaces are preferably formed by chemically charged polymeric membranes, particularly ion-exchange membranes, such as cation and anion-exchange membranes. Preferably, both polymeric membranes, particularly ion-exchange membranes, have a conductive coating or layer on at least one region of either of the polymeric membranes, particularly ion-exchange membranes. Furthermore, each membrane can have a further conductive coating or layer on at least a second region of the membrane, particularly on the second region opposite the first region. By using two charged surfaces of different polarity, species that are anionic at a given pH can be incorporated into one of the surfaces, particularly anion-exchange membranes, while species that are cationic can be incorporated into the other surface, particularly cation-exchange membranes. This can be particularly advantageous when the predetermined pH is selected to be between the isoelectric points of the at least two species to be separated, particularly when it is desired to separate both of these species, or when both of these species are to be understood as impurities in, for example, blood purification, particularly plasma purification or separation of (blood) plasma proteins.

[0058] During step b) of the method of the present disclosure, in such a method using surfaces with different polarities, particularly anion and cation exchange membranes, species with a specific net overall charge of the second polarity are adsorbed to a first porous surface, and species with a specific net overall charge of the first polarity are adsorbed to a second porous surface. For example, species with a cationic overall charge at a predetermined pH are adsorbed to a cation exchange membrane, and species with an anionic overall charge at a predetermined pH are adsorbed to an anion exchange membrane. In the subsequent step d), a release voltage is preferably applied to the first charged porous surface, while no release voltage is applied to the second charged porous surface. This allows only the species previously adsorbed to the first charged porous surface to be desorbed, while the species previously adsorbed to the second porous surface remain adsorbed. Optionally, it may be beneficial to recover the other species remaining adsorbed to the second surface by applying a release voltage to the second charged porous surface, particularly after collecting and / or removing the fluid from which the species is eluted, thereby desorbing the species previously adsorbed to the second charged porous surface. Again, the fluid into which such species are eluted may also be collected and / or removed.

[0059] Combining some of the previously described embodiments can be beneficial, particularly when selectively separating more than two species. For example, in a method using both anion-exchange and cation-exchange membranes, Option I and II can be combined to achieve selective separation. In this regard, a predetermined pH value can be selected such that two species are adsorbed to the same porous surface and a third species is adsorbed to another porous surface. As a result, when the surfaces are eluted separately, the third species is efficiently separated from the other two species. Such an approach uses Option I for selectivity. Option II can be used to achieve selective separation to separate the remaining two species bound to the same porous surface. Thus, by adjusting the release voltage stepwise and selectively desorbing one of the species using a first release voltage that is insufficient to initiate desorption of the other species bound to the surface, the two bound species can be eluted stepwise, thereby selectively separating that species from the other species. Alternatively, additional cycles of the method can be performed to separate these two species bound to the same surface. In this regard, the predetermined pH can be modified between cycles, particularly to modify the net overall charge of the species. Preferably, the pH value is selected so that the net overall charges of these species then have different polarities, so that in the second cycle these species can then be adsorbed onto different surfaces in step b) of the second cycle. As a result, option I was again used for selective separation.

[0060] Furthermore, in such techniques using separately charged porous surfaces, it may be particularly advantageous to provide a conductive coating or layer to be used as an electrode to apply a release voltage between each of the porous surfaces, particularly membranes. This may allow the release voltage to be applied to only a portion of the surface. In particular, the porous surfaces, particularly polymer membranes, may be used in conjunction with the conductive coating or layer as an insulator between the layers.

[0061] In particular, if the at least two charged porous surfaces are differently charged porous surfaces, in particular a first charged porous surface having a first polarity and a second charged porous surface having a second polarity opposite to the first polarity, then every portion of the fluid will pass over all of the porous surfaces. If multiple identical charged porous surfaces are used, each portion of the fluid may pass over every charged porous surface or only some of the charged porous surfaces.

[0062] Preferably, for each elution, fluid is collected and generally removed before the next elution is performed.

[0063] In yet another aspect, advantageous configurations of surfaces, particularly membranes, are provided. Such advantageous configurations include pleated and / or wound configurations of surfaces, particularly membranes. Such configurations are particularly useful for chromatographic methods, including but not limited to those disclosed above. In particular, such configurations may be used as part of any suitable chromatographic device.

[0064] In this regard, a pleated or wound arrangement is created by folding a planar or surface stack into multiple pleats. For a wound arrangement, the planar or surface stack is wound into a series of wound and stacked layers. The pleated or wound arrangement is placed in a housing. Generally, the housing can be selected to have a pleated or wound arrangement that allows fluid to pass over and / or through the surface, preferably the membrane. The housing can provide for flows in different directions, particularly provided by at least one inlet and at least one outlet for supplying (inlet) and removing (outlet) fluid. The pleated or wound arrangement can be arranged in the housing to prevent free movement from the inlet to the outlet and / or to ensure that any flow from the inlet to the outlet passes over and / or past the membrane at least once, preferably multiple times, and / or through and / or through multiple, preferably separate, sections or regions of the stack and / or membrane. The pleated and / or wound arrangements may be arranged in the housing such that the main extension of the arrangement is parallel, perpendicular or oblique to the different flow directions.

[0065] A tube-like structure can serve as a housing for the arrangement. Such a tube-like structure can have a bottom area of ​​any shape, including, but not limited to, a rectangular or circular bottom area. Furthermore, a suitable housing may be in the form of a box-like housing having an inlet and an outlet, with a charged porous surface disposed therebetween. The charged porous surface between the inlet and outlet can be provided laterally. In this regard, the charged porous surface is arranged so that a fluid supplied through the outlet must pass through and / or preferably pass over the charged porous surface, preferably a membrane, at least once, so that free movement from the inlet to the outlet is hindered. Consequently, the inlet must face a first side of the charged porous surface, and the outlet is arranged at a second portion of the charged porous surface opposite, preferably oblique to, the first side.

[0066] However, a circular housing consisting of an inner core and an outer cage with a gap therebetween is preferred in other applications. In this regard, the gap has a gap distance defined by the shortest distance between the core and the cage. A pleated or wound surface, particularly a membrane, is disposed in the gap. Preferably, the inner core and the outer cage are fluid-permeable and / or include inlets / outlets for supplying fluid to the surface and / or removing fluid / eluate. Preferably, the fluid must radially pass through the gap, flowing from the inlet to the outlet. Typically, only one of the inner core and the outer cage has an inlet, while the other has an outlet. In this regard, an inlet can refer to any passage, particularly for supplying fluid, and an outlet can refer to any passage for removal. In particular, the volume inside the core can be used for supply or removal.

[0067] When surfaces of different polarities are used, whether pleated and / or rolled or not, they may preferably be arranged so that the fluid passes over all types of surfaces of different polarities, particularly in succession, and / or the fluid passing from the inlet to the outlet passes over all types of surfaces of different polarities, particularly in succession.

[0068] Preferably, a polymeric film is used to provide and / or as a surface for the pleated and / or wound arrangement. Most preferably, the pleated and / or wound arrangement is formed by more than one polymeric film. At least some or all of these polymeric films are preferably coated with a metal coating or layer on at least one side of the film. Generally, the pleated and / or wound arrangement, preferably a stack of films, comprises at least two conductive coatings or layers, preferably metal coatings.

[0069] In a particularly advantageous embodiment, the pleated and / or wound arrangement comprises at least two polymer membranes, each of which has a metal coating as a conductive coating or layer on one side thereof, and between the coated membranes, in particular, a non-conductive polymer membrane. Preferably, the metal coatings are arranged so that at least two are located on the outermost sides of the stack, in particular on either side of the flow direction within the housing, in particular on the outermost polymer membrane in the stack. In this regard, it is particularly advantageous to pleat and / or wind the membranes and the conductive coating together. In particular, polymer membranes with a metal coating, in particular gold or platinum, can be pleated and / or wound together. Preferably, the wound and / or pleated stack comprises only one type of membrane, uncoated, coated, and / or equipped with binding sites, e.g., they differ only in coating / uncoating and / or binding sites.

[0070] In yet another advantageous embodiment, each polymer film in the stack can have a metal coating as a conductive coating or layer on one or both sides of the respective film. Preferably, some films themselves are non-conductive and act as insulators between the metal coatings or layers. In particular, a release voltage can be applied to selected metal coatings and / or layers only one at a time, and in particular, a release voltage is not applied to at least one more metal coating and / or different pairs of metal coatings, each pair may share one metal coating.

[0071] Preferably, the stack is formed from "identical films", and thus from films of the same type, at least some of which preferably include a conductive coating, or all of which include a conductive coating, and some of which may preferably include a conductive coating, and some of which preferably do not.

[0072] However, in yet another advantageous embodiment, "different membranes" having a polarity different from that of the charged surface, such as cation and anion exchange membranes, can be used in combination. In this regard, it can be beneficial to provide an arrangement in which a voltage, particularly a release voltage, can be applied to one type of membrane separately from other types. In this regard, among any type of membrane, at least one of the membranes of the type has a conductive coating or layer, in particular. At least one insulator, formed, in particular, by a polymer membrane, is provided between these types of conductive coatings or layers. This arrangement can be used to selectively apply a release voltage to selected membranes or selected types of membranes.

[0073] Preferably, for all embodiments of a pleated and / or wound arrangement formed by the stack, the stack as a whole, in particular all the membranes, are pleated and / or wound together with the conductive coating.

[0074] The use of a pleating or winding aid and / or promoter may also be beneficial, in particular to facilitate the preparation of pleats / rolls and, after such pleating / rolling, to maintain the pleat / roll shape prepared by the gathered or wound stack of layers. Such an aid and / or promoter may consist of one or more thermoplastic layers, which are heated during the preparation of the pleats / rolls, in particular to a temperature of 30°C to 100°C and / or above the glass transition temperature, and which retain the pleats and / or rolls, in particular their shape, after said preparation, in particular at temperatures below the aforementioned temperatures. Preferably, the pleating and / or winding aid and / or promoter is porous and / or also permeable to fluids.

[0075] By pleating or rolling a surface, particularly a membrane, a greater surface area per volume can be achieved compared to a non-pleated or non-rolled, and therefore flat, surface, particularly a membrane, especially taking into account the housing.

[0076] The surface area to volume ratio can be influenced by selecting multiple pleat configurations, particularly for housings. In this regard, pleats consist of flat regions located between specific folding points, each flat region having a length, and the pleats preferably have an M-shaped configuration. For each pleat, a first length is preferably selected to exceed at least one dimension of the housing, particularly the width, diameter, etc., particularly for circular housings in which the outer cage and inner core have a gap distance. In this regard, because the circumference of the core is smaller than that of the cage, pleats with a regular surface area, and therefore at least approximately the same length, have a lower, and at least suboptimal, surface area to volume ratio. Therefore, irregular M-shaped pleats are preferred, as these configurations result in a higher, and particularly optimal, surface area to volume ratio. Consequently, such irregular M-shaped pleats include at least one flat region having a second length different from the first length. Most preferably, pleat sets consisting of more than one pleat are used. The pleats in any pleat set preferably include pleats with at least two different irregular M-shaped configurations. In this regard, at least one second length may be smaller than the gap distance or the width of the channel. Most preferably, the set of pleats is particularly regularly repeated within the housing. In particular, for a circular housing, the pleats and / or sets of pleats form particularly complete circles within the housing.

[0077] A circular housing is also beneficial for wound type arrangements: the winding can be formed by winding a surface or stack of surfaces around an inner core of the housing, whereby the inner core specifically supports the winding formed by the winding.

Claims

1. A method for selectively separating amphoteric species, said species consisting of at least two specific species, each specific species having a specific isoelectric point and thereby a specific net overall charge at a given pH value; a) providing an electrically charged, in particular chemically and / or electrically charged, porous surface, said electrically charged porous surface being electrically conductive and / or comprising an electrically conductive coating or layer; b) contacting a fluid having the predetermined pH value with the charged porous surface, the fluid containing the amphoteric species, thereby causing at least a first of the at least two species to adsorb to the charged porous surface, due to an interaction force between the net overall charge of the adsorbed species and the charged porous surface, the interaction force being an attractive force; c) preferably passing at least a portion of said fluid past and / or through said electrically charged porous surface; d) applying a release voltage to the conductive charged porous surface and / or the conductive coating or layer, the release voltage being selected such that the net interaction force between the charged surface and at least a portion of the adsorbed species changes from an attractive force to a repulsive force, thereby releasing the portion of the adsorbed species; The separation I. in step b), by selectively adsorbing at least a portion of said first species while at least one other species of said at least two species is not adsorbed, in particular while the other species of said at least two species is not incorporated; and / or II. i) in step b), by adsorbing at least a portion of the first and at least a portion of the second species of the at least two species, in particular all species of the at least two species; and ii) in step d), by selectively releasing said first species while said at least one second species adsorbed in step b) remains adsorbed, in particular while all other species adsorbed in step b) remain adsorbed, A method characterized by being carried out.

2. During step d), a further fluid is brought into contact with the charged porous surface, in particular having a pH value at said predetermined pH value and / or a pH value which differs from said predetermined pH value by less than 1, in particular less than 0.5, in particular less than 0.2, in particular less than 0.1, and / or which does not contain any species, at least a portion of the further fluid passes over and / or through the porous surface; 10. The method of claim 1, wherein the fluid that has passed over and / or through the porous surface in step d) is collected and contains the species released in step d).

3. 3. The method according to claim 1 or 2, characterized in that throughout all steps b) to d), in particular for all fluids, the pH value is kept and / or constant and / or differs by less than 1, in particular less than 0.5, in particular less than 0.2, in particular less than 0.

1.

4. the species is a vector, at least some of said vectors contain a molecule, thereby providing at least two populations of vectors with different contained molecules, in particular different molecules and / or molecular weights, the vector has a reference isoelectric point and a reference net overall charge at a given pH value; 4. The method according to claim 1, wherein the reference isoelectric point and the reference net overall charge of any specific vector among the vectors containing molecules are shifted by a molecule contained in the specific vector, resulting in a shifted isoelectric point different from the reference isoelectric point and a shifted net overall charge at the predetermined pH value different from the reference overall charge at the predetermined pH value, and wherein the at least two groups of vectors are defined specifically based on the molecules contained in the vectors of each group.

5. 5. The method of claim 4, wherein the vectors of the first vector group contain a first amount of molecules and the vectors of the second vector group contain a second amount of molecules.

6. 6. The method according to claim 5, characterized in that the molecule contained in at least some of the vectors is a genome, in particular RNA, DNA, and that the vectors of one of the at least two groups of vectors each contain a whole genome, and the vectors of at least another of the at least two groups of vectors each contain a partial genome and / or no genome.

7. 7. The method according to claim 1, wherein the predetermined pH is selected so that the polarity of the net overall charge of the first species is opposite to the polarity of the charge of the charged porous surface, and the net overall charge of the second species, in particular all other species, is zero or has the same polarity as the charge of the charged porous surface, thereby allowing in step b) the adsorption of only the first species, in particular no other species.

8. 8. The method according to any one of claims 1 to 7, characterized in that the predetermined pH value is selected such that the polarity of the net overall charge on both the first and second species is of equal polarity, such that the selected pH value is greater than or less than the isoelectric point of both the first and second species, and such that the polarity is opposite to the polarity of the charge on the charged porous surface, thereby allowing in step b) adsorption of both the first and second species onto the charged porous surface.

9. 9. The method of claim 8, wherein in step d) the release voltages are different and / or differently selected for the adsorbed first and second species, and the net interaction force for one of the first and second species is changed to a repulsive force by applying a first release voltage to the conductive surface and / or the conductive coating or layer, while the net interaction force for the other of the first and second species remains attractive, thereby selectively desorbing only the first or second species, while the other remains adsorbed on the charged porous surface.

10. 10. The method of claim 9, wherein after applying the first release voltage to desorb one of the first and second species, the applied voltage is adjusted to a second release voltage, and applying the second release voltage changes the net interaction force in the other of the first and second vector groups from an attractive force to a repulsive force, thereby desorbing the other of the first and second species groups.

11. 11. The method according to any one of claims 1 to 10, characterized in that the porous surface is electrically charged by applying a base voltage, and in that the desorption in step d) is initiated by changing, in particular decreasing, the base voltage to the release voltage, in particular by stepwise changing, in particular decreasing, the voltage to the first release voltage and subsequently to the second release voltage.

12. 12. The method according to any one of claims 1 to 11, characterized in that in step a) at least two charged porous surfaces are provided, said at least two charged porous surfaces comprising at least a first and a second charged porous surface, said first charged porous surface being electrically conductive and / or comprising said electrically conductive coating or layer.

13. 13. The method of claim 12, wherein the second charged porous surface is electrically conductive and / or includes an electrically conductive coating or layer.

14. 14. A method according to claim 12 or 13, characterized in that the at least two charged porous surfaces are arranged so that the superposed layers form a stack.

15. the stack is formed such that the first charged porous surface and / or the conductive coating or layer of the first charged porous surface is located at a first end of the stack; a counter electrode disposed at a second end of the stacked layers opposite the first end; 15. The method of claim 14, wherein the particularly large number of second charged porous surfaces is placed between the first porous surface and the counter electrode, the second charged porous surfaces being particularly not electrically conductive, not comprising a conductive coating or layer, and particularly being chemically charged.

16. no further electrodes are provided between the first and second charged porous surfaces, in particular between all the charged porous surfaces; and / or of the surfaces forming the stack of layers, only the surface located at the first or second end is electrically conductive and / or comprises an electrically conductive layer; and / or at least one charged, particularly non-conductive, surface acts as an insulator between the conductive coating or layer and the counter electrode; and / or 16. The method of claim 15, wherein an insulator is disposed between the conductive coating or layer and the counter electrode.

17. 17. The method according to any one of claims 1 to 16, characterized in that the charged porous surface is formed by at least one polymer membrane having a conductive coating or layer disposed on at least one side of said at least one polymer membrane, and / or the at least two surfaces each comprise at least one polymer membrane.

18. 18. Method according to claim 16 or 17, characterized in that the insulator is formed by at least one, in particular a large number, in particular up to 30, preferably from 5 to 15, additional polymer films and / or non-conductive surfaces of polymer films comprising a conductive coating, in particular the non-conductive surfaces of the films forming the first and second surfaces.

19. 19. The method according to any one of claims 1 to 18, characterized in that the conductive coating or layer is a metal coating or layer, preferably of gold or platinum, in particular consisting of a single metal, preferably gold or platinum, and / or the conductive coating is formed on a non-conductive support, in particular the polymer film of the first and / or second charged porous surface.

20. 20. The method of any one of claims 12 to 19, characterized in that the first porous surface has or is charged with a first polarity and the second porous surface has or is charged with a second polarity, the second polarity being opposite to the first polarity.

21. 21. The method according to claim 20, characterized in that the first and second charged porous surfaces are both formed by chemically charged polymer membranes, in particular ion exchange membranes, and both polymer membranes, in particular ion exchange membranes, have, in particular an electrically conductive coating or layer, on at least one side of either of the polymer membranes, in particular ion exchange membranes.

22. 22. The method of claim 20 or 21, characterized in that in step b) species having a specific net overall charge with the second polarity are adsorbed onto the first porous surface and species having a specific net overall charge with the first polarity are adsorbed onto the second porous surface.

23. 24. The method of claim 23, wherein in step d), a release voltage is applied to the first charged porous surface while no release voltage is applied to the second charged porous surface, thereby desorbing species previously adsorbed on the first charged porous surface while species adsorbed on the second porous surface remain adsorbed, and then optionally a release voltage is applied to the second charged porous surface, thereby desorbing species previously adsorbed on the second charged porous surface.

24. A method according to any one of claims 18 to 23, characterized in that the polymer film and the conductive coating or layer and / or the stack are pleated.

25. 25. The method according to any one of claims 1 to 24, wherein the release voltages, in particular the first and / or second release voltages and / or the base voltage are particularly applied voltages, especially base and / or release voltages in the range of 0.1 to 50 volts, in particular 0.1 to 3 volts.

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