Separation of biomolecules by affinity chromatography

Abstract

The present invention relates to a method for purifying, separating, selecting, and / or isolating biomolecules by affinity chromatography, which provides a stationary phase containing particularly selective binding sites formed by ligands and / or receptors. There is a need to provide a suitable method for purifying, separating, selecting, and / or isolating biomolecules that avoids desorption by changing the pH value using acidic or alkaline buffers and / or by applying salt solutions, thereby eliminating the potential harm caused by degradation of the separated biomolecules. Degradation of separated biomolecules by any means can pose a health risk, particularly in medical applications. This problem can be solved by applying a voltage between two electrodes, particularly where the stationary phase is placed between the electrodes.

Description

[Technical Field]

[0001] The present invention relates to a method for purifying, separating, selecting and / or isolating biomolecules by affinity chromatography, in which a stationary phase is provided containing particularly selective binding sites formed by ligands and / or receptors. Biomolecules contacted with the stationary phase can bind to the particularly selective binding sites by forming a ligand / receptor complex. Elution of the pre-bound biomolecules is then carried out. [Background technology]

[0002] Generally, biomolecules are species in high demand for medical applications, e.g., proteins used as drugs, etc. Therefore, desired biomolecules must be produced with high purity and therefore need to be separated from impurities resulting from their formation. Such separation is often achieved by a bind-elute process, especially using affinity chromatography.

[0003] Affinity chromatography is therefore a method for separating biomolecules based on highly selective macromolecular binding interactions between the biomolecule and a complementary substance.

[0004] Affinity chromatography is based on specific binding interactions between a target analyte (typically dissolved in a mobile phase and therefore fluid) and a binding partner, usually in the form of a ligand or receptor (immobilized on a stationary phase). In a typical affinity chromatography application, the ligand is attached to any solid, insoluble material, usually a polymer. This material is specifically chemically modified to incorporate reactive functional groups with which the ligand or receptor can react, forming a stable covalent bond. Typically, the stationary phase is packed into a column, and a mobile phase, usually in fluid form, containing the target biomolecule is introduced. Biomolecules with a high binding affinity for the ligand / receptor bind to the ligand / receptor and remain bound to the stationary phase. The column is then washed, cleaving the weak interaction with the stationary phase and thereby removing any unwanted, and therefore non-target, biomolecules, while the target biomolecule remains bound. The target biomolecule can then be removed by using a so-called elution buffer, which cleaves the interaction between the bound target biomolecule and the ligand. Thus, the target molecule is recovered in the eluate. Typically, such elution buffers are fluids with a pH value in the acidic or alkaline range. For example, a common elution buffer used is an acetate buffer, e.g., pH 3. Alternatively, or in addition, salt solutions can also be used for elution. Both of these elution methods can cause harsh conditions that can be harmful to any biomolecule, e.g., especially for proteins, and can cause aggregation, fragmentation, or denaturation of the desired species.

[0005] 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 electrodesorbing molecules adsorbed on a charged membrane having a metal coating by applying a voltage of opposite polarity to the charge of the membrane to the coating. [Prior art documents] [Patent documents]

[0006] [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]

[0007] Generally, biomolecules such as proteins are amphoteric species, which usually result in biomolecules with a non-neutral overall charge at certain pH values. Therefore, such biomolecules can generally be bound to charged surfaces by electro-desorption. However, biomolecules with the same polarity of charge are generally either all adsorbed or none at all, which does not typically provide selectivity for specific biomolecules and may require more stringent conditions.

[0008] Based on this, there is a need to provide a suitable method for purifying, separating, selecting and / or isolating biomolecules, which avoids desorption by changing the pH value, in particular using acidic or alkaline buffers, and / or by applying salt solutions, and thus eliminates the potential harm caused by degradation of the separated biomolecules, which, if degraded in any way, could pose a health risk, especially in medical applications.

[0009] This can be achieved by applying a voltage between two electrodes, and in particular by placing the stationary phase between the electrodes. [Means for solving the problem]

[0010] This problem is solved by a method according to claim 1 and an apparatus according to claim 29. The subsequent claims 2 to 28 and 30 to 50 provide advantageous embodiments of the method according to the invention. [Brief explanation of the drawings]

[0011] [Figure 1]1 is a graph showing the results of an experimental procedure to determine the elution voltage required to elute trastuzumab pre-bound to a Protein A ligand immobilized on an affinity membrane. [Figure 2] 2 is a graph showing the results of a subsequent experiment using the same experimental setup as in FIG. 1 to quantitatively analyze trastuzumab elution. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the method of the present invention, a stationary phase for the purification, separation, selection, and / or isolation of biomolecules by affinity chromatography, particularly by a bind-elute process, is provided. Providing such a stationary phase is referred to as "step a)" of the disclosed method. This stationary phase contains particularly selective binding sites formed by ligands and / or receptors. Preferably, only either the ligand or the receptor forms the binding sites. Preferably, the ligands and / or receptors are of a single type, particularly belonging to a single protein class. Suitable stationary phases include, but are not limited to, those formed from gels, resins, or glass fibers. The stationary phase substrate can also be provided by any suitable porous substrate, such as a membrane, particularly a polymeric membrane. Any material capable of forming a suitable stationary phase must be capable of at least containing the particularly selective binding sites formed by the ligands and / or receptors. In particular, these binding sites are accessible to the biomolecules to be purified, separated, selected, and / or isolated using the disclosed method, for example, by contacting the stationary phase with a fluid containing the biomolecules. For this purpose, the ligands and / or receptors forming the binding sites are preferably immobilized on the substrate of the stationary phase. Preferably, the stationary phase is porous, in particular permeable to fluids, such as water and / or alcohols, and the ligands and / or receptors forming the binding sites are preferably immobilized on the inner and / or outer surface of the stationary phase. In this respect, inner surfaces are in particular surfaces located within the pores of the stationary phase. The inner surfaces are preferably accessible to fluids from the outside.

[0013] Most preferably, the stationary phase is formed by at least one porous substrate, in particular a membrane, preferably a polymeric membrane, and the ligands and / or receptors forming the binding sites are immobilized, in particular on the inner and / or outer surface of the at least one porous substrate, in particular a membrane. Consequently, the ligands and / or receptors are located, in particular, inside the porous substrate, in particular a membrane, in particular in the open pores of the porous substrate, in particular a membrane, and / or on the outer surface of the porous substrate, in particular a membrane. Hereinafter, a porous substrate, in particular a membrane, which has and / or contains and / or carries and / or on which the ligands and / or receptors are immobilized, will be referred to as an "affinity substrate, in particular a membrane".

[0014] In particular, instead of a single stationary phase, at least two stationary phases may be provided, which may or may not be identically configured to contain, for example, the same ligand and / or receptor. In particular, the use of more than one stationary phase can increase the binding capacity of the stationary phase as a whole, since more ligands and / or receptors can be provided.

[0015] In the method of the present invention, the second step of the method involves contacting a first fluid containing biomolecules with a stationary phase. This step is hereinafter referred to as "step b)." This allows at least a portion of the biomolecules to bind, particularly to selective binding sites on the stationary phase, by forming ligand / receptor complexes, particularly through non-covalent interactions. The retention of such complexes is based on the sum of the interaction forces between the biomolecule and the binding site. One of the biomolecule and the binding site acts as a ligand, and the other acts as the corresponding receptor. The sum of attractive interaction forces may be inferior to the high binding affinity between the biomolecule and its respective binding site. To achieve such a sum of attractive interaction forces, the respective binding sites and biomolecules must be complementary, e.g., by having complementary three-dimensional shapes, particularly, e.g., by having complementary local spatial configurations.

[0016] Preferably, the ligands and / or receptors used to form the binding sites are antigen ligands and / or receptors. These are preferably proteins or aptamers, particularly oligopeptides or oligonucleotides. They can be preferably immobilized on a stationary phase, particularly on the surface, particularly on the outer and / or inner surface, of at least one affinity substrate, particularly a membrane. For example, techniques for immobilizing such ligands and / or receptors include, but are not limited to, EDC or DCC coupling, and / or radical coupling, particularly by electron beam treatment.

[0017] In this regard, the use of EDC or DCC coupling to arrange ligands and / or receptors that form binding sites typically provides surfaces with fairly regularly oriented binding sites, but typically in relatively small quantities. Notably, EDC or DCC requires an initial grafting step to prepare the surface of the stationary phase, e.g., to attach, e.g., form, amine or carboxy functional groups to the surface. Ligands and / or receptors can then be attached to the surface, e.g., using amination. On the other hand, the use of radical coupling techniques provides a less cumbersome method for immobilizing binding sites on a stationary phase. Stationary phases with binding sites prepared by radical coupling techniques typically contain many more binding sites. However, the binding sites may be arranged in a fairly random orientation.

[0018] The biological molecules to be purified, separated, selected, and / or isolated may include, but are not limited to, proteins, particularly immunoglobulins, or antibodies, particularly natural or monoclonal antibodies, or enzymes, or nucleic acids such as RNA or DNA. In particular, the term "biomolecule" may include viruses. In this regard, for example, a ligand / receptor complex may be formed between a viral envelope protein and a complementary binding site. In particular, the ligands / receptors are specifically selected so that the binding sites provided by these ligands / receptors can efficiently and particularly selectively bind to the biological molecules to be purified, separated, selected, and / or isolated.

[0019] As one example, the binding site formed by an antigen can be used to purify, separate, select, and / or isolate antibodies. Thus, for example, Protein A can be used to purify, separate, select, and / or isolate human immunoglobulin G antibodies (IgG), particularly IgG1, IgG2, and IgG4, due to the high binding affinity between these IgGs and Protein A. In this regard, for example, Protein A as a ligand binds specifically to the Fc receptor of IgG. Therefore, Protein A may be used as a binding site. As another example, Protein G can be similarly used to purify, separate, select, and / or isolate IgG3. Furthermore, as yet another example, DNA or RNA, particularly of different helical structures and / or sequences and / or lengths, can be purified, separated, selected, and / or isolated, for example, using corresponding adaptamers immobilized on a solid phase.

[0020] In particular, in the method of the present invention, the first fluid can contain more than one type of biomolecule, thus at least a first biomolecule and a second biomolecule. For example, the first fluid can be blood. Thus, the terms "first biomolecule" and "second biomolecule" do not refer to two biomolecules of the same type, but to biomolecules of different types. To separate these first and second biomolecules, the ligands and / or receptors acting as binding sites are selected so that the first biomolecule has a higher binding affinity to the ligand and / or receptor compared to the second biomolecule, particularly compared to all other biomolecules contained in the first fluid, and / or the second biomolecule, particularly all other biomolecules different from the first biomolecule, has a lower and / or zero binding affinity to the second biomolecule, particularly all other biomolecules contained in the first fluid. As a result, selectivity is achieved by primarily or solely binding the first biomolecule to the binding site and not binding the second and / or other biomolecules to the provided binding site.

[0021] In step b), the fluid is contacted with the stationary phase, thereby binding the biomolecules to the binding sites, during, until and / or after binding, preferably at least a portion of the first fluid passes through and / or past the stationary phase, and is then removed. Optionally, a second fluid, particularly a second fluid without further biomolecules, is added and is contacted with the stationary phase (together referred to as "optional step c"), and for removal, a wash fluid may be used before contact with the second fluid. The second fluid and wash fluid may be of the same or different species.

[0022] According to the present invention, after the binding of the biomolecules in step b) and, in particular, after the removal of the first fluid and the addition of the second fluid or at least partial removal of the first fluid in step c), the elution of the biomolecules is initiated, hereinafter referred to as "step d)". An eluate is generated which contains, in particular, the eluted biomolecules. The eluate preferably consists of the second fluid and the eluted biomolecules.

[0023] The elution in step d) is carried out by applying a voltage between two electrodes, in particular after contacting the stationary phase with a wash fluid and / or after rinsing the stationary phase and / or while contacting the stationary phase with a second fluid, in particular while there is no applied voltage before step d), in particular while no voltage and / or electric field is applied on the stationary phase in steps a) to c). Preferably, the electrodes are particularly permeable to fluids. In particular, the stationary phase is located between the electrodes, and preferably there is a distance of 5 cm or less, in particular 1 cm or less, between one or both electrodes and the stationary phase and / or between both electrodes. By applying a voltage between the electrodes, an electric field is induced / established near the stationary phase, in particular the stationary phase is located within the electric field, which can cause an interaction between the induced / established electric field and the bound biomolecules and / or ligands / receptors forming the binding sites, respectively. The application of such a voltage and the resulting electric field, respectively, and the interaction between the established electric field and the bound biomolecule and / or the ligand / receptor, respectively, forming the binding site, may in particular reduce the attractive force between the biomolecule and the binding site, or even induce additional repulsive forces between the biomolecule and the binding site, respectively. The electric field may be part of an electromagnetic field, but an electric field generated by a DC voltage is preferred.

[0024] By increasing the applied voltage to a certain level, such repulsive forces, particularly the electric field induced / established by the voltage, can be sufficient to reverse the overall interaction between the biomolecule and the stationary phase, particularly the binding site, from an attractive force to a repulsive force. This effect is based on subtle changes in the conformation of at least one biomolecule and / or binding site, particularly secondary, tertiary, or quaternary conformation, particularly of proteins, which undergo complementary losses due to their interaction with the electric field induced / established by the applied voltage. However, such subtle structural changes do not usually cause denaturation, aggregation, etc. of the biomolecule. In particular, such conformational changes only exist for a limited time and are easily reversed automatically, particularly by the biomolecule losing proximity to the electromagnetic or electric field, e.g., by removing the eluate from the electromagnetic or electric field and / or the stationary phase.

[0025] Preferably, the applied voltage is in the range of 0.1 to 50 volts, particularly 0.1 to 3 volts, particularly 1.5 to 3.0 volts, especially up to 2.5 volts. In particular, voltages above 3 volts are applied for short periods of time, typically less than 2 seconds. The voltage may in particular be DC or AC, although DC or AC with a frequency of less than 10 Hz is generally preferred. Such voltages generally do not damage biomolecules and provide particularly gentle elution conditions.

[0026] While elution by pH adjustment or, in particular, elution using highly concentrated salt solutions, both involve rather harsh conditions that may be harmful to biomolecules, elution by voltage application does not require the use of these common elution principles. Thus, preferably, elution, particularly by total pH adjustment, e.g., by the fluid introduced, is not performed in the method of the present invention, and / or all fluids added during and / or before the elution step, in particular the second fluid and / or the wash fluid, have a pH value equal to that of the first fluid and / or differ by a pH value of less than 1.0, in particular less than 0.5, in particular less than 0.2, in particular less than 0.1. Preferably, all fluids used have a pH value that provides a gentle environment for the biomolecules to be purified, separated, selected, and / or isolated. Thus, pH values ​​that can be considered "neutral," such as a pH value between pH 6 and pH 8, in particular pH 7 and pH 7.8, and most preferably a pH value of approximately pH 7.4, are preferred. 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. Thus, these buffers contain small amounts of salt, the amount of which is small compared to the concentrations of salt solutions commonly used for general salt elution. Notably, these buffers, such as phosphate-buffered saline (PBS), may be particularly isotonic, thus providing osmolality and ion concentrations consistent with those of the human body, which may be particularly beneficial for medical applications. In this regard, commonly used PBS, wash fluids, second fluids, and / or all fluids applied after binding and before and during elution may contain salt concentrations of approximately 150 mM or less. However, in the fluids (washing fluid, second fluid and / or all fluids), particularly buffers with lower concentrations are preferably used, for example, salt concentrations are typically less than 10 mM, particularly less than 5 mM, particularly less than 2 mM, particularly less than 1 mM, particularly less than 0.5 mM, preferably less than 0.25 mM, most preferably around 0.15 mM. Thus, preferably, no salt solution is added, particularly before and / or during the elution step, with a salt concentration exceeding the typical buffer concentrations as described above. The respective "salt-free" fluids are used, particularly for elution of bound biomolecules.In this context, liquids with negligible salt ion concentrations, and in this regard, buffers with salt concentrations consistent with those described, are considered "salt-free." Preferably, phosphate-buffered saline (PBS), particularly PBS with a salt concentration of less than 10 mM, particularly less than 5 mM, particularly less than 2 mM, particularly less than 1 mM, particularly less than 0.5 mM, preferably less than 0.25 mM, and most preferably approximately 0.15 mM, can be used as a buffer, particularly as the second fluid, to maintain a pH within the range of pH 6 to pH 8, particularly pH 7.4, and is considered a "salt-free" solution within the meaning of the present disclosure. PBS used at common concentrations is usually isotonic, particularly having an osmolality and ionic concentration consistent with the human body.

[0027] Using the disclosed method, for example, in an exemplary study, Protein A was used as a binding site for the isolation and extraction of IgG. In this regard, a PBS buffer solution (pH 7.4) of IgG was used. By contacting the solution with a stationary phase in the form of a polymer membrane containing Protein A as binding sites, IgG efficiently and selectively bound to these binding sites. Elution was then performed using additional PBS buffer without additional IgG and applying a voltage of approximately 2.2-2.3 volts. The resulting eluate contained approximately 90% of the IgG previously provided and specifically bound to the binding sites. The same method was repeated several times using the same stationary phase. In each repetition, approximately 90% of the provided / bound IgG was recovered by elution, demonstrating the reliability and durability of the disclosed method.

[0028] In particular, the eluate produced in step d) containing the eluted biomolecules can be further used and / or subjected to further methods, such as performing detection reactions, preparative methods, medical applications, etc., including but not limited to producing very pure drugs or active substances, for example for cancer treatment.

[0029] Preferably, at least one electrode, in particular a plurality of electrodes, preferably all electrodes, are arranged on at least one non-conductive support, in particular formed by a polymer membrane and / or a porous substrate, and / or are formed by a conductive, in particular metallic, coating and / or metallic net arranged on at least one affinity substrate, in particular a membrane. Preferably, at least one affinity substrate, in particular a membrane and / or at least one non-conductive support can act as an insulator between the electrodes. Preferably, gold or platinum is used as the material for the electrode and / or conductive metallic coating. Preferably, the metal used is pure, and / or the coating or layer consists of only a single metal. In particular, the affinity substrate, in particular the membrane, itself is non-conductive, and only the coating is conductive. In particular, only one conductive material and / or metal is used for the electrode and / or conductive coating. In particular, the conductive coating is preferably not a (non-gold and non-platinum) metallic net coated with gold or platinum, and particularly preferably not formed by a metallic net.

[0030] In yet another embodiment, the stationary phase can include at least one electrode, particularly formed by a metal coating and / or a metal net disposed on at least one affinity substrate, particularly a membrane, and / or at least one non-conductive support. The ligands and / or receptors forming binding sites can be immobilized on at least one electrode included in the stationary phase, for example, but not limited to, by covalent or associative binding of the receptor and / or ligand to the metal. In this regard, sulfur-gold bonding can be used to bind the ligand and / or receptor to the electrode. In this regard, binding of the ligand and / or receptor to the metal can be beneficial, particularly for analytical applications, if only a small number of binding sites are required to perform the method. However, for preparation methods in which a large number of binding sites are usually desired, disposing the binding sites on a substrate, particularly a membrane, or on both a substrate, particularly a membrane, and an electrode is preferred, especially when multiple stationary phases are used and disposed together between two electrodes.

[0031] According to an advantageous embodiment, the method can be enhanced by using a stationary phase consisting of at least two specific stationary phases. In particular, each stationary phase is formed by at least one, preferably a number, in particular up to 50, in particular from 5 to 20, preferably less than 10 specific porous substrates, in particular specific permeable membranes. These specific stationary phases preferably contain specific binding sites formed by ligands and / or receptors, whereby the specific binding sites in a first specific stationary phase are formed by ligands and / or receptors of a first kind, and the specific binding sites in a second specific stationary phase are formed by ligands and / or receptors of a second kind. In particular, these first and second ligands and / or receptors may be different, in particular ligands / receptors of different kinds, for example different protein classes. Most preferably, the first and second ligands are selected such that for any two different biomolecules to be separated, purified, selected and / or isolated and / or contained in the first fluid, these different biomolecules have a high binding affinity for only one of the first and second species of ligands and / or receptors, respectively, and a lower, low and / or zero binding affinity for the other of the first and second species of ligands and / or receptors.

[0032] As an example, two specific stationary phases A' and B' can be considered as stationary phases. Specific stationary phase A' can be a ligand and / or receptor A. # and the specific stationary phase B' contains the ligand and / or receptor B # A fluid containing biomolecules A and B is contacted with the stationary phase, and among these, biomolecule A is a ligand / receptor A. # High binding affinity to ligand / receptor B # Biomolecule B has low binding affinity to ligand / receptor A. # Low binding affinity to ligand / receptor B # It has a high binding affinity to the ligand / receptor A. # Biomolecule B is adsorbed only to the ligand receptor B. #Thus, both biomolecules A and B will selectively bind to only one of the specific stationary phases A' or B'.

[0033] In general, several advantageous arrangements can be used, particularly with regard to the arrangement of at least two different specific stationary phases.

[0034] In particular, at least two particularly distinct specific stationary phases can be arranged such that a first and a second particularly distinct specific stationary phase, in particular all the particularly distinct specific stationary phases, are located between a single set of two electrodes, forming stacked layers, in particular constituting a stack of stationary phases. Thus, only two electrodes need be provided to apply a voltage to elute any stationary phase, and / or all stationary phases can be eluted using the same two electrodes, e.g., all stationary phases and / or their ligands and / or receptors are located between the two electrodes.

[0035] Alternatively, a first particularly specific stationary phase can be positioned between a first set of two electrodes, and a second, different particularly specific stationary phase can be positioned between a second set of two electrodes, and in particular, each particularly specific stationary phase can be positioned between two unique sets of electrodes. Such an arrangement can provide the option of applying a voltage to only one set of electrodes at a time, thereby more reliably eluting preferably only one particularly specific stationary phase of the at least two particularly specific stationary phases. In such cases, the first fluid preferably contacts both the first and second stationary phases sequentially.

[0036] In yet another particularly useful arrangement, three sets of electrodes are provided, such that a first particularly specific stationary phase is located between the first and second electrodes of the set of three electrodes, and a second particularly specific stationary phase is located between the second and third electrodes of the set of three electrodes. Thus, one of the electrodes is located between the first and second particularly specific stationary phases and is specifically shared by the first and second stationary phases. In such an arrangement, a voltage can be applied between two of the three selectable electrodes so that a specific electromagnetic field is induced / established around only one of the stationary phases, and thus either the first or second stationary phase, or both the first and second stationary phases. Furthermore, the arrangement can be extended with additional particularly specific stationary phases. In this regard, for example, a third particularly specific stationary phase can be located between the third electrode and an additional fourth electrode. Again, the vicinity of the stationary phase where the electromagnetic field is induced / established can be selected by selecting the electrodes between which a voltage is applied for elution. Using the same concept, the method can be extended by adding additional stationary phases beyond the three specifically selected stationary phases. In such cases, the first fluid is preferably contacted sequentially with the stationary phases, eg, both the first and second stationary phases.

[0037] Preferably, in the elution step d), the applied voltage and / or the electrode and / or electrode set used to apply the voltage are the biomolecules bound to the first, particularly specific, stationary phase in step b) are eluted, and the biomolecules bound to the second, particularly specific, stationary phase in step b), and in particular all other biomolecules bound in step b), remain bound, or The biomolecules bound to the second, particularly different, specific stationary phase in step b) are selected such that they are eluted, while the biomolecules bound to the first, particularly specific stationary phase in step b), and in particular all other biomolecules bound in step b), remain bound.

[0038] Thus, only biomolecules bound to one of the specific stationary phases are eluted, particularly simultaneously. Preferably, after elution of one of the first and second, particularly specific stationary phases, the eluate is removed before eluting the other of the first and second, particularly specific stationary phases, particularly the other, particularly specific stationary phase.

[0039] In yet another advantageous embodiment, the stationary phase, in particular each specific stationary phase, can be formed by at least two, preferably a large number, in particular up to 50, in particular 5 to 20, preferably less than 10 porous substrates, in particular membranes, in particular affinity substrates, in particular membranes. In particular, this can increase the binding capacity of the specific stationary phase. Moreover, the at least two porous substrates, in particular membranes, are permeable substrates, in particular membranes, and can be particularly oriented so that the fluid can pass through the porous substrates, in particular membranes, and between the porous substrates, in particular membranes, that form stacked layers. This allows for good contact between the fluid and the stationary phase.

[0040] In certain further advantageous embodiments, at least two, in particular specific stationary phases, in particular all specific stationary phases, and / or at least two affinity substrates, in particular membranes, and / or specific affinity substrates, in particular membranes, and / or non-conductive supports forming these stationary phases, in particular, can be in a stacked arrangement and / or pleated arrangement and / or wound arrangement and / or separated arrangement. A specific stationary phase is, for example, spaced apart from another stationary phase in the direction of flow of the fluid containing the biomolecules, for example, in particular passed by the first fluid and / or fluids one after the other. The above arrangement concepts are described in more detail further below.

[0041] In particular, every portion of the first fluid passes over every different specific stationary phase. When multiple identical specific stationary phases are used, each portion of the first fluid may pass over every stationary phase or only some of the stationary phases.

[0042] In the stacked arrangement, at least two particularly specific, particularly different, specific stationary phases and / or at least two affinity substrates, particularly membranes, and / or specific affinity substrates, particularly membranes, and / or respective non-conductive supports are arranged as stacked layers constituting a stack. Preferably, the stack is formed by at least two porous substrates, particularly permeable membranes, forming the stacked layers. In particular, the stack is formed so that a first electrode, particularly formed by a conductive, particularly metallic coating or metallic net on a first affinity substrate, particularly membrane, and / or first non-conductive support, is located at a first end of the stack, and a second electrode, particularly formed by a conductive, particularly metallic coating or metallic net on a second affinity substrate, particularly membrane, and / or second non-conductive support, is located at a second end opposite the first end of the stack. The stack comprises at least one affinity substrate, particularly membrane, and / or affinity substrate, particularly membrane, core, including the specific affinity substrate, particularly membrane, disposed between the first and second electrodes, the affinity substrate, particularly membrane, core acting as an insulator between the first and second electrodes.

[0043] In particular, the core of the affinity substrate, particularly the membrane, comprises at least one first specific affinity substrate, particularly the membrane, and at least one second specific affinity substrate, particularly the membrane. Additionally or alternatively, the core of the affinity substrate, particularly the membrane, comprises at least two subcores, which are stacked in a stack, particularly forming a stacked layer, and the subcores are preferably specific subcores. Of these specific subcores, the first specific subcore can preferably comprise a first specific affinity substrate, particularly the membrane, and the second specific subcore can preferably comprise a second, different specific affinity substrate, particularly the membrane.

[0044] In particular, at least one further electrode, particularly provided as a conductive, particularly metallic, coating and / or metallic net disposed on at least one further non-conductive support, particularly formed by a porous substrate, particularly a polymer membrane, and / or on at least one affinity substrate, particularly a membrane, of one subcore, is located between at least two subcores and / or their ligands and / or receptors, particularly between the first and second specific subcores, which are located adjacent to the affinity substrate, particularly a membrane, of another subcore. In particular, the at least one further electrode can selectively apply a release voltage to part of the arrangement, and to any single subcore, in particular selectively inducing / establishing an electromagnetic field around and / or above each subcore in step d), particularly the stationary phase and / or membrane forming, for example, successive subcores.

[0045] In particular, electrodes can be placed between each particular stationary phase and / or layer of stationary phase, in particular affinity substrate, in particular intermembrane, and / or layer of particular stationary phase, in particular particular affinity substrate, in particular intermembrane, and / or layer and / or stationary phase and / or substrate, in particular intermembrane, forming the subcore and / or their ligands and / or receptors.

[0046] With respect to the separate arrangement of the particularly specific stationary phases, at least two particularly specific stationary phases are preferably arranged side by side in the flow direction of the first fluid, and / or the first fluid passes over the at least two particularly specific stationary phases in succession. Preferably, the at least two stationary phases are two different specific stationary phases.

[0047] In yet another embodiment, a pleated and / or wound arrangement of a stationary phase is provided, which is preferably formed by a substrate, particularly a membrane, particularly stacked layers of membranes, including at least two or all of the electrodes. In such an arrangement, preferably a stack of stationary phase and electrodes is used, which are pleated and / or wound together. Such an arrangement is particularly useful for chromatographic methods, including, but not limited to, those disclosed above. In particular, such an arrangement can be used as part of any suitable chromatographic device.

[0048] In this regard, the pleated arrangement is produced by folding the stationary phase, or preferably a stack of stationary phases including at least two or all of the electrodes, in particular a substrate, preferably a membrane, into multiple pleats. For a wound arrangement, the stationary phase or a stack of stationary phases is wound, preferably into a series of wound and stacked layers. The pleated or wound arrangement is placed in a housing. In general, the housing can be selected to have a pleated and / or wound arrangement such that the fluid passes over and / or through the stationary phase, in particular the substrate, preferably a membrane. The housing can provide for flow in different directions, in particular by at least one inlet and at least one outlet for supplying (inlet) and removing (outlet) fluids. The pleated or wound arrangement can then 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 and / or past the stationary phase, in particular the substrate, preferably 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 substrate, preferably the membrane, preferably passing and / or through all the different specific phases. The pleated and / or wound arrangement can for example be arranged in the housing so that the main extension of the arrangement is parallel, perpendicular or oblique to the different flow directions.

[0049] 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, providing an inlet and an outlet, between which the stationary phase is placed. The stationary phase between these inlet and outlet can be fed laterally. In this regard, the stationary phase is arranged so that a fluid fed through the outlet must pass through and / or preferably pass over the stationary phase, in particular the substrate, 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 stationary phase, and the outlet must be positioned at a second portion of the stationary phase opposite, preferably diagonally opposite, the first side.

[0050] However, a circular housing consisting of an inner core and an outer cage with a gap therebetween is preferred in other application scenarios. In this regard, the gap is the gap distance defined by the shortest distance between the core and the cage. A pleated or wound surface, particularly a substrate, preferably a membrane, is placed 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 arrangement, 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 mean any passage, particularly a passage through which a fluid can pass, for supply, and an outlet can mean any passage for removal. In particular, the volume inside the core can be used for supply or removal.

[0051] When "different types" of stationary phases are used, each containing a specific ligand / receptor for each type, whether pleated and / or wound or not, they may preferably be arranged so that the fluid passes over all types of stationary phases, particularly in succession, and / or the fluid passing from the inlet to the outlet passes over all types of stationary phases, particularly in succession.

[0052] Preferably, porous substrates, particularly most preferably polymeric membranes, are used to form the stationary phase in a pleated and / or wound configuration. Most preferably, the pleated and / or wound configuration is formed by more than one porous substrate, particularly polymeric membranes. At least some or all of these porous substrates, preferably polymeric membranes, are preferably coated with a metal coating or layer on at least one side of the membrane. Generally, the pleated and / or wound configuration, preferably a stack of membranes, comprises at least two conductive coatings or layers, preferably metal coatings.

[0053] In a particularly advantageous embodiment, the pleated and / or wound arrangement comprises at least two porous substrates, preferably polymeric membranes, each of which has a conductive coating or metal coating as a layer on one side thereof, forming a stack, and each further, particularly non-conductive, porous substrate, preferably polymeric membrane, forming at least one stationary phase between the coated membranes. Preferably, at least two of the metal coatings are arranged so that they are located on the outermost side of the stack, one on each side of the stack, particularly the outermost porous substrate, preferably polymeric membrane, particularly relative to the flow direction in the housing. In this regard, it is particularly advantageous to pleat and / or wind the membrane and the conductive coating together, particularly the stationary phase, together with the electrodes. In particular, porous substrates, preferably polymeric membranes, with a metal coating, particularly of gold or platinum, and porous substrates without a coating can be pleated and / or wound together. Preferably, the wound and / or pleated stack comprises only one membrane, uncoated, coated, and / or bound with binding sites, e.g., they differ only in coating / uncoating and / or binding sites.

[0054] In yet another advantageous embodiment, each porous substrate, preferably a polymeric membrane, of the stack can have a metal coating as a conductive coating or layer on one or both sides of the respective porous substrate, preferably a membrane. Preferably, some porous substrates, preferably membranes, themselves are non-conductive and act as insulators between the metal coatings or layers. In particular, the voltage for elution can be applied to selected metal coatings and / or layers only one at a time, and in particular the voltage for elution can be applied to at least different metal coatings and / or different pairs of metal coatings, each pair sharing one metal coating.

[0055] Preferably, the stack is formed from "comparable substrates, in particular membranes", thus from the same type of porous substrate, preferably membranes, of which at least some preferably comprise a conductive coating, or all comprise a conductive coating. Preferably, some may comprise a conductive coating, while others preferably do not, and in particular the latter preferably comprise binding sites, while the former may or may not comprise binding sites.

[0056] However, in yet another advantageous embodiment, different specific stationary phases having different ligands / receptors immobilized on substrates, preferably membranes, can be used in combination. In particular, these ligands / receptors are specific to different substrates, preferably membranes. In this regard, it may be advantageous to provide an arrangement that allows applying a voltage for elution to one specific stationary phase type, separate from other types. In this regard, of any type of substrate, preferably membrane, at least one of these substrates, preferably membranes, of the type has, in particular, a conductive coating or layer. Between these types of conductive coatings or layers, at least one insulator, formed, in particular, by a porous substrate, preferably a polymeric membrane, is provided. This arrangement can be used to selectively apply a release voltage to selected substrates, preferably membranes, and / or selected types of substrates, preferably membranes.

[0057] Preferably, for all embodiments of a pleated and / or wound arrangement formed by the stack, the stack as a whole, in particular all substrates, preferably membranes, are pleated and / or wound and the conductive coatings are pleated and / or wound together.

[0058] The use of a pleating and / 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.

[0059] By pleating or rolling the electrode and / or stationary phase, particularly the porous substrate, preferably the membrane, a greater surface area per volume can be achieved, especially taking into account the housing, compared to a non-pleated or non-rolled, and therefore flat, electrode and / or stationary phase, particularly the substrate, preferably the membrane.

[0060] The surface area-to-volume ratio can be influenced by selecting multiple pleat configurations, particularly for housings. In this regard, pleats consist of flat areas located between specific folding points, each flat area having a length, and the pleats preferably have an M-shaped configuration. For each pleat, the first length is preferably selected to exceed at least one extension of the housing, particularly the width, diameter, etc., especially 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 area with a second length different from the first length. Most preferably, pleat sets consisting of more than one pleat are used. Such 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.

[0061] A circular housing is also beneficial for wound 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.

[0062] Experimental Section: Further experimental results are presented below, in which the monoclonal antibody trastuzumab was purified, separated, selected, and / or isolated using affinity membranes with immobilized ligands in the form of Protein A on the membrane forming binding sites. The stationary phase was provided as an arrangement consisting of an affinity membrane placed between two porous electrodes. These electrodes were formed by coating a metal onto another non-conductive support in the form of a polymeric membrane.

[0063] FIG. 1 shows the results of the experimental procedure to determine the elution voltage required to elute trastuzumab that was pre-bound to a Protein A ligand immobilized on an affinity membrane.

[0064] In this regard, Figure 1 shows the continuous measurement of absorbance over time of the fluid passing through the affinity membrane using UV detection a short distance after passing through the membrane (solid line).

[0065] The affinity membrane was loaded / fed with an ultrapure aqueous solution of trastuzumab (feed in Figure 1). (on) The leakage of trastuzumab from the membrane (A break The increase in absorbance (see the onset of the peak) indicates that the membrane is fully loaded. As a result, the loading / feeding of trastuzumab to the membrane is completed in the feed (off) Subsequently, low-concentration phosphate saline buffer (PBS) (approximately 12 μM total phosphate, 0.15 mM sodium chloride) was continuously passed through the membrane to remove residual trastuzumab that was not bound to the affinity membrane.

[0066] Then, an elution voltage was applied to the affinity membrane, and the voltage was increased stepwise (see the time for each voltage). After applying a voltage of 1500 mV, the first small peak A elut 1500 was observed, which indicated the start of elution of trastuzumab. However, when a voltage of 1500 mV was applied, the elution of trastuzumab was slight. When the voltage was increased to 2000 mV, the elution increased significantly (absorbance peak A elut 2000However, if the voltage is further increased to 2500 mV, the absorbance A elut 2500 Another peak occurred at 0.05 V, indicating that voltages above 2000 mV were required in each experimental setup to achieve maximum elution of trastuzumab.

[0067] Subsequently, when the voltage was turned off to zero, a slight increase in absorbance was observed, which was attributed to the transient adsorption of trastuzumab during the application of voltage to the energized electrode, possibly due to electrosorption.

[0068] Then, pH-triggered elution was performed by passing the affinity membrane through a sodium acetate (NaAc) buffer solution at pH 3.5 (1 M NaAc). elut pH3.5 The absorbance peak may be due to trastuzumab bound to the affinity membrane, which does not elute upon application of the elution voltage, but may also be due to residual trastuzumab pre-adsorbed on other parts of the experimental setup, such as the hose surface, the membrane housing, or other parts of the experimental opening.

[0069] FIG. 2 shows a subsequent experiment using the same experimental setup to quantitatively analyze trastuzumab elution.

[0070] Similar to the experiment shown in Figure 1, the membrane was again fed with trastuzumab until it was fully loaded, as indicated by UV detection of the fluid that had passed through the affinity membrane (not shown in Figure 2). The mass of trastuzumab bound to the binding sites of the affinity membrane was calculated to be approximately 480 μg (based on the trastuzumab concentration in the feed solution, the feed rate, and the time required to fully load the membrane). The affinity membrane used (effective diameter: 22 mm; thickness: 0.2 mm) had an effective bed volume of approximately 75 μl. Therefore, the binding capacity of the membrane used was approximately 6.4 mg / cm. 3 It was.

[0071] Elution was initiated by applying an elution voltage of 2500 mV, as determined in a previous experiment, while continuously passing low-concentration phosphate saline buffer (PBS) (approximately 12 μM total phosphate, 0.15 mM sodium chloride) through the membrane. After application of the elution voltage, peak A, the largest peak at approximately 13 bed volumes, was detected within UV detection. elut volt occurred immediately (this time includes the delay of the eluted trastuzumab moving around the affinity membrane and into the UV spectrometer). The eluted peak was analyzed in the UV and had a full width at half maximum of approximately 9 bed volumes (FWHM in Figure 2). elut (shown as ). Thus, the applied voltage initiated rapid elution, resulting in a narrow elution peak of only a few bed volumes. As a result, the described method is effective for producing concentrated biomolecule solutions.

[0072] After the elution peak dropped off sharply, the voltage was switched on and off. Switching the voltage on and off resulted in a small increase in absorbance in the UV detector. Similar to the experiment in Figure 1, this further increase may be due to transient binding of trastuzumab to the electrode, particularly via electrosorption, after it had been eluted from the binding sites.

[0073] For qualitative analysis, pH elution of the affinity membrane using sodium acetate (NaAc) buffer at pH 3.5 (1 M NaAc) was performed, and a separate elution peak A was detected by UV detection. elut pH3.5 Peak A elut volt and A elut pH3.5 By comparing the results, especially in terms of the area under the curve, it can be concluded that approximately 95% of the trastuzumab applied to the affinity membrane was eluted by applying an elution voltage. Again, the trastuzumab eluted by pH elution, rather than by applying an elution voltage, may originate from residual trastuzumab previously adsorbed on other parts of the experimental equipment, such as the hose surface, the membrane housing, or other parts of the experimental opening.

Claims

1. 1. A method for purifying, separating, selecting and / or isolating biomolecules by affinity chromatography, comprising: a) providing a stationary phase, said stationary phase containing selective binding sites formed in particular by ligands and / or receptors; b) contacting a first fluid containing biomolecules with the stationary phase, thereby binding at least some of the biomolecules to the particularly selective binding sites of the stationary phase by forming a ligand / receptor complex, particularly via non-covalent interactions, wherein one of the biomolecule and the binding site acts as the ligand and the other of the biomolecule and the binding site acts as the receptor; c) preferably passing at least a portion of said first fluid past and / or through said stationary phase and / or optionally contacting a second fluid, particularly one free of biomolecules, with said stationary phase; d) eluting the biomolecules previously bound in step b); Including, A method characterized in that the elution in step d) is carried out by applying a voltage between two electrodes, at least one of which is liquid permeable.

2. 2. The method according to claim 1, wherein the stationary phase is formed in particular by at least one porous affinity substrate, in particular a membrane, at least one of the porous affinity substrates being liquid-permeable, and wherein the ligands and / or receptors forming the binding sites are immobilized on the at least one porous affinity substrate, in particular a membrane, in particular on the inner and / or outer surface of the at least one affinity substrate, in particular a membrane.

3. 2. The method according to claim 1, wherein at least one of the electrodes is formed by at least one electrically conductive, in particular metallic, coating and / or metallic net, in particular said at least one coating and / or net being formed and / or arranged on at least one non-conductive carrier, in particular formed by a porous substrate, in particular a polymer membrane.

4. 4. The method according to claim 3, wherein at least one of the electrodes, in particular the conductive coating and / or metallic net forming the electrode, consists of a metal, in particular a metal coating, preferably gold or platinum, in particular a single metal, preferably gold or platinum.

5. The method described in claim 3, wherein the at least one conductive coating and / or metal net forming the electrode and / or the at least one non-conductive carrier are liquid permeable.

6. 4. The method according to claim 2 or 3, wherein the stationary phase is arranged on the at least one affinity substrate, in particular a membrane, and / or on the at least one non-conductive support, and comprises at least one, preferably at least two or all of the electrodes, in particular formed by a metal coating and / or the metallic net, and wherein ligands and / or receptors, in particular forming binding sites, are immobilized on the at least one electrode comprised in the stationary phase.

7. 4. The method according to claim 2 or 3, wherein the at least one porous affinity substrate, in particular a membrane, and / or the at least one non-conductive support acts as an insulator between the electrodes and / or an insulator is provided between the electrodes, in particular formed by the at least one affinity substrate, in particular a membrane and / or the at least one non-conductive support.

8. 2. The method of claim 1, wherein the ligand and / or receptor is an antigen ligand and / or receptor and / or is a protein or an aptamer, in particular an oligopeptide or an oligonucleotide.

9. 2. The method of claim 1, wherein the biological molecule to be purified, separated, selected and / or isolated is a protein, in particular an immunoglobulin, or an antibody, in particular a natural or monoclonal antibody, or an enzyme, or a nucleic acid; and / or wherein the first fluid contains at least a first biological molecule and a second biological molecule, and wherein the ligands and / or receptors forming the binding sites are selected such that the first biological molecule has a higher binding affinity to the ligand and / or receptor compared to the second biological molecule, in particular compared to all other biological molecules contained in the first fluid, and / or such that the binding affinity of the second biological molecule, in particular all other biological molecules contained in the first fluid that are different from the first biological molecule, is low and / or zero.

10. 2. The method according to claim 1, wherein, in particular during elution by voltage application and / or for at least a short time during elution by voltage application, elution by pH value correction is not carried out, and / or in particular during elution by voltage application and / or for at least a short time during elution by voltage application, any fluid added in the elution step has a pH value equal to that of the first fluid and / or differs in pH value by less than 1.0, in particular less than 0.5, in particular less than 0.2, in particular less than 0.1 compared to the pH value of the first fluid.

11. 2. The method of claim 1, wherein no fluid with a salt concentration greater than 150 mM is added for elution of the bound biomolecules, and / or the voltage applied for elution is in the range of 0.1 to 50 volts, particularly 0.1 to 3 volts, particularly 1.5 to 3.0 volts, particularly up to 2.5 volts.

12. The method of claim 1, wherein no voltage is applied before step d) and / or between steps a) and c).

13. 1. An affinity chromatography device for purifying, separating, selecting and / or isolating biomolecules, comprising a stationary phase, characterized in that the stationary phase comprises at least one porous affinity substrate, in particular formed by at least one porous affinity substrate, arranged between at least two electrodes, in particular in a stacked arrangement, at least one of the electrodes being liquid permeable and the at least one affinity substrate comprising at least one substrate as stationary phase, and binding sites formed by ligands and / or receptors being immobilized on the porous affinity substrate.

14. 14. The device according to claim 13, wherein the at least one porous affinity substrate is at least one membrane, in particular a polymer membrane, and / or the at least one electrode is formed by at least one conductive, in particular metallic, coating and / or metallic net, in particular the at least one coating and / or net being formed and / or arranged on at least one non-conductive carrier, in particular formed by a porous substrate, in particular a polymer membrane, and / or on the at least one affinity substrate, in particular a membrane.

15. 15. Apparatus according to claim 13 or 14, wherein the electrodes are configured to have a voltage applied between them preferably in the range of 0.1 to 50 volts, in particular 0.1 to 3 volts, in particular 1.5 to 3.0 volts, in particular up to 2.5 volts, and / or the electrodes are configured to exhibit an electric field on the stationary phase when a voltage is applied to the electrodes, and / or at least one of the electrodes, in particular the conductive coating and / or metallic net forming the electrode, consists of a metal, in particular a metal coating, preferably gold or platinum, in particular consists of a single metal, preferably gold or platinum.

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